Curcumin, a natural phenol that is present in turmeric has been shown to sensitize tu‐ mor cells to several anti-cancer drugs via modulation of NF-κB and histone deacetylase. Curcumin suppresses activation of NF-κB through IkB kinase (IKK) activity inhibition [69]. In a xenograft model, curcumin plus paclitaxel significantly suppressed the inci‐ dence of breast cancer metastasis in lung tissue, and also demonstrated in these lung tis‐ sues was the reduction of the p65 subunit of NF-κB [70]. By combining compounds which can either directly or indirectly inhibit the NF-KB signaling pathway concomitant with chemotherapy, the resulting synergistic treatment may allow lower doses of the tox‐ ic chemotherapeutic agents to be used, improving patient responses [71]. These data help to demonstrate that down regulation of the NF-κB pathway could lead to the tumor cells

**Figure 3.** Model showing the effects of NF-κB on DNA damage and repair

activation of the NF-κB pathways which directly induced a transcriptional repression of the collagen gene expression [63]. Taken together, it could be hypothesized that the elevation of NF-κB transcriptional activity may contribute to the decrease in DNA repair capacity of skin cells and thereby lead to accelerated skin aging. Since NF-κB is activated by DNA damage, there is a potential for a vicious circle to take place as more NF-κB may decrease the capacity

of the cell to repair damages and lead to a longer persistence of the DNA damages.

ly mediate cell survival, proliferation, invasion, and metastasis [65].

**chemotherapy**

220 New Research Directions in DNA Repair

chemoresistant tumors [68]

**7. NF-κB and the development of resistance to alkylating agent-based**

In addition to the putative role of NF-κB and the decreased DNA repair capacity of skin cells leading to skin aging, NF-κB regulation of DNA repair may also contribute to chemore‐ sistance. Studies of chemotherapeutic resistance have shown a significant correlation exists between NF-κB activation and the decreased effectiveness of some chemotherapeutic agents. Agents such as taxol and irradiation treatments upregulate the transcription factor NF-κB which leads to promoting survival and chemoresistance in solid tumor cancers [64]. The mechanism for this chemoresistance is through the activation NF-κB which can subsequent‐

Sphingosine kinase may be of therapeutic interest in the context of inflammatory disease and drug resistant cancers. Sphingolipid metabolism has been shown to be aberrant in breast cancer tumor samples, resulting from an increase of sphingosine kinase expression [66]. The sphingosine kinase cascade pathway was first linked to the NF-κB pathway in 1998 via demonstration that TNF induced adhesion was mediated through sphingosine kinase signaling, which links to downstream NF-κB activation [67]. Using a novel selective Sphk2 inhibitor, ABC294640, Antoon et al. demonstrated inhibition of NF-κB activation via inhibi‐ tion of Sphk2 [68]. In vivo testing in a well-established immunocompromised xenograft model for tumor growth, demonstrated that this inhibitor showed lower proliferation of cancerous cells, and no tumor growth when compared to control. This establishes the under‐ lying pathways including the inhibition of NF-κB activation, as viable target for otherwise

Curcumin, a natural phenol that is present in turmeric has been shown to sensitize tu‐ mor cells to several anti-cancer drugs via modulation of NF-κB and histone deacetylase. Curcumin suppresses activation of NF-κB through IkB kinase (IKK) activity inhibition [69]. In a xenograft model, curcumin plus paclitaxel significantly suppressed the inci‐ dence of breast cancer metastasis in lung tissue, and also demonstrated in these lung tis‐ sues was the reduction of the p65 subunit of NF-κB [70]. By combining compounds which can either directly or indirectly inhibit the NF-KB signaling pathway concomitant with chemotherapy, the resulting synergistic treatment may allow lower doses of the tox‐ ic chemotherapeutic agents to be used, improving patient responses [71]. These data help to demonstrate that down regulation of the NF-κB pathway could lead to the tumor cells becoming more susceptible to current chemotherapies, and allow for lower doses of these therapies, leading to better patient outcomes.

## **8. Summary: The regulation of DNA damage and DNA repair by NF-κB**

Skin is under continuous assault from a variety of damaging environmental factors includ‐ ing ultraviolet irradiation and atmospheric pollutants. Extrinsic factors, particularly sun‐ light, have been demonstrated to accelerate the intrinsic aging process by increasing free radical production and decreasing antioxidant protections which can result in DNA damage and can affect the repair of damaged DNA. The age-related accumulation of somatic dam‐ age is worsened by sun exposure, leading to an increased incidence of skin disorders, skin cancer and potentially skin aging. New findings on the molecular mechanisms involved in the regulation of DNA damage and the subsequent repair of damaged DNA in the skin can help identify new targets to modulate DNA repair activity and thereby have a significant effect on skin physiology. The NF-κB pathway is a key regulator of inflammatory mediators in skin cells and has been reported to be the final common pathway for the conversion of environmental insults into inflammation in the skin. Through the ability to regulate process‐ es that result in increased DNA damage and decrease the repair of damaged DNA, the NFκB pathway may be a primary pathway linking inflammation and DNA damage. Pharmacological inhibition of NF-κB therefore may provide protection to skin from the nu‐ merous external aggressions encountered daily and reduce the DNA damage to oxidatively challenged and aging skin by increasing endogenous DNA repair processes.

[8] Kleijer, W.J., et al., Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA Re‐

Regulation of DNA Repair Process by the Pro-Inflammatory NF-κB Pathway

http://dx.doi.org/10.5772/54341

223

[9] Robbins, J.H., et al., Xeroderma pigmentosum. An inherited diseases with sun sensi‐ tivity, multiple cutaneous neoplasms, and abnormal DNA repair. Ann Intern Med,

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[16] Niedernhofer, L.J., Tissue-specific accelerated aging in nucleotide excision repair de‐

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## **Acknowledgements**

The authors would like to thank Dr. Paul Khavari ( Department of Dermatology, Stanford University) and Hélène Wong (Johnson and Johnson) for discussions on NF-κB regulation and DNA Damage

## **Author details**

Simarna Kaur1 , Thierry Oddos2 , Samantha Tucker-Samaras1 and Michael D. Southall1

1 Johnson & Johnson Skin Research Center, CPPW, a Division of Johnson & Johnson Con‐ sumer Companies, Inc. Skillman, New Jersey, USA

2 Johnson & Johnson Skin Research Center, CPPW, a Division of Johnson & Johnson Con‐ sumer Companies, Inc. Skillman, New Jersey, France

### **References**

[8] Kleijer, W.J., et al., Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA Re‐ pair (Amst), 2008. 7(5): p. 744-50.

Pharmacological inhibition of NF-κB therefore may provide protection to skin from the nu‐ merous external aggressions encountered daily and reduce the DNA damage to oxidatively

The authors would like to thank Dr. Paul Khavari ( Department of Dermatology, Stanford University) and Hélène Wong (Johnson and Johnson) for discussions on NF-κB regulation

, Samantha Tucker-Samaras1

1 Johnson & Johnson Skin Research Center, CPPW, a Division of Johnson & Johnson Con‐

2 Johnson & Johnson Skin Research Center, CPPW, a Division of Johnson & Johnson Con‐

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**Section 2**

**Interface with Replication, Transcription,**

**Telomeres, and Cell Cycle Regulation**

**Interface with Replication, Transcription, Telomeres, and Cell Cycle Regulation**

**Chapter 9**

**Relation of the Types of**

Magdalena Kowalewicz-Kulbat

Additional information is available at the end of the chapter

properly cited.

Dorota Rybaczek and

http://dx.doi.org/10.5772/54020

**1. Introduction**

damage.

breaks (SSBs).

**DNA Damage to Replication Stress and**

**the Induction of Premature Chromosome Condensation**

Any integrated view of the diversity of biochemical reactions involved in the faithful rep‐ lication of eukaryotic chromosomes and their accurate mitotic segregation is not possible without careful consideration of the molecular mechanisms that are responsible for repair‐ ing damaged DNA. In order to arrange and order the sequence of events, in which the various levels of organization are only stages of the same molecular pathway, there is a need for both a timely switching on of numerous genes and the precise cooperation of large numbers of proteins. An important clue concerning the nature of the competitive in‐ teraction between these different elements comes from looking at the response to DNA

The present chapter is a review of the types of DNA damage generated under stressful conditions and experimental approaches to the relation of these types of DNA damage to hydroxyurea treatment and caffeine-induced premature chromosome condensation (PCC). In this chapter, an attempt is also made to explain the molecular base of DNA damage and to present experimental procedures allowing the illustration of DNA damages at the cell level, especially with the use of histochemical and immunocytochemical methods. It will be experimentally shown, among others, that replication stress mainly leads to the generation of double-strand breaks in DNA (DSBs), while the breakage of restrictive inter‐ actions of checkpoints during PCC induction results in the accumulation of single-strand

and reproduction in any medium, provided the original work is properly cited.

© 2013 Rybaczek and Kowalewicz-Kulbat; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Chapter 9**

## **Relation of the Types of DNA Damage to Replication Stress and the Induction of Premature Chromosome Condensation**

Dorota Rybaczek and Magdalena Kowalewicz-Kulbat

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54020

## **1. Introduction**

Any integrated view of the diversity of biochemical reactions involved in the faithful rep‐ lication of eukaryotic chromosomes and their accurate mitotic segregation is not possible without careful consideration of the molecular mechanisms that are responsible for repair‐ ing damaged DNA. In order to arrange and order the sequence of events, in which the various levels of organization are only stages of the same molecular pathway, there is a need for both a timely switching on of numerous genes and the precise cooperation of large numbers of proteins. An important clue concerning the nature of the competitive in‐ teraction between these different elements comes from looking at the response to DNA damage.

The present chapter is a review of the types of DNA damage generated under stressful conditions and experimental approaches to the relation of these types of DNA damage to hydroxyurea treatment and caffeine-induced premature chromosome condensation (PCC). In this chapter, an attempt is also made to explain the molecular base of DNA damage and to present experimental procedures allowing the illustration of DNA damages at the cell level, especially with the use of histochemical and immunocytochemical methods. It will be experimentally shown, among others, that replication stress mainly leads to the generation of double-strand breaks in DNA (DSBs), while the breakage of restrictive inter‐ actions of checkpoints during PCC induction results in the accumulation of single-strand breaks (SSBs).

properly cited.

© 2013 Rybaczek and Kowalewicz-Kulbat; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. The types and molecular base of DNA damage**

DNA can be damaged by the action of endogenous (intrinsic) or exogenous (extrinsic) stress factors. The endogenous factors include, among others, errors generated during replication and reactive oxygen species (ROS). The exogenous (environmental) factors are divided into (i) physical factors, e.g. UV and ionizing radiation (X, γ); (ii) chemical factors, i.e. mutagenic polycyclic aromatic hydrocarbons (PAH), nitrosamines, dioxins, analogues of bases and al‐ kylating agents; and (iii) biological factors, such as viruses.

**3. Replication stress and activation of checkpoint signaling pathways**

2002; comp. Rybaczek & Kowalewicz-Kulbat, 2011)].

**Figure 1.** The three major S-phase checkpoints within the cell cycle

Under the conditions of replication stress, the rate of DNA synthesis is slowed down and the possibility of entry into mitosis is blocked until the expression of specific genes and acti‐ vation of repair factors. The control over DNA synthesis then involves a system of intra-S phase checkpoint, activated after the detection of DNA damage - in particular double strand breaks (DSBs) or single-strand breaks (SSBs) [Figure 1; (Bartek et al., 2004; Osborn et al.,

Relation of the Types of DNA Damage to Replication Stress and the Induction of Premature Chromosome

Condensation

233

http://dx.doi.org/10.5772/54020

Further stages of the cell cycle are blocked until the repair of detected damage (Adamsen et al., 2011; Herrick & Bensimon, 2008). It has also been shown that any disruption of structural nature (e.g. DSB or SSB) induces a slowdown in the replication fork movement and further DNA damage, e.g. through the influence of replication inhibitors, may result in total inhibi‐ tion of the cycle in the intra-S phase checkpoint (Blow & Hodgson, 2002; Elledge, 1996). Then checkpoint sensory factors trigger a signal transduction cascade, delivering a signal of DNA damage to effector proteins via transmitters (Mordes & Cortez, 2008; Nojima, 2006).

Thus, the detection of DSBs activates an ATM-dependent pathway (*Ataxia Telangiectasia Mu‐ tated*) and a slightly more slowly activated parallel ATR-dependent pathway (*Ataxia Telan‐*

Stress-induced damage includes spontaneous depurination and deamination, oxidation, for‐ mation of DNA adducts induced by alkylating agents, formation of cyclobutane dimers, sin‐ gle- and double-strand damage, as well as errors made during replication, repair, reverse transcription and recombination. DNA is also subject to covalent modifications that may af‐ fect nitrogen bases and lead to changes in base pairing between DNA strands, or even en‐ tirely preventing base pairing. Genomic instability may also be associated with chromosomal rearrangements which result from changes that occur in the *trans* position (in‐ cluding replication, DNA repair and S phase checkpoint pathways) or from changes that act in the *cis* position, i.e. in the regions of chromosomal instability, known as hotspots, for ex‐ ample breaks or fragile sites and highly transcribed DNA sequences (Aguilera & Gómez-González, 2008).

Plants, due to their 'settled' lifestyles are exposed to many environmental factors that cause disturbances in the cell cycle. They are often threatened by excessive salinity, drought, ex‐ treme low or high temperatures, as well as fungal or bacterial infections (Vashisht & Tuteja, 2006). Each of these burdens leads to the mobilization of defense responses: (1) activation of cell cycle checkpoints and DNA repair factors, (2) inhibition of cell growth, or (3) initiation of the apoptosis pathway (Deckert et al., 2009 and references therein).

Recognition of double-stranded breaks depends on the MRN complex (Mre11-Rad50-Nbs1), necessary for binding chromatin-remodeling factors (Schiller et al., 2012). MRN complex acts as a stabilizing platform for broken endings of DNA molecules. It binds to the sites of dam‐ age and ATM kinase, and promotes phosphorylation of histone H2A (H2AX-Ser139) and the processing of DNA. Processing of ends can either rely on their alignment, necessary to con‐ tinue the connection through the induction of non-homologous end joining, or long singlestranded fragments for homologous recombination. Eukaryotic organisms use many types of DNA repair: (i) 3'-5' exonuclease activity of DNA polymerase; (ii) reversion repair (RR); (iii) mismatch repair (MMR); (iv) base excision repair (BER); (v) nucleotide excision repair (NER), (vi) non-homologous end joining (NHEJ); (vii) homologous recombination (HR); (viii) translesion synthesis (TLS). The methods also include: photoreactivation; methylgua‐ nine methyltransferase (MGMT), catalyzing the reaction of demethylation of methylated guanine bases; double strand break repair (DSBR); synthesis-dependent strand annealing (SDSA) and break-induced replication (BIR).

## **3. Replication stress and activation of checkpoint signaling pathways**

**2. The types and molecular base of DNA damage**

kylating agents; and (iii) biological factors, such as viruses.

González, 2008).

232 New Research Directions in DNA Repair

DNA can be damaged by the action of endogenous (intrinsic) or exogenous (extrinsic) stress factors. The endogenous factors include, among others, errors generated during replication and reactive oxygen species (ROS). The exogenous (environmental) factors are divided into (i) physical factors, e.g. UV and ionizing radiation (X, γ); (ii) chemical factors, i.e. mutagenic polycyclic aromatic hydrocarbons (PAH), nitrosamines, dioxins, analogues of bases and al‐

Stress-induced damage includes spontaneous depurination and deamination, oxidation, for‐ mation of DNA adducts induced by alkylating agents, formation of cyclobutane dimers, sin‐ gle- and double-strand damage, as well as errors made during replication, repair, reverse transcription and recombination. DNA is also subject to covalent modifications that may af‐ fect nitrogen bases and lead to changes in base pairing between DNA strands, or even en‐ tirely preventing base pairing. Genomic instability may also be associated with chromosomal rearrangements which result from changes that occur in the *trans* position (in‐ cluding replication, DNA repair and S phase checkpoint pathways) or from changes that act in the *cis* position, i.e. in the regions of chromosomal instability, known as hotspots, for ex‐ ample breaks or fragile sites and highly transcribed DNA sequences (Aguilera & Gómez-

Plants, due to their 'settled' lifestyles are exposed to many environmental factors that cause disturbances in the cell cycle. They are often threatened by excessive salinity, drought, ex‐ treme low or high temperatures, as well as fungal or bacterial infections (Vashisht & Tuteja, 2006). Each of these burdens leads to the mobilization of defense responses: (1) activation of cell cycle checkpoints and DNA repair factors, (2) inhibition of cell growth, or (3) initiation

Recognition of double-stranded breaks depends on the MRN complex (Mre11-Rad50-Nbs1), necessary for binding chromatin-remodeling factors (Schiller et al., 2012). MRN complex acts as a stabilizing platform for broken endings of DNA molecules. It binds to the sites of dam‐ age and ATM kinase, and promotes phosphorylation of histone H2A (H2AX-Ser139) and the processing of DNA. Processing of ends can either rely on their alignment, necessary to con‐ tinue the connection through the induction of non-homologous end joining, or long singlestranded fragments for homologous recombination. Eukaryotic organisms use many types of DNA repair: (i) 3'-5' exonuclease activity of DNA polymerase; (ii) reversion repair (RR); (iii) mismatch repair (MMR); (iv) base excision repair (BER); (v) nucleotide excision repair (NER), (vi) non-homologous end joining (NHEJ); (vii) homologous recombination (HR); (viii) translesion synthesis (TLS). The methods also include: photoreactivation; methylgua‐ nine methyltransferase (MGMT), catalyzing the reaction of demethylation of methylated guanine bases; double strand break repair (DSBR); synthesis-dependent strand annealing

of the apoptosis pathway (Deckert et al., 2009 and references therein).

(SDSA) and break-induced replication (BIR).

Under the conditions of replication stress, the rate of DNA synthesis is slowed down and the possibility of entry into mitosis is blocked until the expression of specific genes and acti‐ vation of repair factors. The control over DNA synthesis then involves a system of intra-S phase checkpoint, activated after the detection of DNA damage - in particular double strand breaks (DSBs) or single-strand breaks (SSBs) [Figure 1; (Bartek et al., 2004; Osborn et al., 2002; comp. Rybaczek & Kowalewicz-Kulbat, 2011)].

**Figure 1.** The three major S-phase checkpoints within the cell cycle

Further stages of the cell cycle are blocked until the repair of detected damage (Adamsen et al., 2011; Herrick & Bensimon, 2008). It has also been shown that any disruption of structural nature (e.g. DSB or SSB) induces a slowdown in the replication fork movement and further DNA damage, e.g. through the influence of replication inhibitors, may result in total inhibi‐ tion of the cycle in the intra-S phase checkpoint (Blow & Hodgson, 2002; Elledge, 1996). Then checkpoint sensory factors trigger a signal transduction cascade, delivering a signal of DNA damage to effector proteins via transmitters (Mordes & Cortez, 2008; Nojima, 2006).

Thus, the detection of DSBs activates an ATM-dependent pathway (*Ataxia Telangiectasia Mu‐ tated*) and a slightly more slowly activated parallel ATR-dependent pathway (*Ataxia Telan‐* *giectasia mutated – Rad3-related*). The target substrate for both these sensory kinases is Cdc25 phosphatase (Cortez, 2003). The function of ATR kinase is not limited solely to the transmis‐ sion of signals in response to DNA double breaks in the S phase checkpoint. This enzyme is activated during each S phase and plays an active role in regulating the initiation of DNA replication under physiological conditions. In addition, it is involved in the recognition of single-stranded DNA molecules (ssDNA; Shechter et al., 2004). ATR occurs in a durable complex with ATR-interacting protein (ATRIP), focusing in the area of the nucleus in re‐ gions corresponding to the sites of DNA damage (Myers et al., 2007). Research carried out on cytoplasmic extracts of *Xenopus* oocytes revealed that ATR associates with chromatin during DNA replication, and dissociates after its completion (Freire et al., 2006; Harper & Elledge, 2007; reviewed by Marheineke & Hyrien, 2004). The association of ATR and DNA breaks is also a result of the elimination of the replication factor A (RPA), while its appear‐ ance is independent of the presence of α-type DNA polymerase. Therefore it seems that the "recruitment" of ATR occurs after a partial generation of replication forks in the *origin* re‐ gion, but before Polα association (Luciani et al., 2004; Namiki & Zaou, 2006; Zou & Elledge, 2003). Although ATR-ATRIP complexes can bind to certain DNA structures, their participa‐ tion in the activation of cell responses to replication stress is not possible without the partici‐ pation of two other factors: replication factor C (RFC) and proliferating-cell-nuclear-antigenlike proteins (PCNA-like). During replication, RFC recognizes the binding sites between primers/starters of RNA and DNA matrix and assembles PCNA, a toroidal homotrimer pro‐ tein encircling DNA – also known as a "sliding clamp" which determines the processivity of the related DNA polymerases (Majka & Burgers, 2004; Tan et al., 2012). In the cells of *S. pombe*, Rad17 (RFC1 factor and four small subunits RFC2-5) and Rad9/Hus1/Rad1 (PCNAlike 9-1-1 complex), participate not only in the functional organization of the intra-S phase checkpoint, but also other cell cycle checkpoints whose function is to monitor the structural DNA damage [e.g. G2 (Majka et al., 2006, reviewed by Lin & Dutta, 2007)]. Recruitment of PCNA-like complexes to the sites of DNA damage in a molecule is, perhaps, independent of the activation of ATR and Chk1 (Niimi et al., 2008; Scorah et al., 2008), but is an important element of the mechanism signaling the appearance of structural disorders. In the cells of *S. pombe* and in mammals, Rad17 and Hus1 are factors determining the possibility of phos‐ phorylation of Chk1 kinase by ATR. Rad17 is also a substrate of ATR. Although both these proteins bind to chromatin in intact cells, phosphorylation of Rad17 by ATR significantly in‐ creases with the increasing volume of PCNA-like complexes, following the occurrence of DNA conformational disorders. It therefore appears that the first stage of the then triggered signaling pathway is the independent localization of Rad17 and ATR-ATRIP complexes in the regions of damage; the next stage is a Rad17-dependent assembly of PCNA-like com‐ plexes around the DNA. PCNA-like complexes enable the activation of ATR molecules and consequently - the phosphorylation of ATR substrates located within chromatin, such as Rad17 and Rad9 (Majka et al., 2006; Niida & Nakanishi, 2006). In addition to ATM and ATR kinases in humans, and their homologues in yeast cells, the PIKK family of signaling pro‐ teins includes also DNA-dependent protein kinase (DNA-PK). This enzyme consists of a DNA-PK catalytic subunit (DNA-PKCS,) and a heterodimeric subunit Ku70-Ku80. DNA-PKCS is a DNA-dependent serine-threonine kinase, showing a relatively weak ability to

bind to DNA free ends; however, this affinity is enhanced and stabilizes under the influence of heterodimer Ku70-Ku80. It is believed that DNA-PK participates primarily in the repair of double-strand breaks (DSBs) by non-homologous end-joining [NHEJ (Müller et al., 2007; Pa‐

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Replication protein A (RPA) binds to all single-strand DNAs in the nucleus, including the parts of ssDNA formed during DNA replication and repair (Costanzo et al., 2003). The asso‐ ciation of RPA and ssDNA (RPA-ssDNA) is an important component of signaling and the place to which the ATR molecule binds (this mechanism occurs both in human cells and in *S. cerevisiae*; Zou & Elledge, 2003). However, recognition of RPA-ssDNA structures and re‐ cruitment of other proteins to these complexes occur through the activity of ATRIP which occurs in conjunction with the ATR kinase. Biochemical studies indicate that ATRIP binds to the N-terminal part of the large subunit of RPA via its conserved acidic alpha-helix domain (Ball et al., 2007). The RPA-ssDNA complex is not a sufficient stimulus for binding the ATR-ATRIP complex and does not activate ATR. The induction and transmission of the signal "down" depends on ATR-ATRIP interaction with another protein complex, i.e. 9-1-1, which recognizes the DNA end adjacent to the RPA-coated ssDNA. The 9-1-1 complex is also re‐ sponsible for recruiting TopBP1 protein, the main activator of ATR-ATRIP complex in the cells of vertebrates (Kumagai et al., 2006). In addition, the RPA-ssDNA platform recruits RAD17 and claspin, proteins strongly interacting with ATR, leading to the phosphorylation of ATR substrates, including Chk1 kinase (Bartek et al., 2004). Thus the presence of RPA is crucial for the specific recruitment of signaling factors to the 5' end of the damaged DNA (Ellison & Stillman, 2003). In this case, it is single-strand DNA fragments that are responsi‐ ble for the activation of the checkpoint. Structures of this type are generated as a result of impaired DNA polymerase activity during replication, during the formation of double strand DNA breaks, at the ends of telomeres, and even during DNA repair via nucleotide excision. All of these factors activate the ATR kinase to recruit repair proteins (Byun et al., 2005; Cimprich & Cortez, 2008; Nedelcheva et al., 2005). Recent studies have shown that for the effective recruitment and signaling in response to DNA damage, ATR kinase requires continuous cooperation with its sister sensory ATM kinase, showing some similarity in structure and function (Cimprich & Cortez, 2008). These kinases also share phosphorylation

substrates, e.g. H2AX histones (Burma et al., 2001; Ward & Chen, 2001).

**checkpoint**

**4. Premature chromosome condensation and overriding of cell cycle**

The initiation of mitotic chromosome condensation in normal cells is preceded by the completion of all processes related to DNA replication and repair of abnormal DNA structures generated during the S phase. The main task of the checkpoint in G2 phase is to block cell entry into mitosis in the event of an anomaly in the genetic material. The common elements of the biochemical pathway that control the G2/M transition and of the S-phase checkpoint, are ATM and ATR kinases, and their role is to maintain the MPF complex, i.e. M-phase promoting factor (CDK1 kinase with cyclin B) in an inactive state

welczak & Turchi, 2008; Shimura et al., 2007)].

bind to DNA free ends; however, this affinity is enhanced and stabilizes under the influence of heterodimer Ku70-Ku80. It is believed that DNA-PK participates primarily in the repair of double-strand breaks (DSBs) by non-homologous end-joining [NHEJ (Müller et al., 2007; Pa‐ welczak & Turchi, 2008; Shimura et al., 2007)].

*giectasia mutated – Rad3-related*). The target substrate for both these sensory kinases is Cdc25 phosphatase (Cortez, 2003). The function of ATR kinase is not limited solely to the transmis‐ sion of signals in response to DNA double breaks in the S phase checkpoint. This enzyme is activated during each S phase and plays an active role in regulating the initiation of DNA replication under physiological conditions. In addition, it is involved in the recognition of single-stranded DNA molecules (ssDNA; Shechter et al., 2004). ATR occurs in a durable complex with ATR-interacting protein (ATRIP), focusing in the area of the nucleus in re‐ gions corresponding to the sites of DNA damage (Myers et al., 2007). Research carried out on cytoplasmic extracts of *Xenopus* oocytes revealed that ATR associates with chromatin during DNA replication, and dissociates after its completion (Freire et al., 2006; Harper & Elledge, 2007; reviewed by Marheineke & Hyrien, 2004). The association of ATR and DNA breaks is also a result of the elimination of the replication factor A (RPA), while its appear‐ ance is independent of the presence of α-type DNA polymerase. Therefore it seems that the "recruitment" of ATR occurs after a partial generation of replication forks in the *origin* re‐ gion, but before Polα association (Luciani et al., 2004; Namiki & Zaou, 2006; Zou & Elledge, 2003). Although ATR-ATRIP complexes can bind to certain DNA structures, their participa‐ tion in the activation of cell responses to replication stress is not possible without the partici‐ pation of two other factors: replication factor C (RFC) and proliferating-cell-nuclear-antigenlike proteins (PCNA-like). During replication, RFC recognizes the binding sites between primers/starters of RNA and DNA matrix and assembles PCNA, a toroidal homotrimer pro‐ tein encircling DNA – also known as a "sliding clamp" which determines the processivity of the related DNA polymerases (Majka & Burgers, 2004; Tan et al., 2012). In the cells of *S. pombe*, Rad17 (RFC1 factor and four small subunits RFC2-5) and Rad9/Hus1/Rad1 (PCNAlike 9-1-1 complex), participate not only in the functional organization of the intra-S phase checkpoint, but also other cell cycle checkpoints whose function is to monitor the structural DNA damage [e.g. G2 (Majka et al., 2006, reviewed by Lin & Dutta, 2007)]. Recruitment of PCNA-like complexes to the sites of DNA damage in a molecule is, perhaps, independent of the activation of ATR and Chk1 (Niimi et al., 2008; Scorah et al., 2008), but is an important element of the mechanism signaling the appearance of structural disorders. In the cells of *S. pombe* and in mammals, Rad17 and Hus1 are factors determining the possibility of phos‐ phorylation of Chk1 kinase by ATR. Rad17 is also a substrate of ATR. Although both these proteins bind to chromatin in intact cells, phosphorylation of Rad17 by ATR significantly in‐ creases with the increasing volume of PCNA-like complexes, following the occurrence of DNA conformational disorders. It therefore appears that the first stage of the then triggered signaling pathway is the independent localization of Rad17 and ATR-ATRIP complexes in the regions of damage; the next stage is a Rad17-dependent assembly of PCNA-like com‐ plexes around the DNA. PCNA-like complexes enable the activation of ATR molecules and consequently - the phosphorylation of ATR substrates located within chromatin, such as Rad17 and Rad9 (Majka et al., 2006; Niida & Nakanishi, 2006). In addition to ATM and ATR kinases in humans, and their homologues in yeast cells, the PIKK family of signaling pro‐ teins includes also DNA-dependent protein kinase (DNA-PK). This enzyme consists of a DNA-PK catalytic subunit (DNA-PKCS,) and a heterodimeric subunit Ku70-Ku80. DNA-PKCS is a DNA-dependent serine-threonine kinase, showing a relatively weak ability to

234 New Research Directions in DNA Repair

Replication protein A (RPA) binds to all single-strand DNAs in the nucleus, including the parts of ssDNA formed during DNA replication and repair (Costanzo et al., 2003). The asso‐ ciation of RPA and ssDNA (RPA-ssDNA) is an important component of signaling and the place to which the ATR molecule binds (this mechanism occurs both in human cells and in *S. cerevisiae*; Zou & Elledge, 2003). However, recognition of RPA-ssDNA structures and re‐ cruitment of other proteins to these complexes occur through the activity of ATRIP which occurs in conjunction with the ATR kinase. Biochemical studies indicate that ATRIP binds to the N-terminal part of the large subunit of RPA via its conserved acidic alpha-helix domain (Ball et al., 2007). The RPA-ssDNA complex is not a sufficient stimulus for binding the ATR-ATRIP complex and does not activate ATR. The induction and transmission of the signal "down" depends on ATR-ATRIP interaction with another protein complex, i.e. 9-1-1, which recognizes the DNA end adjacent to the RPA-coated ssDNA. The 9-1-1 complex is also re‐ sponsible for recruiting TopBP1 protein, the main activator of ATR-ATRIP complex in the cells of vertebrates (Kumagai et al., 2006). In addition, the RPA-ssDNA platform recruits RAD17 and claspin, proteins strongly interacting with ATR, leading to the phosphorylation of ATR substrates, including Chk1 kinase (Bartek et al., 2004). Thus the presence of RPA is crucial for the specific recruitment of signaling factors to the 5' end of the damaged DNA (Ellison & Stillman, 2003). In this case, it is single-strand DNA fragments that are responsi‐ ble for the activation of the checkpoint. Structures of this type are generated as a result of impaired DNA polymerase activity during replication, during the formation of double strand DNA breaks, at the ends of telomeres, and even during DNA repair via nucleotide excision. All of these factors activate the ATR kinase to recruit repair proteins (Byun et al., 2005; Cimprich & Cortez, 2008; Nedelcheva et al., 2005). Recent studies have shown that for the effective recruitment and signaling in response to DNA damage, ATR kinase requires continuous cooperation with its sister sensory ATM kinase, showing some similarity in structure and function (Cimprich & Cortez, 2008). These kinases also share phosphorylation substrates, e.g. H2AX histones (Burma et al., 2001; Ward & Chen, 2001).

## **4. Premature chromosome condensation and overriding of cell cycle checkpoint**

The initiation of mitotic chromosome condensation in normal cells is preceded by the completion of all processes related to DNA replication and repair of abnormal DNA structures generated during the S phase. The main task of the checkpoint in G2 phase is to block cell entry into mitosis in the event of an anomaly in the genetic material. The common elements of the biochemical pathway that control the G2/M transition and of the S-phase checkpoint, are ATM and ATR kinases, and their role is to maintain the MPF complex, i.e. M-phase promoting factor (CDK1 kinase with cyclin B) in an inactive state (Raleigh & Connell, 2000). Both in animal cells and in yeast, the activation of the CDK2 cyclin B complex, induced by phosphatase Cdc25, is a necessary condition for the initia‐ tion of mitotic chromosome condensation. The activation of ATM and ATR kinases during the G2 phase causes a cascade of phosphorylation. Similar to DNA replication, the substrates of these sensory kinases are the kinases Chk2 (for ATM) and Chk1 (for ATR). Chk1 kinase (active form) phosphorylates Cdc25 phosphatase by blocking its enzymatic activity (Cdc25 is then not able to carry out the activating dephosphorylation of CDK1 kinase; De Veylder et al., 2003). Phosphorylation of the phosphatase Cdc25 can lead to its degradation through ubiquitin-dependent proteolysis, or to association with 14-3-3 pro‐ tein and consequently to its removal from the nucleus (Boutros et al., 2006). At the same time, ATM and ATR kinases induce gene expression of Wee1 kinase (responsible for blocking cell cycle progression in G2 phase), thus gaining the time required to repair de‐ fective DNA structures. Probably, the activation of Wee1 kinase also involves the activity of kinases Chk1 and Chk2 (De Schutter et al., 2007). In animal cells, ATM kinase also acti‐ vates the p53 pathway. This factor is involved, among others, in the regulation of re‐ sponses to replication stress, altered DNA structure, oxidative stress and osmotic shock, and disturbances in the integrity of cell membranes. Because of its multiple functions in cell cycle regulation, p53 has been termed 'the guardian of the genome' (Han et al., 2008).

**Figure 3.** Feulgen-stained root meristem cells of *Vicia faba*: (A) hydroxyurea-treated (2.5 mM, 24 h); (B) caffeine-in‐ duced PCC (2.5 mM HU for 24 h → the mixture of 2.5 mM HU and 5 mM CF for 8 h). The array of aberrations in serie 'A' included a relatively small number of breakpoints per cell nucleus (≤ 5). The full array of aberrations (≥ 25 per cell nu‐ cleus) in serie 'B' included chromosomal breaks, irregular condensation/decondensation of chromatin, lost and lag‐ ging chromatids and chromosomes as well as segregation defects. Micronucleus formation (arrows), were found significantly increased in comparison either with the control or HU treatment (comp. Rybaczek & Kowalewicz-Kulbat, 2011; Rybaczek et al., 2008). The mitotic index was calculated as the percent ratio between the number of dividing cells and the entire meristematic cell population. Index of aberrations was calculated as the percent ratio between the number of cells showing chromosome aberrations and all mitotic cells. PCC index was calculated as the percent ratio between the number of cells showing chromosome aberrations typical of premature mitosis and all mitotic cells. Ex‐ perimental procedure of Feulgen staining: root tips were fixed in cold absolute ethanol and glacial acetic acid (3:1, v/v) for 1 h, washed several times with ethanol, rehydrated, hydrolysed in 4 M HCl (1.5 h), and stained with Schiff's reagent (pararosaniline; Sigma-Aldrich) according to standard methods. After rinsing in SO2-water (3 times) and distil‐ led water, 1.5 mm long apical segments were cut off, placed in a drop of 45% acetic acid, and squashed onto micro‐ scope slides. Following freezing with dry ice, coverslips were removed and the dehydrated dry slides were embedded in Canada Baume. Slides were analysed under the light microscope to count mitotic cells that had characteristic fea‐

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tures of either normal mitosis or PCC. *Bar* 20 μm

**Figure 2.** Overview of the induction of premature chromosome condensation (PCC)

Relation of the Types of DNA Damage to Replication Stress and the Induction of Premature Chromosome Condensation http://dx.doi.org/10.5772/54020 237

(Raleigh & Connell, 2000). Both in animal cells and in yeast, the activation of the CDK2 cyclin B complex, induced by phosphatase Cdc25, is a necessary condition for the initia‐ tion of mitotic chromosome condensation. The activation of ATM and ATR kinases during the G2 phase causes a cascade of phosphorylation. Similar to DNA replication, the substrates of these sensory kinases are the kinases Chk2 (for ATM) and Chk1 (for ATR). Chk1 kinase (active form) phosphorylates Cdc25 phosphatase by blocking its enzymatic activity (Cdc25 is then not able to carry out the activating dephosphorylation of CDK1 kinase; De Veylder et al., 2003). Phosphorylation of the phosphatase Cdc25 can lead to its degradation through ubiquitin-dependent proteolysis, or to association with 14-3-3 pro‐ tein and consequently to its removal from the nucleus (Boutros et al., 2006). At the same time, ATM and ATR kinases induce gene expression of Wee1 kinase (responsible for blocking cell cycle progression in G2 phase), thus gaining the time required to repair de‐ fective DNA structures. Probably, the activation of Wee1 kinase also involves the activity of kinases Chk1 and Chk2 (De Schutter et al., 2007). In animal cells, ATM kinase also acti‐ vates the p53 pathway. This factor is involved, among others, in the regulation of re‐ sponses to replication stress, altered DNA structure, oxidative stress and osmotic shock, and disturbances in the integrity of cell membranes. Because of its multiple functions in cell cycle regulation, p53 has been termed 'the guardian of the genome' (Han et al., 2008).

236 New Research Directions in DNA Repair

**Figure 2.** Overview of the induction of premature chromosome condensation (PCC)

**Figure 3.** Feulgen-stained root meristem cells of *Vicia faba*: (A) hydroxyurea-treated (2.5 mM, 24 h); (B) caffeine-in‐ duced PCC (2.5 mM HU for 24 h → the mixture of 2.5 mM HU and 5 mM CF for 8 h). The array of aberrations in serie 'A' included a relatively small number of breakpoints per cell nucleus (≤ 5). The full array of aberrations (≥ 25 per cell nu‐ cleus) in serie 'B' included chromosomal breaks, irregular condensation/decondensation of chromatin, lost and lag‐ ging chromatids and chromosomes as well as segregation defects. Micronucleus formation (arrows), were found significantly increased in comparison either with the control or HU treatment (comp. Rybaczek & Kowalewicz-Kulbat, 2011; Rybaczek et al., 2008). The mitotic index was calculated as the percent ratio between the number of dividing cells and the entire meristematic cell population. Index of aberrations was calculated as the percent ratio between the number of cells showing chromosome aberrations and all mitotic cells. PCC index was calculated as the percent ratio between the number of cells showing chromosome aberrations typical of premature mitosis and all mitotic cells. Ex‐ perimental procedure of Feulgen staining: root tips were fixed in cold absolute ethanol and glacial acetic acid (3:1, v/v) for 1 h, washed several times with ethanol, rehydrated, hydrolysed in 4 M HCl (1.5 h), and stained with Schiff's reagent (pararosaniline; Sigma-Aldrich) according to standard methods. After rinsing in SO2-water (3 times) and distil‐ led water, 1.5 mm long apical segments were cut off, placed in a drop of 45% acetic acid, and squashed onto micro‐ scope slides. Following freezing with dry ice, coverslips were removed and the dehydrated dry slides were embedded in Canada Baume. Slides were analysed under the light microscope to count mitotic cells that had characteristic fea‐ tures of either normal mitosis or PCC. *Bar* 20 μm

In a cell there are also mechanisms responsible for DNA damage tolerance (DDT), which al‐ low the completion of the replication of genetic material despite the damage to DNA that blocks replicase complex. In addition, disruption of the efficiency of the intra-S phase check‐ point, following the action of chemical agents, leads to the induction of premature chromo‐ some condensation (PCC; Figure 2), specifically via overriding of the control over the stability of the genome, even despite the uncompleted S phase and not implemented postreplication repair processes in G2 phase (Figure 3A). The successive phases of prematurely initiated mitosis follow an aberration course because the unreplicated regions of the genome are manifested in the form of losses or breaks in chromosomes [(Figure 3B) comp. Rybaczek et al., 2008; Rybaczek, 2011]. Caffeine (CF) is a particularly effective PCC inducer. It blocks the activity of kinases ATM/ATR (Cortez, 2003), by which they can not phosphorylate their downstream kinases (i.e. Chk1 and Chk2; Rybaczek & Kowalewicz-Kulbat, 2011; Rybaczek et al., 2007) and, consequently, catalytic activity of Cdc25 phosphatases is maintained - phos‐ phatases which serve as inducers of complexes CDK1-cyclin B (MPF; M-phase Promoting Factor) and trigger mitotic phosphorylations (Gotoh & Durante, 2006; Rybaczek & Kowale‐ wicz-Kulbat, 2011).

histone H2A, and thus switching off the signal of the DNA structure checkpoint and - after the completion of repair - restoration of the correct chromatin structure. The results of test‐ ing using antibodies recognizing phosphorylated histone H2AX (α-H2AXS139) - microscopic images of immunofluorescence in meristematic root cells of *Allium porum*, *Vicia faba*, *Rapha‐ nus sativum*, and HeLa cells, and strong signals obtained using a Western blot – provide, above all, the next example of homology of organization of cellular systems in animals and plants - the similarities in their structural elements, systems, and hence, similarities of bio‐ chemical regulatory mechanisms (Rybaczek & Kowalewicz-Kulbat, 2011; Rybaczek & Mas‐ zewski, 2007a). Our studies have shown that a significant level of Ser139 phosphorylation in histone H2AX appears after hydroxyurea treatment, as it was the case with phosphoryla‐ tions of Chk1 serines 317 and 345. Correlation of immunolabeling using anti-Chk1 (Ser317) and anti-H2AX (Ser139) antibodies, especially evident at the boundaries of nucleolar and perinucleolar regions of chromatin, seems to indicate that both regions overlap with the areas of an increased activity of Chk1 kinase (Rybaczek & Maszewski, 2007b). It was also concluded that as opposed to *V. faba* and *A.porrum* (both representing a 'reticulate' type of DNA package) the diffuse chromatin in chromocentric cell nuclei of *R. sativus* may be more vulnerable both to generate DSBs and to recruit repair factors (Rybaczek & Maszewski, 2007a). The formation of histone H2AX foci phosphorylated at Ser139 is therefore a sensitive test showing the presence of structural damage to the genome (Figure 4A, B). An equally sensitive test detecting single-strand DNA damage is labeling nuclei by antibodies recogniz‐ ing single-stranded DNA (anti-single-stranded DNA, Figure 4A, B) or antibodies recogniz‐

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ing *PARP2* gene product, i.e. Poly(ADP-Ribose) Polymerase-2 (PARP-2;Figure 5A, B).

mM caffeine (under constant sustained hydroxyurea stress; U = 8.61; P ≤ 0.001).

the hyperexpression of *cdc25* genes (Forbes et al. 1998).

Comparisons of means were made using nonparametric Mann-Whitney U tests, due to the fact that some series had a skewed distribution (Figure 4A). The following has been indicat‐ ed: (i) a significant increase in the DSB series compared to SSB in the control series (U = 6.23; P ≤ 0.001), (ii) a significant increase in the DSB series compared to SSB after a 24-hour activi‐ ty of 2.5 mM hydroxyurea (U = 8.61; P ≤ 0.001), and (iii) a significant increase in SSB com‐ pared to DSB in the series in which PCC induction was performed under the influence of 5

Additionally, the presence of double-stranded breaks (DSBs) in the nuclei of cells undergo‐ ing PCC suggests also that premature entry into mitosis occurs before the completion of DNA repair (Rybaczek et al. 2007; Rybaczek et al. 2008). The key target of S-M checkpoint is the activity of the cyclin B/Cdk1 complexes (MPF), but similar effects can result from the change in the activity balance of protein kinases and phosphatases brought about, e.g. by

PARP activation is an immediate cellular response to chemical or radiation-induced DNA SSB damage. PARP-2 is a nuclear protein whose main role is to detect and signal SSB to the enzy‐ matic machinery involved in the SSB repair. Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a Poly(ADP-Ribose) chain (PAR) as a sig‐ nal for other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins such as X-ray cross-complementing gene 1 (XRCC1). After re‐ pairing, the PAR chains are degraded via PAR glycohydrolase [(PARG) Isabelle et al., 2010].

The overriding of the checkpoint function induced by the action of caffeine leads to the se‐ lective sensibilization of pro-oncogenic cells deprived of p53 protein and tumorous cells to the action of antineoplastic factors and the effect of ionizing radiation (Yao et al. 1996). The test results obtained by Wang and co-workers (1999) show that the effectiveness disturbance of the S-M control system induced by caffeine in *S. pombe* cells is connected with the activa‐ tion of Cdc2 kinase (due to the removal of phosphate group from Tyr15 within the ATPbinding pocket) and with the septation process that during a normal course of cell cycle of *S. pombe* results from the transfer through mitosis.

## **5. Labeling of DNA damages following hydroxyurea-induced stress and caffeine-induced premature chromosome condensation**

One of the basic protective mechanisms of the replicative apparatus are foci concentrating molecules of phosphorylated histones H2AX (Rybaczek & Maszewski, 2007a; Rybaczek & Maszewski, 2007b). The generation of γ-H2AX molecules as a result of exposure to stressors is a rapid process. Half of the γ-H2AX histones appear as early as after 1 min of irradiation and a maximum level is reached with 3 to 10 minutes of exposure; then, in terms of 1 Gy radiation, γ-phosphorylation concerns approximately 1% of histone H2AX molecules, which is equivalent to about 2x106 base pairs of DNA in the region of the double-strand break (DSB). It is assumed that each grouping of these molecules determines a single DSB region (Paull et al., 2000; Rogakou et al., 1998). Phosphorylated histone H2AX binds cohesin and chromatin-modifying complex NuA4. The acetylation of histones follows, which allows con‐ nection of the INO80 complex, which removes histones in the area of the damaged DNA, thereby creating single-strand regions. This greatly simplifies the recruitment of proteins of the pathway of response to DNA damage and repair proteins. Then TIP60 complex is con‐ nected, followed by the removal of dimers H2AX/H2B and insertion of non-phosphorylated histone H2A, and thus switching off the signal of the DNA structure checkpoint and - after the completion of repair - restoration of the correct chromatin structure. The results of test‐ ing using antibodies recognizing phosphorylated histone H2AX (α-H2AXS139) - microscopic images of immunofluorescence in meristematic root cells of *Allium porum*, *Vicia faba*, *Rapha‐ nus sativum*, and HeLa cells, and strong signals obtained using a Western blot – provide, above all, the next example of homology of organization of cellular systems in animals and plants - the similarities in their structural elements, systems, and hence, similarities of bio‐ chemical regulatory mechanisms (Rybaczek & Kowalewicz-Kulbat, 2011; Rybaczek & Mas‐ zewski, 2007a). Our studies have shown that a significant level of Ser139 phosphorylation in histone H2AX appears after hydroxyurea treatment, as it was the case with phosphoryla‐ tions of Chk1 serines 317 and 345. Correlation of immunolabeling using anti-Chk1 (Ser317) and anti-H2AX (Ser139) antibodies, especially evident at the boundaries of nucleolar and perinucleolar regions of chromatin, seems to indicate that both regions overlap with the areas of an increased activity of Chk1 kinase (Rybaczek & Maszewski, 2007b). It was also concluded that as opposed to *V. faba* and *A.porrum* (both representing a 'reticulate' type of DNA package) the diffuse chromatin in chromocentric cell nuclei of *R. sativus* may be more vulnerable both to generate DSBs and to recruit repair factors (Rybaczek & Maszewski, 2007a). The formation of histone H2AX foci phosphorylated at Ser139 is therefore a sensitive test showing the presence of structural damage to the genome (Figure 4A, B). An equally sensitive test detecting single-strand DNA damage is labeling nuclei by antibodies recogniz‐ ing single-stranded DNA (anti-single-stranded DNA, Figure 4A, B) or antibodies recogniz‐ ing *PARP2* gene product, i.e. Poly(ADP-Ribose) Polymerase-2 (PARP-2;Figure 5A, B).

In a cell there are also mechanisms responsible for DNA damage tolerance (DDT), which al‐ low the completion of the replication of genetic material despite the damage to DNA that blocks replicase complex. In addition, disruption of the efficiency of the intra-S phase check‐ point, following the action of chemical agents, leads to the induction of premature chromo‐ some condensation (PCC; Figure 2), specifically via overriding of the control over the stability of the genome, even despite the uncompleted S phase and not implemented postreplication repair processes in G2 phase (Figure 3A). The successive phases of prematurely initiated mitosis follow an aberration course because the unreplicated regions of the genome are manifested in the form of losses or breaks in chromosomes [(Figure 3B) comp. Rybaczek et al., 2008; Rybaczek, 2011]. Caffeine (CF) is a particularly effective PCC inducer. It blocks the activity of kinases ATM/ATR (Cortez, 2003), by which they can not phosphorylate their downstream kinases (i.e. Chk1 and Chk2; Rybaczek & Kowalewicz-Kulbat, 2011; Rybaczek et al., 2007) and, consequently, catalytic activity of Cdc25 phosphatases is maintained - phos‐ phatases which serve as inducers of complexes CDK1-cyclin B (MPF; M-phase Promoting Factor) and trigger mitotic phosphorylations (Gotoh & Durante, 2006; Rybaczek & Kowale‐

The overriding of the checkpoint function induced by the action of caffeine leads to the se‐ lective sensibilization of pro-oncogenic cells deprived of p53 protein and tumorous cells to the action of antineoplastic factors and the effect of ionizing radiation (Yao et al. 1996). The test results obtained by Wang and co-workers (1999) show that the effectiveness disturbance of the S-M control system induced by caffeine in *S. pombe* cells is connected with the activa‐ tion of Cdc2 kinase (due to the removal of phosphate group from Tyr15 within the ATPbinding pocket) and with the septation process that during a normal course of cell cycle of *S.*

**5. Labeling of DNA damages following hydroxyurea-induced stress and**

One of the basic protective mechanisms of the replicative apparatus are foci concentrating molecules of phosphorylated histones H2AX (Rybaczek & Maszewski, 2007a; Rybaczek & Maszewski, 2007b). The generation of γ-H2AX molecules as a result of exposure to stressors is a rapid process. Half of the γ-H2AX histones appear as early as after 1 min of irradiation and a maximum level is reached with 3 to 10 minutes of exposure; then, in terms of 1 Gy radiation, γ-phosphorylation concerns approximately 1% of histone H2AX molecules, which

(DSB). It is assumed that each grouping of these molecules determines a single DSB region (Paull et al., 2000; Rogakou et al., 1998). Phosphorylated histone H2AX binds cohesin and chromatin-modifying complex NuA4. The acetylation of histones follows, which allows con‐ nection of the INO80 complex, which removes histones in the area of the damaged DNA, thereby creating single-strand regions. This greatly simplifies the recruitment of proteins of the pathway of response to DNA damage and repair proteins. Then TIP60 complex is con‐ nected, followed by the removal of dimers H2AX/H2B and insertion of non-phosphorylated

base pairs of DNA in the region of the double-strand break

**caffeine-induced premature chromosome condensation**

wicz-Kulbat, 2011).

238 New Research Directions in DNA Repair

*pombe* results from the transfer through mitosis.

is equivalent to about 2x106

Comparisons of means were made using nonparametric Mann-Whitney U tests, due to the fact that some series had a skewed distribution (Figure 4A). The following has been indicat‐ ed: (i) a significant increase in the DSB series compared to SSB in the control series (U = 6.23; P ≤ 0.001), (ii) a significant increase in the DSB series compared to SSB after a 24-hour activi‐ ty of 2.5 mM hydroxyurea (U = 8.61; P ≤ 0.001), and (iii) a significant increase in SSB com‐ pared to DSB in the series in which PCC induction was performed under the influence of 5 mM caffeine (under constant sustained hydroxyurea stress; U = 8.61; P ≤ 0.001).

Additionally, the presence of double-stranded breaks (DSBs) in the nuclei of cells undergo‐ ing PCC suggests also that premature entry into mitosis occurs before the completion of DNA repair (Rybaczek et al. 2007; Rybaczek et al. 2008). The key target of S-M checkpoint is the activity of the cyclin B/Cdk1 complexes (MPF), but similar effects can result from the change in the activity balance of protein kinases and phosphatases brought about, e.g. by the hyperexpression of *cdc25* genes (Forbes et al. 1998).

PARP activation is an immediate cellular response to chemical or radiation-induced DNA SSB damage. PARP-2 is a nuclear protein whose main role is to detect and signal SSB to the enzy‐ matic machinery involved in the SSB repair. Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a Poly(ADP-Ribose) chain (PAR) as a sig‐ nal for other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins such as X-ray cross-complementing gene 1 (XRCC1). After re‐ pairing, the PAR chains are degraded via PAR glycohydrolase [(PARG) Isabelle et al., 2010].

**Figure 4.** Immunolabeling indices (%) estimated for *Vicia faba* stained with anti-ssDNA [red, TRITC-labeled] and anti-H2AX(Ser139) [green, FITC-labeled] antibodies. *Columns*, mean from five independent experiments; *bars*, SD. For im‐ munocytochemical detection of single-standed DNA and phospho-H2AX histone cells were fixed for 45 min in 4% formaldehyde buffered with PBS. Excised apical parts of roots were then placed in a citric acid-buffered digestion solu‐ tion (pH 5.0; 37°C for 45 min) containing 2.5% pectinase (Fluka), 2.5% cellulase (Onozuka R-10; Serva) and 2.5% pec‐ toliase (ICN). The cells were pre-treated in a blocking buffer (10% horse serum, 1% bovine serum albumin; BSA, 0.02% NaN3, 1 x PBS) for 1 h at room temperature to minimize the non-specific adsorption of the antibodies to the coverslip, and were incubated overnight in a humidified atmosphere (4°C) with primary antibody. Mouse monoclonal antibody to single-stranded DNA was used at 1:200 (MILLIPORE), rabbit polyclonal antibody to phospho-H2AX (Ser139) was used at 1:750 (CELL SIGNALING). Secondary antibodies, including FITC-conjugated goat anti-rabbit (for H2AX), and TRITC-conjugated goat anti-mouse antibodies (for ssDNA), were used at 1:1000 for 1 h at room temperature in the dark. Secondary antibodies were from Sigma-Aldrich. The labeling index was calculated as the ratio of immunofluor‐ escence-labeled cells to all cells in a meristematic population. *Bar* 20 μm

**Figure 5.** Fig. 5. Immunolabeling indices (%) estimated for *Vicia faba* stained with anti-PARP-2 antibody [green, Dy‐

488] and DAPI [blue]. *Columns*, mean from five independent experiments; *bars*, SD. For immunocytochemical PARP-2 (Poly[ADP-Ribose] Polymerase-2) cells were fixed for 45 min in 4% formaldehyde buffered with PBS. Excised apical parts of roots were then placed in a citric acid-buffered digestion solution (pH 5.0; 37°C for 45 min) containing 2.5% pectinase (Fluka), 2.5% cellulase (Onozuka R-10; Serva) and 2.5% pectoliase (ICN). The cells were pre-treated in a blocking buffer (10% horse serum, 1% bovine serum albumin; BSA, 0.02% NaN3, 1 x PBS) for 1 h at room temperature to minimize the non-specific adsorption of the antibodies to the coverslip, and were incubated overnight in a humidi‐ fied atmosphere (4°C) with primary antibody. Rabbit polyclonal antibodies specific to PARP-2 were purchased from AGRISERA (at a dilution of 1:50). Bound primary antibodies were detected with secondary goat anti-rabbit IgG Dy‐

Relation of the Types of DNA Damage to Replication Stress and the Induction of Premature Chromosome

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488 antibody (AGRISERA; at a dilution of 1:1000, for 1 h at 18°C). Nuclear DNA was stained with 4',6-diamidi‐ no-2-phenyl-indole (DAPI, 0.4 μg/ml; Sigma-Aldrich). The labeling index was calculated as the ratio of

Nonparametric Kruskal-Wallis tests were used for analysis of variance (H = 78.9; P ≤ 0.001; Figure 5A). Comparisons between groups were made using post hoc tests (Figure 5A). A

immunofluorescence-labeled cells to all cells in a meristematic population. *Bar* 20 μm

Light®

Light®

Relation of the Types of DNA Damage to Replication Stress and the Induction of Premature Chromosome Condensation http://dx.doi.org/10.5772/54020 241

**Figure 5.** Fig. 5. Immunolabeling indices (%) estimated for *Vicia faba* stained with anti-PARP-2 antibody [green, Dy‐ Light® 488] and DAPI [blue]. *Columns*, mean from five independent experiments; *bars*, SD. For immunocytochemical PARP-2 (Poly[ADP-Ribose] Polymerase-2) cells were fixed for 45 min in 4% formaldehyde buffered with PBS. Excised apical parts of roots were then placed in a citric acid-buffered digestion solution (pH 5.0; 37°C for 45 min) containing 2.5% pectinase (Fluka), 2.5% cellulase (Onozuka R-10; Serva) and 2.5% pectoliase (ICN). The cells were pre-treated in a blocking buffer (10% horse serum, 1% bovine serum albumin; BSA, 0.02% NaN3, 1 x PBS) for 1 h at room temperature to minimize the non-specific adsorption of the antibodies to the coverslip, and were incubated overnight in a humidi‐ fied atmosphere (4°C) with primary antibody. Rabbit polyclonal antibodies specific to PARP-2 were purchased from AGRISERA (at a dilution of 1:50). Bound primary antibodies were detected with secondary goat anti-rabbit IgG Dy‐ Light® 488 antibody (AGRISERA; at a dilution of 1:1000, for 1 h at 18°C). Nuclear DNA was stained with 4',6-diamidi‐ no-2-phenyl-indole (DAPI, 0.4 μg/ml; Sigma-Aldrich). The labeling index was calculated as the ratio of immunofluorescence-labeled cells to all cells in a meristematic population. *Bar* 20 μm

**Figure 4.** Immunolabeling indices (%) estimated for *Vicia faba* stained with anti-ssDNA [red, TRITC-labeled] and anti-H2AX(Ser139) [green, FITC-labeled] antibodies. *Columns*, mean from five independent experiments; *bars*, SD. For im‐ munocytochemical detection of single-standed DNA and phospho-H2AX histone cells were fixed for 45 min in 4% formaldehyde buffered with PBS. Excised apical parts of roots were then placed in a citric acid-buffered digestion solu‐ tion (pH 5.0; 37°C for 45 min) containing 2.5% pectinase (Fluka), 2.5% cellulase (Onozuka R-10; Serva) and 2.5% pec‐ toliase (ICN). The cells were pre-treated in a blocking buffer (10% horse serum, 1% bovine serum albumin; BSA, 0.02% NaN3, 1 x PBS) for 1 h at room temperature to minimize the non-specific adsorption of the antibodies to the coverslip, and were incubated overnight in a humidified atmosphere (4°C) with primary antibody. Mouse monoclonal antibody to single-stranded DNA was used at 1:200 (MILLIPORE), rabbit polyclonal antibody to phospho-H2AX (Ser139) was used at 1:750 (CELL SIGNALING). Secondary antibodies, including FITC-conjugated goat anti-rabbit (for H2AX), and TRITC-conjugated goat anti-mouse antibodies (for ssDNA), were used at 1:1000 for 1 h at room temperature in the dark. Secondary antibodies were from Sigma-Aldrich. The labeling index was calculated as the ratio of immunofluor‐

escence-labeled cells to all cells in a meristematic population. *Bar* 20 μm

240 New Research Directions in DNA Repair

Nonparametric Kruskal-Wallis tests were used for analysis of variance (H = 78.9; P ≤ 0.001; Figure 5A). Comparisons between groups were made using post hoc tests (Figure 5A). A statistically significant increase in the fluorescence labeling index of the anti-PARP2 in series HU and PCC was observed relative to the control, as well as a significantly higher labeling index for HU compared to the PCC series (Figure 5A).

either SSBs or DSBs) is that they involve large protein complexes, and that the repaired DNA is subject to many structural changes not only initially but also during repair itself (e.g. unwinding or nucleolytic processing). Finally, control systems of higher plant cell cy‐ cles involve regulatory factors related to the "permanently embryonic" nature of meristemat‐ ic zones, autotrophic metabolism, spatial stabilization, the presence of cellulose wall and the resulting specific intertissue dependencies (Jacobs, 1992). Hopefully, cutting-edge research techniques will soon make it possible to reveal many of the still unknown mechanisms of

Relation of the Types of DNA Damage to Replication Stress and the Induction of Premature Chromosome

Condensation

243

http://dx.doi.org/10.5772/54020

The instability of the genome, visible in chromosome mutations and rearrangements, is usu‐ ally associated with a pathological disorders, but is also of key importance for evolution. Processes that make up the cell cycle (replication, chromatin condensation, anaphase-telo‐ phase chromosome segregation and cytokinesis) occur in a sequential manner and are sub‐ ject to precise control. However, the cell cycle includes several functionally different cycles that are inherently related to the cell cycle but independent of each other, for example, nu‐ clear DNA cycle, nuclear membrane cycle, nucleolus cycle, microtubular cycle, a cycle of bi‐ osynthesis and segregation of cell organelles, and the use of sucrose like highly-energetic substances. Despite the enormous diversity of processes occurring in the cell cycle, the mechanisms responsible for the integrity of the genome exhibit a remarkable homology and coherence of action in reducing the effects of DNA damage. This results in the evolutionary development of organisms and an increase in their productivity in the expansion to new and

The work was funded by "POMOST" fellowship from the Foundation for Polish Science (the

1 Department of Cytophysiology, Faculty of Biology and Environmental Protection, Univer‐

2 Department of Immunology and Infectious Biology, University of Łódź, Łódź, Poland

and Magdalena Kowalewicz-Kulbat2

DNA repair and to formulate really definite conclusions.

**7. Conclusion**

more demanding environments.

contract no. POMOST/2011-4/8).

**Acknowledgement**

**Author details**

Dorota Rybaczek1

sity of Łódź, Łódź, Poland

In summary, this chapter aims to review how the nature of the damage to nucleobases influ‐ ences DNA repair with regards to DSB and SSB generation (Figures 4, 5). Reports, literature and our own research results show histone H2AX phosphorylated at Ser139 is the marker of double-strand breaks (Figure 4A, C). It was shown that rapid and sensitive detection of sin‐ gle-strand damage is possible thanks to immunocytochemical reaction performed using commercially available antibodies recognizing ssDNA (anti-ssDNA, MILLIPORE, Figure 4B, C), or another similarly useful SSBs marker, Poly(ADP-Ribose) Polymerase-2 (AGRISERA, Figure 5A, B). We demonstrate that replication stress leads mainly to the generation of dou‐ ble-strand breaks in DNA (DSBs), while the breakage of restrictive interactions of check‐ points during PCC induction results in the accumulation of single-strand breaks (SSBs).

## **6. Future perspectives and the key questions that remain unanswered**

The formation of DNA damage is a continuous process. Out of necessity, it must be per‐ ceived in terms of temporal and spatial chromatin dynamics, and as coupled with the activa‐ tion of checkpoints (Zhou & Elledge, 2000; Liu et al., 2006). The consequence of this activation is possibly the most efficient (i.e. fast and effective) initiation of the repair process‐ es. Maintaining the efficiency is important, as any decrease in DNA repair efficiency, for ex‐ ample resulting from mutations in genes encoding repair proteins, may lead to neoplasia.

Most recent studies on DNA repair have been aimed at achieving various strategic objec‐ tives, most often concerned with strengthening the effects of widely understood radio and chemotherapy (Legerski, 2010). Thoms and Bristow (2010) describe the achievement of the "therapeutic ratio" as the primary aim of their investigations. Other researchers emphasize the benefits of mathematical methods in either future experimental studies of DNA repair or clinical studies of drug resistance (Lavi et al., 2012).

DNA repair processes have been studied using (i) different experimental systems, e.g. *in vi‐ tro* model (Garner & Costanzo, 2009), (ii) different cell types, e.g. human stem cells (Rocha et al., 2013) or even neurons (McMurray, 2005); (iii) model organisms, e.g. *Arabidopsis thaliana* cells, *Xenopus laevis* egg cell free extract (Garner & Costanzo, 2009); (iv) different proteins e.g. cyclin-dependent kinases (CDKs; Yata & Esashi, 2009), histone variants (Shi & Ober‐ doerffer, 2012) or cell cycle checkpoints connected proteins (Liu et al., 2006); as well as (v) the context of chromatin condensation (Shi & Oberdoerffer, 2012).

Most (although not all) molecular mechanisms involved in DNA repair appear to be evolu‐ tionarily conservative. However, many important questions still remain unanswered. This is particularly evident in studies on chromatin adopting different conformations and damaged - with varying intensity - by various factors and various states of condensation. This variety makes it difficult to draw definite conclusions with regard to the processes of DNA repair in chromatin fibres. In addition, the common features of almost all types of repair (concerning either SSBs or DSBs) is that they involve large protein complexes, and that the repaired DNA is subject to many structural changes not only initially but also during repair itself (e.g. unwinding or nucleolytic processing). Finally, control systems of higher plant cell cy‐ cles involve regulatory factors related to the "permanently embryonic" nature of meristemat‐ ic zones, autotrophic metabolism, spatial stabilization, the presence of cellulose wall and the resulting specific intertissue dependencies (Jacobs, 1992). Hopefully, cutting-edge research techniques will soon make it possible to reveal many of the still unknown mechanisms of DNA repair and to formulate really definite conclusions.

## **7. Conclusion**

statistically significant increase in the fluorescence labeling index of the anti-PARP2 in series HU and PCC was observed relative to the control, as well as a significantly higher labeling

In summary, this chapter aims to review how the nature of the damage to nucleobases influ‐ ences DNA repair with regards to DSB and SSB generation (Figures 4, 5). Reports, literature and our own research results show histone H2AX phosphorylated at Ser139 is the marker of double-strand breaks (Figure 4A, C). It was shown that rapid and sensitive detection of sin‐ gle-strand damage is possible thanks to immunocytochemical reaction performed using commercially available antibodies recognizing ssDNA (anti-ssDNA, MILLIPORE, Figure 4B, C), or another similarly useful SSBs marker, Poly(ADP-Ribose) Polymerase-2 (AGRISERA, Figure 5A, B). We demonstrate that replication stress leads mainly to the generation of dou‐ ble-strand breaks in DNA (DSBs), while the breakage of restrictive interactions of check‐ points during PCC induction results in the accumulation of single-strand breaks (SSBs).

**6. Future perspectives and the key questions that remain unanswered**

The formation of DNA damage is a continuous process. Out of necessity, it must be per‐ ceived in terms of temporal and spatial chromatin dynamics, and as coupled with the activa‐ tion of checkpoints (Zhou & Elledge, 2000; Liu et al., 2006). The consequence of this activation is possibly the most efficient (i.e. fast and effective) initiation of the repair process‐ es. Maintaining the efficiency is important, as any decrease in DNA repair efficiency, for ex‐ ample resulting from mutations in genes encoding repair proteins, may lead to neoplasia.

Most recent studies on DNA repair have been aimed at achieving various strategic objec‐ tives, most often concerned with strengthening the effects of widely understood radio and chemotherapy (Legerski, 2010). Thoms and Bristow (2010) describe the achievement of the "therapeutic ratio" as the primary aim of their investigations. Other researchers emphasize the benefits of mathematical methods in either future experimental studies of DNA repair or

DNA repair processes have been studied using (i) different experimental systems, e.g. *in vi‐ tro* model (Garner & Costanzo, 2009), (ii) different cell types, e.g. human stem cells (Rocha et al., 2013) or even neurons (McMurray, 2005); (iii) model organisms, e.g. *Arabidopsis thaliana* cells, *Xenopus laevis* egg cell free extract (Garner & Costanzo, 2009); (iv) different proteins e.g. cyclin-dependent kinases (CDKs; Yata & Esashi, 2009), histone variants (Shi & Ober‐ doerffer, 2012) or cell cycle checkpoints connected proteins (Liu et al., 2006); as well as (v)

Most (although not all) molecular mechanisms involved in DNA repair appear to be evolu‐ tionarily conservative. However, many important questions still remain unanswered. This is particularly evident in studies on chromatin adopting different conformations and damaged - with varying intensity - by various factors and various states of condensation. This variety makes it difficult to draw definite conclusions with regard to the processes of DNA repair in chromatin fibres. In addition, the common features of almost all types of repair (concerning

index for HU compared to the PCC series (Figure 5A).

242 New Research Directions in DNA Repair

clinical studies of drug resistance (Lavi et al., 2012).

the context of chromatin condensation (Shi & Oberdoerffer, 2012).

The instability of the genome, visible in chromosome mutations and rearrangements, is usu‐ ally associated with a pathological disorders, but is also of key importance for evolution. Processes that make up the cell cycle (replication, chromatin condensation, anaphase-telo‐ phase chromosome segregation and cytokinesis) occur in a sequential manner and are sub‐ ject to precise control. However, the cell cycle includes several functionally different cycles that are inherently related to the cell cycle but independent of each other, for example, nu‐ clear DNA cycle, nuclear membrane cycle, nucleolus cycle, microtubular cycle, a cycle of bi‐ osynthesis and segregation of cell organelles, and the use of sucrose like highly-energetic substances. Despite the enormous diversity of processes occurring in the cell cycle, the mechanisms responsible for the integrity of the genome exhibit a remarkable homology and coherence of action in reducing the effects of DNA damage. This results in the evolutionary development of organisms and an increase in their productivity in the expansion to new and more demanding environments.

## **Acknowledgement**

The work was funded by "POMOST" fellowship from the Foundation for Polish Science (the contract no. POMOST/2011-4/8).

## **Author details**

Dorota Rybaczek1 and Magdalena Kowalewicz-Kulbat2

1 Department of Cytophysiology, Faculty of Biology and Environmental Protection, Univer‐ sity of Łódź, Łódź, Poland

2 Department of Immunology and Infectious Biology, University of Łódź, Łódź, Poland

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**Chapter 10**

**p21CDKN1A and DNA Repair Systems: Recent Findings**

After exposure to genotoxic agents, cells activate DNA damage response pathways consist‐ ing of a signaling cascade (cell cycle checkpoints), and of DNA repair processes able to rec‐

DNA repair is characterized by an impressive high number of different proteins necessary to perform specialized biochemical reactions, which are different according to the type of le‐ sion to be repaired [2]. Thus, the nucleotide excision repair (NER) mechanism will repair bulky lesions, such as the cyclobutane pyrimidine dimers (CPDs) produced by UV-C irradi‐ ation, or other types of adducts produced by the interaction of chemicals with DNA. Base excision repair (BER) is instead involved in the removal of bases damaged by alkylating, or oxidative agents, while the repair of single and double strand breaks is performed through the pathway of homologous recombination, or via the non homologous end-joning (NHEJ) repair. In addition, cells repair errors introduced during DNA replication with the mecha‐

Among the many factors involved in these defense processes against DNA damage, p21CDKN1A protein – known also as p21(WAF1/CIP1/SDI1) – plays a key role in several fundamental biological processes, such as cell cycle control, DNA replication/repair, gene transcription, apoptosis, and cell motility [3-6]. This protein is a cyclin-dependent kinase (CDK) inhibitor belonging to the Cip/Kip family; it was first described as a potent inhibitor of cell prolifera‐ tion and DNA replication, both in physiological conditions and after DNA damage [7,8]. Homologs are found in several organisms, including *Xenopus* (Xic1), *Drosophila* (Dacapo), as well as *C. Elegans* (CKI-1). In mammals, p21 was previously known as CDK-interacting pro‐

> © 2013 Tillhon et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**and Future Perspectives**

Lucia A. Stivala and Ennio Prosperi

http://dx.doi.org/10.5772/54173

nism of mismatch repair (MMR).

**1. Introduction**

Micol Tillhon, Ornella Cazzalini, Ilaria Dutto,

Additional information is available at the end of the chapter

ognize and remove a great number of DNA lesions [1].

## **p21CDKN1A and DNA Repair Systems: Recent Findings and Future Perspectives**

Micol Tillhon, Ornella Cazzalini, Ilaria Dutto, Lucia A. Stivala and Ennio Prosperi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54173

## **1. Introduction**

[56] Schiller, C.B., Lammens, K., Guerini, I., Coordes, B., Feldmann, H., Schlauderer, F., Möckel, C., Schele, A., Strässer, K., Jackson, S.P. & Hopfner, K.P. (2012) Structure of Mre11-Nbs1 complex yields insights into ataxia-telangiectasia-like disease mutations

[57] Scorah, J., Dong, M-Q., Yates, III jr, Scott, M., Gillespie, D. & McGowan, Ch. (2008) A conserved PCNA-interacting protein sequence in Chk1 is required for checkpoint

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[60] Shimura, T., Martin, M.M., Torres, M.J., Gu, C., Pluth, J.M., DiBernardi, M.A., McDo‐ nald, J.S. & Aladjem, M.J. (2007) DNA-PK is involved in repairing a transient surge of DNA breaks induced by deceleration of DNA replication. *J Mol Biol* 367, 665-680.

[61] Tan, Z., Wortman, M., Dillehay, K.L., Seibel, W.L., Evelyn, C.R., Smith, S.J., Malkas, L.H., Zheng, Y., Lu, S. & Dong, Z. (2012) Small-molecule targeting of proliferating cell nuclear antigen chromatin association inhibits tumor cell growth. *Mol Pharmacol*

[62] Thoms, J. & Bristow, R.G. (2010) DNA repair targeting and radiotherapy: a focus on

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[64] Wang, S.-W., Norbury, C., Harris, A.L. &Toda, T. (1999) Caffeine can override the S-

[65] Ward, I.M. & Chen, J. (2001) Histone H2AX is phosphorylated in an ATR-dependent

[66] Yao, T., Utsunomiya, T., Nagai, E., Oya, M. & Tsuneyoshi, M. (1996) p53 expression patterns in colorectal adenomas and early carcinomas: a special reference to de‐

[67] Yata, K. & Esashi, F. (2009) Dual role of CDKs in DNA repair: To be, or not to be.

[68] Zhou, B.B. & Elledge, S.J. (2000) The DNA damage response: putting checkpoints in

[69] Zou, L. & Elledge, S.J. (2003) Sensing DNA damage through ATRIP recognition of

manner in response to replicational stress. *J Biol Chem* 276, 47759-47762.

pressed adenoma and non-polyploid carcinoma. *Phatol Int* 46, 962-967.

and DNA damage signaling. *Nat Struct Mol Biol* 19, 693-700.

function. *J Biol Chem* 283: 1725-17259.

repair. *Biochim Biophys Acta* 1819, 811-819.

the therapeutic ratio. *Semin Radiat Oncol* 20, 217-222.

M checkpoint in fission yeast. *J Cell Sci* 112, 927-937.

81, 811-819.

248 New Research Directions in DNA Repair

*DNA Repair* 8, 6-18.

perspective. *Nature* 408, 433-439.

RPA-ssDNA complexes. *Science* 300, 1542-1548.

After exposure to genotoxic agents, cells activate DNA damage response pathways consist‐ ing of a signaling cascade (cell cycle checkpoints), and of DNA repair processes able to rec‐ ognize and remove a great number of DNA lesions [1].

DNA repair is characterized by an impressive high number of different proteins necessary to perform specialized biochemical reactions, which are different according to the type of le‐ sion to be repaired [2]. Thus, the nucleotide excision repair (NER) mechanism will repair bulky lesions, such as the cyclobutane pyrimidine dimers (CPDs) produced by UV-C irradi‐ ation, or other types of adducts produced by the interaction of chemicals with DNA. Base excision repair (BER) is instead involved in the removal of bases damaged by alkylating, or oxidative agents, while the repair of single and double strand breaks is performed through the pathway of homologous recombination, or via the non homologous end-joning (NHEJ) repair. In addition, cells repair errors introduced during DNA replication with the mecha‐ nism of mismatch repair (MMR).

Among the many factors involved in these defense processes against DNA damage, p21CDKN1A protein – known also as p21(WAF1/CIP1/SDI1) – plays a key role in several fundamental biological processes, such as cell cycle control, DNA replication/repair, gene transcription, apoptosis, and cell motility [3-6]. This protein is a cyclin-dependent kinase (CDK) inhibitor belonging to the Cip/Kip family; it was first described as a potent inhibitor of cell prolifera‐ tion and DNA replication, both in physiological conditions and after DNA damage [7,8]. Homologs are found in several organisms, including *Xenopus* (Xic1), *Drosophila* (Dacapo), as well as *C. Elegans* (CKI-1). In mammals, p21 was previously known as CDK-interacting pro‐

© 2013 Tillhon et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

tein 1 (CIP1), wild type p53-activated fragment (WAF1), senescent cell-derived inhibitor 1 (SDI1), and melanoma differentiation-associated protein 6 (MDA-6); all these names have been substituted by a new terminology including all CDK inhibitors, and p21 is now named CDKN1A.

common malignancies occurring in these animals were hemopoietic (B-cell lymphoma), en‐ dothelial, and epithelial tumors [21]. In addition, accelerated tumor formation and an in‐ creased capacity of tumor metastasis, respectively induced by urethane or by gamma radiation, were found in p21-/- mice [22,23]. Accelerated tumorigenesis, and promotion of lung metastasis was also found in correlation with cytoplasmic p21 in the mammary epiteli‐ um of mice expressing the MMTV/*neu* oncogene [24]. Tumor suppression functions of p21 were also confirmed by studies in the skin and in the colon of p21-deficient mice [25,26]. Furthermore, spontaneous tumor formation in p21-null mice was also found to occur in combination with other knock-out genetic backgrounds, such as Muc2-/- (mice lacking mucin

p21CDKN1A and DNA Repair Systems: Recent Findings and Future Perspectives

http://dx.doi.org/10.5772/54173

251

In addition to enhanced tumor formation, further investigations showed that loss of p21 caused exhaustion of blood stem cells [29], and induced development of Systemic Lupus Er‐ ythematosus in female animals [30]. Thus, the results obtained from transgenic mice, clearly indicated the tumor suppressor role of p21, although other studies have provided contrast‐ ing results [6,31]. As an example, p21-null mice crossed with knock-in PML-RAR mice, showed an oncogenic role of p21 in maintaining self-renewal of leukemic stem cells [32]. The dual behaviour of p21 most probably occurs because of its participation in several cellular

An important aspect for determining the target of p21 activity is the intracellular localiza‐ tion. Early studies indicated that lack of p21 expression, or cytoplasmic localization of the protein, promoted anchorage-independent growth, and drug resistance [5,6,31]. Human p21 protein is located predominantly in the nucleus; however, it is also present in the nucleolus and in the cytoplasm. In the nucleus, in addition to inhibit CDK2 and binding to PCNA, p21 may also associate with transcriptional regulators [4]. In the nucleolus, p21 was found to colocalize with cyclin E [33], and to accumulate after DNA damage, as a consequence of inhibi‐ tion of nuclear export [34]. Interestingly, growing body of evidence indicates that the cytoplasmic localization of p21 is linked to drug resistance [6,31], thus suggesting that in this compartment the protein may have a tumor-promoting function [35]. Cellular localization of p21 is regulated mainly by post-translation modifications. In fact, nuclear translocation ap‐ pears to be counteracted by different kinases phosphorylating Thr145 and Ser146 residues located near the NLS region of p21 [36-38]. These modifications are responsible for cytoplas‐ mic localization of p21, as well as for the loss of interaction with PCNA [39]. An important role in p21 phosphorylation is played by AKT1/PKB, which also mediates stability of the protein [36,37]. Another relevant modification of p21 (i.e ubiquitination) regulating its deg‐ radation, has been shown to occur predominantly in the nucleus, because p21 mutant in the

A summary of the most important functions performed by p21 protein is reported in the fol‐

As the principal mediator of cell cycle arrest in response to DNA damage, p21 not only acts by inactivating G1-phase cyclins/CDKs complexes, but also by inhibiting cell cycle progres‐

2), and Apc1638+/- (mutant allele of the adenomatosis polyposis gene) mice [27,28].

processes, and it is dependent on different factors [6,31].

NLS region exhibited enhanced stability [40].

lowing paragraphs.

**Cell-cycle regulation**

Due to the lack of a defined tertiary structure, p21 protein may adopt an extended confor‐ mation [9], which may explain its ability to interact with a number of proteins involved in several important biological processes [3-6] (Figure 1).

**Figure 1.** Schematic structure of p21 protein showing the regions responsible for binding to Cyclins, CDK and PCNA. Below the N- and C-terminal regions are indicated the processes in which they are involved, respectively.

## **2. p21 biology and functions**

The main role of p21 is cell-cycle regulation, performed by inhibiting the activity of cyclin-CDK complexes thanks to direct interaction through specific sequences (termed CDK and Cy motifs) in the N-terminal domain of the protein [10-13]. Cell cycle progression may be also regulated, independently of cyclins and CDKs, thanks to the strong affinity binding to proliferating cell nuclear antigen (PCNA) [14-17], a protein playing a central role in DNA replication and repair, as well as in other processes of DNA metabolism [18,19]. This associ‐ ation may interfere with PCNA-dependent enzyme activities involved in DNA synthesis [18,19]. In contrast with the negative cell-cycle regulation, p21 may also serve as an assem‐ bly factor for cyclin D-CDK4/6 complexes, thus promoting cyclin D-dependent events, and downstream activation of cyclin E-CDK2 [7,8].

*CDKN1A* gene inactivation studies performed with experimental models, and in particular with knock-out mice, have confirmed the tumor suppressor functions of this protein [20,21]. The p21-null mice showed a normal development and did not show any spontaneous tumor formation until 7-month of age [20]. However, embryonic fibroblasts derived from these ani‐ mals were deficient in G1 checkpoint arrest following DNA damage [20]. Subsequent stud‐ ies in this model were extended to a longer time frame and the observations reported that p21-deficient mice developed spontaneous tumors at a median age of 16 months. The most common malignancies occurring in these animals were hemopoietic (B-cell lymphoma), en‐ dothelial, and epithelial tumors [21]. In addition, accelerated tumor formation and an in‐ creased capacity of tumor metastasis, respectively induced by urethane or by gamma radiation, were found in p21-/- mice [22,23]. Accelerated tumorigenesis, and promotion of lung metastasis was also found in correlation with cytoplasmic p21 in the mammary epiteli‐ um of mice expressing the MMTV/*neu* oncogene [24]. Tumor suppression functions of p21 were also confirmed by studies in the skin and in the colon of p21-deficient mice [25,26]. Furthermore, spontaneous tumor formation in p21-null mice was also found to occur in combination with other knock-out genetic backgrounds, such as Muc2-/- (mice lacking mucin 2), and Apc1638+/- (mutant allele of the adenomatosis polyposis gene) mice [27,28].

In addition to enhanced tumor formation, further investigations showed that loss of p21 caused exhaustion of blood stem cells [29], and induced development of Systemic Lupus Er‐ ythematosus in female animals [30]. Thus, the results obtained from transgenic mice, clearly indicated the tumor suppressor role of p21, although other studies have provided contrast‐ ing results [6,31]. As an example, p21-null mice crossed with knock-in PML-RAR mice, showed an oncogenic role of p21 in maintaining self-renewal of leukemic stem cells [32]. The dual behaviour of p21 most probably occurs because of its participation in several cellular processes, and it is dependent on different factors [6,31].

An important aspect for determining the target of p21 activity is the intracellular localiza‐ tion. Early studies indicated that lack of p21 expression, or cytoplasmic localization of the protein, promoted anchorage-independent growth, and drug resistance [5,6,31]. Human p21 protein is located predominantly in the nucleus; however, it is also present in the nucleolus and in the cytoplasm. In the nucleus, in addition to inhibit CDK2 and binding to PCNA, p21 may also associate with transcriptional regulators [4]. In the nucleolus, p21 was found to colocalize with cyclin E [33], and to accumulate after DNA damage, as a consequence of inhibi‐ tion of nuclear export [34]. Interestingly, growing body of evidence indicates that the cytoplasmic localization of p21 is linked to drug resistance [6,31], thus suggesting that in this compartment the protein may have a tumor-promoting function [35]. Cellular localization of p21 is regulated mainly by post-translation modifications. In fact, nuclear translocation ap‐ pears to be counteracted by different kinases phosphorylating Thr145 and Ser146 residues located near the NLS region of p21 [36-38]. These modifications are responsible for cytoplas‐ mic localization of p21, as well as for the loss of interaction with PCNA [39]. An important role in p21 phosphorylation is played by AKT1/PKB, which also mediates stability of the protein [36,37]. Another relevant modification of p21 (i.e ubiquitination) regulating its deg‐ radation, has been shown to occur predominantly in the nucleus, because p21 mutant in the NLS region exhibited enhanced stability [40].

A summary of the most important functions performed by p21 protein is reported in the fol‐ lowing paragraphs.

#### **Cell-cycle regulation**

tein 1 (CIP1), wild type p53-activated fragment (WAF1), senescent cell-derived inhibitor 1 (SDI1), and melanoma differentiation-associated protein 6 (MDA-6); all these names have been substituted by a new terminology including all CDK inhibitors, and p21 is now named

Due to the lack of a defined tertiary structure, p21 protein may adopt an extended confor‐ mation [9], which may explain its ability to interact with a number of proteins involved in

**Figure 1.** Schematic structure of p21 protein showing the regions responsible for binding to Cyclins, CDK and PCNA.

The main role of p21 is cell-cycle regulation, performed by inhibiting the activity of cyclin-CDK complexes thanks to direct interaction through specific sequences (termed CDK and Cy motifs) in the N-terminal domain of the protein [10-13]. Cell cycle progression may be also regulated, independently of cyclins and CDKs, thanks to the strong affinity binding to proliferating cell nuclear antigen (PCNA) [14-17], a protein playing a central role in DNA replication and repair, as well as in other processes of DNA metabolism [18,19]. This associ‐ ation may interfere with PCNA-dependent enzyme activities involved in DNA synthesis [18,19]. In contrast with the negative cell-cycle regulation, p21 may also serve as an assem‐ bly factor for cyclin D-CDK4/6 complexes, thus promoting cyclin D-dependent events, and

*CDKN1A* gene inactivation studies performed with experimental models, and in particular with knock-out mice, have confirmed the tumor suppressor functions of this protein [20,21]. The p21-null mice showed a normal development and did not show any spontaneous tumor formation until 7-month of age [20]. However, embryonic fibroblasts derived from these ani‐ mals were deficient in G1 checkpoint arrest following DNA damage [20]. Subsequent stud‐ ies in this model were extended to a longer time frame and the observations reported that p21-deficient mice developed spontaneous tumors at a median age of 16 months. The most

Below the N- and C-terminal regions are indicated the processes in which they are involved, respectively.

several important biological processes [3-6] (Figure 1).

**2. p21 biology and functions**

downstream activation of cyclin E-CDK2 [7,8].

CDKN1A.

250 New Research Directions in DNA Repair

As the principal mediator of cell cycle arrest in response to DNA damage, p21 not only acts by inactivating G1-phase cyclins/CDKs complexes, but also by inhibiting cell cycle progres‐ sion through other mechanisms. These possibly include direct interaction with PCNA to in‐ hibit DNA replication, and indirect effects mediated by interaction with other cell cycle regulators. In addition, p21 has been shown to play a role in the maintenance of G2-phase arrest, through multiple mechanisms [3,5,6].

Cyclin B-CDK1 complex has a relatively low affinity for p21 when compared with the other cyclin-CDK complexes [60], and a low amount of cyclin B/CDK1 was found to be associated with p21 after activation of the G2 checkpoint [61]. However, p21 has been demonstrated to contribute to CDK1 inactivation by inhibiting the CDK-activating kinase (CAK) and, conse‐ quently, the CDK1-activating Thr161 phosphorylation. Thus, p21/CAK pathway appears to be essential in sustaining the G2 arrest in response to DNA damage [61]. Other likely targets of p21 in G2 phase are cyclin A-CDK1/2 complexes [62,63]. As an additional mechanism of G2 arrest, p21 was also suggested to mediate nuclear retention of cyclin B1-CDK1 complex in response to genotoxic stress, thus preventing its activation by Cdc25 and CAK [64]. Recent‐ ly, it has been also proposed that p21 contributes to G2 arrest by mediating cyclin B degrada‐ tion in response to DNA damage [65]. Furthermore, a new p21-dependent mechanism to maintain G2 arrest after DNA damage has been shown to involve Emi1 protein, an inhibitor of the Anaphase Promoting Complex (APC) whose destruction controls progression through mitosis to G1 phase [66]. It has been reported that p21 down-regulates Emi1 in cells arrested in G2 by DNA damage, thereby contributing to APC activation and degradation of key substrates, including cyclins A2 and B1. Thus, p21 controls positively this checkpoint

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Another important function of p21 is related to the control of basal proliferation in specific cell types. In particular, the stem cell self-renewal of keratinocytes [67], of the haematopoiet‐ ic system [29], and of the mouse forebrain and hyppocampus [68,69], have been shown to depend on p21 protein. In fact, studies in CDKN1A knock-out mice showed that p21 re‐ stricts the self-renewal potential of stem cell population, and promotes their irreversible commitment to differentiation [67]. In the absence of p21, an increase in stem cell prolifera‐ tion with a consequent exhaustion of the population was observed in different cell types [67-70]. Interestingly, p21 is also able to maintain the self-renewal potential of leukemic stem cells, and to protect them from DNA damage accumulation, thereby demonstrating an onco‐

Cell quiescence and senescence are other processes in which p21 plays a fundamental role by keeping cells arrested in G0, or G0-like state, in order to prevent untimely DNA replica‐ tion [71,72]. Accordingly, loss of p21 has been shown to facilitate cell cycle entry from a qui‐ escence state, at the expense of replication stress [73]. Interestingly, lack of p21 expression has been found to link cell cycle control with appendage regeneration in mice, since p21-/-

p21 also plays a complex role in cell differentiation. In fact, its expression is induced in dif‐ ferentiating cells of the skin and of the intestinal epithelium, as well as in cultured epider‐ mal cells, while down-regulation has been observed at late stages of differentiation [75,76]. However, p21 appears to play a positive role in promoting differentiation of human pro‐ myelocytic leukaemia cells [77], mouse skeletal muscle and cartilage cells [78,79], and oligo‐ dendrocytes [80]. The whole body of evidence indicates that p21 plays either positive or negative roles in differentiation, independently of cell cycle control, but depending on cell type and specific stage of differentiation. This regulatory function may involve specific in‐

animals showed a phenotype similar to that of regenerating mouse strains [74].

teractions of p21 with critical regulators of differentiation [3,6].

preventing G2-arrested cells from entering mitosis [66].

genic activity of the protein [32].

The demonstration that p21 is involved in cell response to DNA damage, mediated through transcriptional activation by p53, was first obtained in mammalian cells [41,42]. The main role of p21 in the G1 checkpoint resides in its ability to inhibit the activity of cyclin E, and cyclin A/CDK2 complexes required for the G1/S phase transition, thereby contributing to G1 phase arrest [43]. Accordingly, mouse embryonic fibroblasts (MEFs) obtained from p21-null mice fail to arrest in G1 phase, in response to DNA damage [20,44]. Recently, it has been demonstrated that CDK2-/- MEFs, as well as regenerating liver cells in CDK2-/- mice, are able to arrest at the G1/S checkpoint in response to γ-irradiation. This response has been found to depend on the ability of CDK1 to substitute for CDK2, and on p21, which may associate with, and inhibit nuclear CDK1 at the G1/S transition [45].