It will be worth investigating as to how translocations are generated in compromised cases of NHEJ. One theory proposed by our group states that molecules such as translin, trax and their partners/interactors who do not appear to directly function in either of the predomi‐ nant repair pathways, NHEJ or HR, might somehow deceptively lead the cells into misre‐ pair functions and leading to chromosomal translocations. A possible mechanism that could function like the NHEJ would involve recruitment of translin and parallel factors onto dam‐ aged DNA ends, rejoining the staggered DNA DSBs followed by ligation of broken ends. The result would be a DNA sequence comprising of deletions and insertions at the repaired breakpoint junctions [141]. The mechanism is analogous to the study which demonstrated XRCC4-DNA Ligase IV complex as the most critical factor in rejoining the broken DNA ends though NHEJ. However, the recruitment and assembly of the NHEJ core factors was

Various biochemical and genetics studies have demonstrated that even in the absence of one or more core components of NHEJ, broken ends of DNA are joined. These mechanisms are referred to as alternative end-joining (EJ) or back-up pathways [143]. In this case, the rejoin‐ ing of DNA DSBs occurs at slower kinetics and can be erroneous which is incompatible with the concept of HR mechanism. Hence, it can be inferred that there are two possible signaling cascades in an event of DNA DSB, once which is the classical NHEJ (C-NHEJ), also known as DNA-PK – dependent NHEJ (D-NHEJ) involving Ku-DNA-PK complex as well as XRCC4-DNA Ligase IV complex which is effective in class switch recombination (CSR) in normal B lymphocytes. The other is the back-up NHEJ (B-NHEJ) which takes over the repair

in rejoining the broken chromosomes furthering the mechanism of NHEJ.

strikingly diverse from the known classical hierarchy of the molecules [142].

stage for deregulated proliferation [138].

**11. Translin and NHEJ**

#### *10.5.6. Leukemia*

Chronic myelogenic leukemia, associated with unregulated growth of myeloid cells in bone marrow is the result of a somatic gene rearrangement forming a fusion by two-way ex‐ change between 2 genes; BCR on chromosome 22 and ABL on chromosome 9 to form BCR-ABL. Consequently, t(9;22)(q34;q11) is the chromosomal translocation and the small derivative chromosome 22 product is well known as the Philadelphia or Ph chromosome [132]. Clinical studies also demonstrated these breakpoints in most patients with topoiso‐ merase II inhibitor therapy-related acute myeloid leukemia (tAML) [133]. Breakpoint se‐ quence patterns on this region of the BCR gene shared 80% identity with the translin consensus recognition sites. These were also positively identified in acute lymphoblastic leu‐ kemia cases with BCR-ABL hybrid. Alu sequences, the most repetitive regions of the human genome possess a high frequency of involvement in BCR recombination. Surprisingly, they also shared a close homology to translin consensus sequences, thereby indicating that the protein might be able to bind to one of the most ubiquitous regions of the genome [134].

#### *10.5.7. Lymphoma*

Burkitt's lymphoma cells that are deficient in component(s) of NHEJ pathway exhibit a large number of translocations resembling the classic translocations [135]. Further investigation may lead to a novel pathway employing translin and interactors for rejoining the break‐ points at these junctions that resemble translin recognition motifs.

#### *10.5.8. Carcinoma*

Translin-like protein was also detected in the proteomic analysis of human colorectal carci‐ noma cell lines along with other proteins, such as endothelial cell growth factor 1 (plateletderived), rhotekin protein (RTKN), septin 1, cyclin dependent kinase 1, and sialic acid binding Ig-like lectin 11, tyrosinase-related protein. All of these are known to be involved in cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, which is associat‐ ed with distinct aspects of tumour metastasis [136].

#### *10.5.9. Dysgerminoma*

Dysgerminoma, arising from gonad cells, is a rare form of ovarian tumor in adolescent women [137]. RNF139/TRC8 is a potential tumor suppressor gene and its post-transcription‐ al regulation is disrupted by a balanced translocation t(8;22)(q24.13;q11.21). Translin was found to be involved in posttranscriptional regulation of TRC8, which could be related to the interaction between translin and TRC8 to dysgerminoma. Thereby, a model was pro‐ posed wherein one copy of TRC8 was disrupted by palindrome-mediated translocation fol‐ lowed by further loss of TCR8 expression through suppression by translin, thus setting the stage for deregulated proliferation [138].

Monosomy 1p36 is the most common terminal deletion in newborns [139]. Two interstitial deletions were further discovered within the same chromosome generating balanced recip‐ rocal translocation t(1;9)(p36.3;q34). Alignments of these junctions did not exhibit any se‐ quence similarities suggesting the involvement of NHEJ in the ligation of broken ends. Further analysis of the breakpoint regions, even from solid tumors, revealed sequences simi‐ lar to that of translin consensus binding motifs, GCCCWSSW [140]. Although the translin recognition sequences are frequent in the human genome, due to their repetitive nature, DNA breakage can still not be considered a random event. These results could support the hypothesis that either the translin-binding sites are more prone to breakage or are involved in rejoining the broken chromosomes furthering the mechanism of NHEJ.

## **11. Translin and NHEJ**

analysis revealed more than 70% homologous sequences possessing translin-binding motifs adjacent to TLS/CHOP breakpoint junctions. Also, topoisomerase II consensus cleavage sites were found at these regions suggesting a role of the enzyme in creating staggered ends and recruiting one of the several factors such as translin in the process of chromosomal translo‐ cation. [130]. Furthermore, sequences highly homologous to consensus translin-binding mo‐ tifs were also found at the breakpoints generated by translocation t(X;18) in synovial

Chronic myelogenic leukemia, associated with unregulated growth of myeloid cells in bone marrow is the result of a somatic gene rearrangement forming a fusion by two-way ex‐ change between 2 genes; BCR on chromosome 22 and ABL on chromosome 9 to form BCR-ABL. Consequently, t(9;22)(q34;q11) is the chromosomal translocation and the small derivative chromosome 22 product is well known as the Philadelphia or Ph chromosome [132]. Clinical studies also demonstrated these breakpoints in most patients with topoiso‐ merase II inhibitor therapy-related acute myeloid leukemia (tAML) [133]. Breakpoint se‐ quence patterns on this region of the BCR gene shared 80% identity with the translin consensus recognition sites. These were also positively identified in acute lymphoblastic leu‐ kemia cases with BCR-ABL hybrid. Alu sequences, the most repetitive regions of the human genome possess a high frequency of involvement in BCR recombination. Surprisingly, they also shared a close homology to translin consensus sequences, thereby indicating that the protein might be able to bind to one of the most ubiquitous regions of the genome [134].

Burkitt's lymphoma cells that are deficient in component(s) of NHEJ pathway exhibit a large number of translocations resembling the classic translocations [135]. Further investigation may lead to a novel pathway employing translin and interactors for rejoining the break‐

Translin-like protein was also detected in the proteomic analysis of human colorectal carci‐ noma cell lines along with other proteins, such as endothelial cell growth factor 1 (plateletderived), rhotekin protein (RTKN), septin 1, cyclin dependent kinase 1, and sialic acid binding Ig-like lectin 11, tyrosinase-related protein. All of these are known to be involved in cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, which is associat‐

Dysgerminoma, arising from gonad cells, is a rare form of ovarian tumor in adolescent women [137]. RNF139/TRC8 is a potential tumor suppressor gene and its post-transcription‐

points at these junctions that resemble translin recognition motifs.

ed with distinct aspects of tumour metastasis [136].

sarcoma [131].

22 New Research Directions in DNA Repair

*10.5.6. Leukemia*

*10.5.7. Lymphoma*

*10.5.8. Carcinoma*

*10.5.9. Dysgerminoma*

It will be worth investigating as to how translocations are generated in compromised cases of NHEJ. One theory proposed by our group states that molecules such as translin, trax and their partners/interactors who do not appear to directly function in either of the predomi‐ nant repair pathways, NHEJ or HR, might somehow deceptively lead the cells into misre‐ pair functions and leading to chromosomal translocations. A possible mechanism that could function like the NHEJ would involve recruitment of translin and parallel factors onto dam‐ aged DNA ends, rejoining the staggered DNA DSBs followed by ligation of broken ends. The result would be a DNA sequence comprising of deletions and insertions at the repaired breakpoint junctions [141]. The mechanism is analogous to the study which demonstrated XRCC4-DNA Ligase IV complex as the most critical factor in rejoining the broken DNA ends though NHEJ. However, the recruitment and assembly of the NHEJ core factors was strikingly diverse from the known classical hierarchy of the molecules [142].

Various biochemical and genetics studies have demonstrated that even in the absence of one or more core components of NHEJ, broken ends of DNA are joined. These mechanisms are referred to as alternative end-joining (EJ) or back-up pathways [143]. In this case, the rejoin‐ ing of DNA DSBs occurs at slower kinetics and can be erroneous which is incompatible with the concept of HR mechanism. Hence, it can be inferred that there are two possible signaling cascades in an event of DNA DSB, once which is the classical NHEJ (C-NHEJ), also known as DNA-PK – dependent NHEJ (D-NHEJ) involving Ku-DNA-PK complex as well as XRCC4-DNA Ligase IV complex which is effective in class switch recombination (CSR) in normal B lymphocytes. The other is the back-up NHEJ (B-NHEJ) which takes over the repair task on the occasion of deficiency of the core factors such as Ku heterodimer, DNA-PK and XRCC4-DNA Ligase IV [144]. Boboila et. al. have demonstrated that CSR is mediated by al‐ ternative end-joining (A-EJ) in the event of combined deficiency of Ku 70 and DNA Ligase IV. IgH-c-myc chromosomal translocations were also augmented in this case [145]. Another study demonstrated that the characteristics of translocation breakpoint junctions in wildtype mammalian cells and those deficient in XRCC4-DNA Ligase IV were similar, further implying that A-EJ pathway could be the primary mediator of chromosomal translocation in mammalian cells [146].

might be involved in the control of recombination at microsatellites and in the maintenance of telomeres which are highly repetitive structures. The binding of translin to oligonucleoti‐ des *in vitro* has been demonstrated to increase the extension of telomeres [96]. Amplification of telomerase and increased telomere length is associated with the invasive and metastatic potential of murine and human tumors [149]. Translin transcripts are also at an elevated lev‐

Recombination Hot-Spots and Defense Players – Maintenance of Genomic Integrity

http://dx.doi.org/10.5772/54016

25

Other than DNA/RNA regulation, translin might also be considered a responsible factor in

Long-term administration of imipramine, an anti-depressant drug, downregulated translin presumably playing a vital role in the segregation of chromosomes and cytokinesis as well as accelerating cell proliferation [152]. tsnΔ and traxΔ cells were not responsive to several DNA damaging agents indicating that neither protein was required for recovery from DNA damage, dispelling the suggestion that these proteins are evolutionarily conserved due to a fundamental role in the DNA damage response [153]. The finding that trax and translin seem to regulate cell proliferation in higher eukaryotes, but not in *S. pombe*, where the bio‐ chemical function is conserved, indicates that there is not a clear correlation between the conserved biochemical function and regulation of cell proliferation, suggesting that the two are not linked. Further analysis in this simple eukaryote will provide insight into the nature

Trax harbors a nuclear localization signal and interacts with translin to transport it to the nucleus when required. Once in the nucleus, translin–trax can interact with DNA to carry out the repair function along with several other co-factors. Thereafter, trax dissociates from the complex, exchanging translin for C1D, and freeing translin to interact with mRNAs marked for export from the nucleus via translin's nuclear export signal [60, 86]. Once trans‐ lin has re-entered the cytoplasm, it can remain bound to the mRNA until a cellular signal for release and subsequent translation of the message has been received (Figure 3). The ability to act as a shuttling protein is a hallmark of the RNA-binding proteins that traffic mRNAs in neuronal cells [154]. Based on studies of translin-trax involved in dendritic targeting of BDNF mRNA, it is conceivable that heteromeric translin/trax complexes mediate dendritic trafficking of mRNAs, but that its nuclease activity is suppressed during mRNA transport [155] and activated when functioning as components of RISC complex. Accordingly, it will be of interest in future studies to test these models of translin's dual role in mRNA transport

The influence of translin on proliferation, DNA repair, chromosome segregation and cytoki‐ nesis, RNA stability and transport, and translation of proteins as well as telomere elongation

el in mouse lung adenocarocinoma indicating an early event in carcinogenesis [150].

one of the benchmark obese phenotypes in mice [151].

**13. Future directives**

of this process.

and silencing.

may be critical in tumor formation and progression.

All of these recent evidences suggest that chromosomal translocations are rather suppressed when canonical NHEJ in involved in repair of DNA DSBs. But they become more common when A-EJ takes over. One of the speculation is that there is a rare probability for concurrent DSBs as one is usually repaired and restored to original chromosomal configuration by im‐ mediate sensing of Ku-DNA-PK-XRCC4-DNA Ligase IV complex before the next one oc‐ curs. Thus the temporal opportunity for translocations to occur is reduced [147]. On the occasion of inefficient C-NHEJ, the rejoining is slower by A-EJ, widening the time frame be‐ fore each DSB closure, thus increasing the chance of two or multiple DSBs at the same time, leading to chromosomal translocations. However, which enzymes participate in this path‐ way is not quite certain. There are mounting evidences depicting Mre11-Rad50-NBS1, pol β, PARP, PALF and DNA Ligase I or III as some of the players carrying out A-EJ. However, no set rules governing the hierarchy of this mechanism are brought to light. Several of these translocations possess translin recognition motifs. Therefore, one theory could be postulat‐ ed, wherein, translin might be the early sensory molecule binding to recognition sequences and recruiting the downstream nucleases and ligases.

Several other pathways which are ATM-dependent or MRN-dependent are also highlighted by other groups. Since any physiological mechanism is less likely to be exclusively inde‐ pendent, there is a high probability of cross-talk even among the DSB repair pathways: HR, C-NHEJ, A-EJ, or other pathways involving the core and alternative components. However, this discussion is beyond the scope of this review.

## **12. Conclusions**

Detailed analysis of breakpoint junction consensus sequences suggested that they were not simple and could possess a diverse amount of variations. Translin has been found to bind at translocation break points and proposed to be involved in DNA recombination and repair and in the regulation of telomere length [148]. Surprisingly, AT and GC repeat sequences which had almost no homology with known breakpoint sequences such as ATGCAG and GCCC(A/T)(G/C)(G/C)(A/T) showed a high binding affinity to translin. Translin also binds d(GT)n and (TTAGGG)n overhangs linked to Ds DNA which forms unusual structures such as DNA quadruplexes or that inhibits their binding to the protein unless unwound and the binding domains are accessible per octamer [96]. This leads to a proposition that translin might be involved in the control of recombination at microsatellites and in the maintenance of telomeres which are highly repetitive structures. The binding of translin to oligonucleoti‐ des *in vitro* has been demonstrated to increase the extension of telomeres [96]. Amplification of telomerase and increased telomere length is associated with the invasive and metastatic potential of murine and human tumors [149]. Translin transcripts are also at an elevated lev‐ el in mouse lung adenocarocinoma indicating an early event in carcinogenesis [150].

Other than DNA/RNA regulation, translin might also be considered a responsible factor in one of the benchmark obese phenotypes in mice [151].

## **13. Future directives**

task on the occasion of deficiency of the core factors such as Ku heterodimer, DNA-PK and XRCC4-DNA Ligase IV [144]. Boboila et. al. have demonstrated that CSR is mediated by al‐ ternative end-joining (A-EJ) in the event of combined deficiency of Ku 70 and DNA Ligase IV. IgH-c-myc chromosomal translocations were also augmented in this case [145]. Another study demonstrated that the characteristics of translocation breakpoint junctions in wildtype mammalian cells and those deficient in XRCC4-DNA Ligase IV were similar, further implying that A-EJ pathway could be the primary mediator of chromosomal translocation in

All of these recent evidences suggest that chromosomal translocations are rather suppressed when canonical NHEJ in involved in repair of DNA DSBs. But they become more common when A-EJ takes over. One of the speculation is that there is a rare probability for concurrent DSBs as one is usually repaired and restored to original chromosomal configuration by im‐ mediate sensing of Ku-DNA-PK-XRCC4-DNA Ligase IV complex before the next one oc‐ curs. Thus the temporal opportunity for translocations to occur is reduced [147]. On the occasion of inefficient C-NHEJ, the rejoining is slower by A-EJ, widening the time frame be‐ fore each DSB closure, thus increasing the chance of two or multiple DSBs at the same time, leading to chromosomal translocations. However, which enzymes participate in this path‐ way is not quite certain. There are mounting evidences depicting Mre11-Rad50-NBS1, pol β, PARP, PALF and DNA Ligase I or III as some of the players carrying out A-EJ. However, no set rules governing the hierarchy of this mechanism are brought to light. Several of these translocations possess translin recognition motifs. Therefore, one theory could be postulat‐ ed, wherein, translin might be the early sensory molecule binding to recognition sequences

Several other pathways which are ATM-dependent or MRN-dependent are also highlighted by other groups. Since any physiological mechanism is less likely to be exclusively inde‐ pendent, there is a high probability of cross-talk even among the DSB repair pathways: HR, C-NHEJ, A-EJ, or other pathways involving the core and alternative components. However,

Detailed analysis of breakpoint junction consensus sequences suggested that they were not simple and could possess a diverse amount of variations. Translin has been found to bind at translocation break points and proposed to be involved in DNA recombination and repair and in the regulation of telomere length [148]. Surprisingly, AT and GC repeat sequences which had almost no homology with known breakpoint sequences such as ATGCAG and GCCC(A/T)(G/C)(G/C)(A/T) showed a high binding affinity to translin. Translin also binds d(GT)n and (TTAGGG)n overhangs linked to Ds DNA which forms unusual structures such as DNA quadruplexes or that inhibits their binding to the protein unless unwound and the binding domains are accessible per octamer [96]. This leads to a proposition that translin

mammalian cells [146].

24 New Research Directions in DNA Repair

**12. Conclusions**

and recruiting the downstream nucleases and ligases.

this discussion is beyond the scope of this review.

Long-term administration of imipramine, an anti-depressant drug, downregulated translin presumably playing a vital role in the segregation of chromosomes and cytokinesis as well as accelerating cell proliferation [152]. tsnΔ and traxΔ cells were not responsive to several DNA damaging agents indicating that neither protein was required for recovery from DNA damage, dispelling the suggestion that these proteins are evolutionarily conserved due to a fundamental role in the DNA damage response [153]. The finding that trax and translin seem to regulate cell proliferation in higher eukaryotes, but not in *S. pombe*, where the bio‐ chemical function is conserved, indicates that there is not a clear correlation between the conserved biochemical function and regulation of cell proliferation, suggesting that the two are not linked. Further analysis in this simple eukaryote will provide insight into the nature of this process.

Trax harbors a nuclear localization signal and interacts with translin to transport it to the nucleus when required. Once in the nucleus, translin–trax can interact with DNA to carry out the repair function along with several other co-factors. Thereafter, trax dissociates from the complex, exchanging translin for C1D, and freeing translin to interact with mRNAs marked for export from the nucleus via translin's nuclear export signal [60, 86]. Once trans‐ lin has re-entered the cytoplasm, it can remain bound to the mRNA until a cellular signal for release and subsequent translation of the message has been received (Figure 3). The ability to act as a shuttling protein is a hallmark of the RNA-binding proteins that traffic mRNAs in neuronal cells [154]. Based on studies of translin-trax involved in dendritic targeting of BDNF mRNA, it is conceivable that heteromeric translin/trax complexes mediate dendritic trafficking of mRNAs, but that its nuclease activity is suppressed during mRNA transport [155] and activated when functioning as components of RISC complex. Accordingly, it will be of interest in future studies to test these models of translin's dual role in mRNA transport and silencing.

The influence of translin on proliferation, DNA repair, chromosome segregation and cytoki‐ nesis, RNA stability and transport, and translation of proteins as well as telomere elongation may be critical in tumor formation and progression.

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**Figure 3.** Proposed model for shuttling mechanism of Translin-Trax complex

## **Acknowledgements**

I would like to thank the Journal of Acta Crystallographica Section D, Biological Crystallog‐ raphy and Dr. Masataka Kasai, for granting me permission to reproduce the crystal struc‐ ture of Translin, in order to better emphasize the significance of the oligomeric state. RPK is also greatly appreciative of Dr. Vinay Kumar and his group, BARC, India, for consenting to the re-use of the crystal structure of the *Drosophila* mutant translin and its oligomeric status from his publication, Gupta et. al., (2008) FEBS Journal. I would also like to thank Dr. Sunil Saroj, Emory University, USA, for his kind help in graphic designing of the model.

## **Author details**

Radhika Pankaj Kamdar1 and Basuthkar J. Rao2

1 Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA

2 Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, Ma‐ harashtra, India

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**Figure 3.** Proposed model for shuttling mechanism of Translin-Trax complex

I would like to thank the Journal of Acta Crystallographica Section D, Biological Crystallog‐ raphy and Dr. Masataka Kasai, for granting me permission to reproduce the crystal struc‐ ture of Translin, in order to better emphasize the significance of the oligomeric state. RPK is also greatly appreciative of Dr. Vinay Kumar and his group, BARC, India, for consenting to the re-use of the crystal structure of the *Drosophila* mutant translin and its oligomeric status from his publication, Gupta et. al., (2008) FEBS Journal. I would also like to thank Dr. Sunil

1 Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia,

2 Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, Ma‐

Saroj, Emory University, USA, for his kind help in graphic designing of the model.

and Basuthkar J. Rao2

**Acknowledgements**

26 New Research Directions in DNA Repair

**Author details**

harashtra, India

USA

Radhika Pankaj Kamdar1

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[146] Simsek D, Jasin M. Alternative end-joining is suppressed by the canonical NHEJ component Xrcc4-ligase IV during chromosomal translocation formation. Nature

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**Chapter 2**

**The Role of Multimerization During**

**Non-Homologous End Joining**

Michelle Rubin, Jonathan Newsome and

Additional information is available at the end of the chapter

spontaneous mutations that can lead to tumorigenesis [5].

In order to sustain life, cells must protect their genetic information from the constant threat posed by mutagenic agents such as ultraviolet light, irradiation or reactive oxy‐ gen species, as well as from mistakes introduced during the replication of their ge‐ nomes [1]. To deal with this problem, natural selection has favored a system that repairs the damage caused by these DNA lesions while allowing the highly infrequent but steady production of mutations that constitute the source for adaptive changes dur‐ ing evolution. To repair damaged DNA, cells have developed a myriad of highly speci‐ alized pathways that recognize and repair specific types of injuries produced by specific types of mutagenic events [2, 3]. For instance, while base excision repair detects and re‐ pairs chemically damaged nucleotide bases typically produced by oxygen radicals or al‐ kylating agents, nucleotide excision repair is responsible for the removal of thymine dimers caused by ultraviolet light exposure. Additionally, the mismatch repair pathway specializes in fixing errors introduced during DNA replication. More globally, these pathways are part of the DNA damage response (DDR), a signal transduction cascade coordinated by the ATM/ATR kinases in mammalian cells that halts cell cycle progres‐ sion while DNA is being repaired, and it can trigger apoptosis when the damage is deemed non-repairable [4]. The importance of these pathways is underlined by their high conservation, both in prokaryotes and eukaryotes, and it is emphasized by the role that they play in disease when impaired. Malfunctioning DNA repair pathways are as‐ sociated with several disorders such as Xeroderma pigmentosum or Nijmegen syn‐ drome as well as with increased cancer risk, as they boost the formation of

> © 2013 Rubin 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,

© 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,

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

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

Albert Ribes-Zamora

**1. Introduction**

http://dx.doi.org/10.5772/53982

**Chapter 2**

## **The Role of Multimerization During Non-Homologous End Joining**

Michelle Rubin, Jonathan Newsome and Albert Ribes-Zamora

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53982

## **1. Introduction**

In order to sustain life, cells must protect their genetic information from the constant threat posed by mutagenic agents such as ultraviolet light, irradiation or reactive oxy‐ gen species, as well as from mistakes introduced during the replication of their ge‐ nomes [1]. To deal with this problem, natural selection has favored a system that repairs the damage caused by these DNA lesions while allowing the highly infrequent but steady production of mutations that constitute the source for adaptive changes dur‐ ing evolution. To repair damaged DNA, cells have developed a myriad of highly speci‐ alized pathways that recognize and repair specific types of injuries produced by specific types of mutagenic events [2, 3]. For instance, while base excision repair detects and re‐ pairs chemically damaged nucleotide bases typically produced by oxygen radicals or al‐ kylating agents, nucleotide excision repair is responsible for the removal of thymine dimers caused by ultraviolet light exposure. Additionally, the mismatch repair pathway specializes in fixing errors introduced during DNA replication. More globally, these pathways are part of the DNA damage response (DDR), a signal transduction cascade coordinated by the ATM/ATR kinases in mammalian cells that halts cell cycle progres‐ sion while DNA is being repaired, and it can trigger apoptosis when the damage is deemed non-repairable [4]. The importance of these pathways is underlined by their high conservation, both in prokaryotes and eukaryotes, and it is emphasized by the role that they play in disease when impaired. Malfunctioning DNA repair pathways are as‐ sociated with several disorders such as Xeroderma pigmentosum or Nijmegen syn‐ drome as well as with increased cancer risk, as they boost the formation of spontaneous mutations that can lead to tumorigenesis [5].

© 2013 Rubin 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. © 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.

For any type of cell, one of the more toxic DNA injuries is the double-strand break (DSB). This form of lesion can arise as a consequence of mechanical stress, exposure to irradiation or as a result of a replication fork encountering a single-strand nick [6]. DSBs can induce translocations, aneuploidy and global genome instability that can ulti‐ mately render cells either unviable or tumorigenic [5]. To repair a DSB, mammalian cells can take advantage of the presence of homologous chromosomal copies and use homologous recombination (HR) to faithfully amend the break [7]. In the cell cycle phases where identical chromosomal copies are not available, the preferred repair path‐ way is non-homologous end joining (NHEJ) which seams the two ends of the break with mostly minimal alteration of the DNA sequence [1, 8-10]. NHEJ requires the com‐ pletion of three major steps: (1) protection and synapsis of both DNA ends, (2) process‐ ing of the DNA termini and (3) the final ligation of the ends [9, 10]. The DNA-PK complex, formed by the Ku heterodimer and DNA-PKcs, is responsible for the initial protection and synapses of the ends and recruits other NHEJ factors to the DSB. These factors include the nuclease Artemis and polymerases Polμ and Polλ that will remove and add nucleotides to replace possible damaged bases generated during the breaking process. The final ligation step is performed by DNA ligase IV (LigIV), whose recruit‐ ment to DSBs depends on its close association with XRCC4, a process aided by XLF. The regulation of these steps is still not clear but it is known that DNA-PKcs phosphor‐ ylates several NHEJ factors and can induce its own removal from DSBs, and that ubiq‐ uitination also plays a role in disassembling complexes once the damage has been repaired [11-14]. Furthermore, ATM can phosphorylate NHEJ factors, although their role during NHEJ remains to be elucidated [15].

complexes form depending on the type of damage, the location of the break or the cell cycle phase when the injury occurs [10]. One of the emerging themes in the field is the assembly of NHEJ factors as multimers at DSB. This multimerization has been reported for several NHEJ proteins and occurs with varying degrees of complexity. The simplest form of multimerization is seen with DNA-PKcs, where monomers bound to opposing ends of a DSB can dimerize and effectively synapse the DNA break [19]. Similarly, two Ku heterodimers are capable of forming a heterotetramer that can tether the ends of a DSB. More intricate multimerization can be observed in the MRN heterotrimer, a com‐ plex composed of Mre11, Rad50 and Nbs1 that can form either heterohexamers, where two molecules of each subunit combine into a larger structure, or multimers of four MRN complexes at DSBs [20]. Most recently, a complex multimerization of NHEJ pro‐ teins has been observed in the form of long filaments created by the polymerization of multiple alternating copies of XLF and XRCC4 homodimers [21-23]. Combined, this da‐ ta suggests that formation of multimers is a constant theme in the assembly of NHEJ proteins at DSBs. Below we review current literature on this topic, identifying questions that remain to be answered while laying out possible new research directions. While NHEJ can be divided into classical NHEJ (c-NHEJ) or alternative NHEJ (a-NHEJ) de‐ pending on the factors required for completion, here we focus on c-NHEJ and will refer

The Role of Multimerization During Non-Homologous End Joining

http://dx.doi.org/10.5772/53982

43

Once a break forms, one of the first responders is the Ku heterodimer, an abundant protein (~400,000 molecules per cell) formed by the Ku70 and Ku80 subunits [24]. Ku is an obligate dimer as in the absence of one subunit the other subunit disappears from cell extracts, pre‐ sumably due to lack of proper folding [25, 26]. Despite showing low sequence similarities, both Ku70 and Ku80 contain nearly identical domains and have very similar secondary and tertiary structures. Both subunits share a vonWillebrand domain (vWA, also referred to as the a/b domain) in their N-termini followed by a central dimerization domain that can also bind DNA [27]. The only divergence between both subunits is the presence of a C-terminus SAP domain (SAF-A/B, Acinus and PIAS) exclusively in Ku70, which is replaced in KU80 by

The structure of Ku shows a quasi-symmetrical configuration with both central domains dimerizing and forming a DNA binding ring flanked by the two vWA domains on oppo‐ site sides of the ring [27]. This creates a toroidal structure with a basket-like shape that can thread onto DNA (Figure 1A). Ku binds duplex DNA ends with great affinity (Kd~10-9 M) and in a sequence independent manner, hence its role as the first DSB recog‐ nition factor [29-31]. Ku needs at least 14bp to bind DNA and since the DNA binding ring is preformed, Ku requires a free end to associate with DNA [27]. Consistent with this, the affinity of Ku to circular DNA is orders of magnitude lower compared with linear DNA [32]. Similarly, Ku's affinity to single-stranded DNA (ssDNA) is lower than to doublestranded DNA (dsDNA), which presumably favors HR over NHEJ once resection of ends

a domain that is involved in recruiting DNA-PKcs to DSBs [24, 27, 28].

to it as NHEJ.

**2.1. The Ku heterodimer**

becomes too extensive to hold Ku [7].

During evolution, the end protection properties of some NHEJ factors were recycled into protecting the natural ends of chromosomes. How telomeres manage to harbor NHEJ factors to protect their ends while preventing them from triggering end-to-end fusions is still an open question [16]. Later on, NHEJ was again recycled into joining physiologically program‐ med DSBs that occur during V(D)J recombination and class switch recombination (CSR) in B- and T- lymphocytes. These pathways ensure antigen-binding diversity in antibodies as well as the presence of different antibody isotypes capable of binding different downstream effectors. Consistent with a role of NHEJ in these pathways, mutations in several NHEJ fac‐ tors are associated with diseases in which the immune system is compromised. For instance, mutations in XLF, DNA-PKcs and Artemis are present in patients suffering from severe combined immunodeficiency or SCID [17].

#### **2. Multimerization of NHEJ factors**

For the last two decades, research in NHEJ has mostly focused on the identification of genes involved in this pathway and the dissection of their enzymatic activities [9]. The structures of most NHEJ factors have been solved and this is starting to unravel how NHEJ is regulated throughout the cell cycle [18]. Despite these advances, we are still lacking a clear model of how all these factors assemble at DSBs and whether different complexes form depending on the type of damage, the location of the break or the cell cycle phase when the injury occurs [10]. One of the emerging themes in the field is the assembly of NHEJ factors as multimers at DSB. This multimerization has been reported for several NHEJ proteins and occurs with varying degrees of complexity. The simplest form of multimerization is seen with DNA-PKcs, where monomers bound to opposing ends of a DSB can dimerize and effectively synapse the DNA break [19]. Similarly, two Ku heterodimers are capable of forming a heterotetramer that can tether the ends of a DSB. More intricate multimerization can be observed in the MRN heterotrimer, a com‐ plex composed of Mre11, Rad50 and Nbs1 that can form either heterohexamers, where two molecules of each subunit combine into a larger structure, or multimers of four MRN complexes at DSBs [20]. Most recently, a complex multimerization of NHEJ pro‐ teins has been observed in the form of long filaments created by the polymerization of multiple alternating copies of XLF and XRCC4 homodimers [21-23]. Combined, this da‐ ta suggests that formation of multimers is a constant theme in the assembly of NHEJ proteins at DSBs. Below we review current literature on this topic, identifying questions that remain to be answered while laying out possible new research directions. While NHEJ can be divided into classical NHEJ (c-NHEJ) or alternative NHEJ (a-NHEJ) de‐ pending on the factors required for completion, here we focus on c-NHEJ and will refer to it as NHEJ.

#### **2.1. The Ku heterodimer**

For any type of cell, one of the more toxic DNA injuries is the double-strand break (DSB). This form of lesion can arise as a consequence of mechanical stress, exposure to irradiation or as a result of a replication fork encountering a single-strand nick [6]. DSBs can induce translocations, aneuploidy and global genome instability that can ulti‐ mately render cells either unviable or tumorigenic [5]. To repair a DSB, mammalian cells can take advantage of the presence of homologous chromosomal copies and use homologous recombination (HR) to faithfully amend the break [7]. In the cell cycle phases where identical chromosomal copies are not available, the preferred repair path‐ way is non-homologous end joining (NHEJ) which seams the two ends of the break with mostly minimal alteration of the DNA sequence [1, 8-10]. NHEJ requires the com‐ pletion of three major steps: (1) protection and synapsis of both DNA ends, (2) process‐ ing of the DNA termini and (3) the final ligation of the ends [9, 10]. The DNA-PK complex, formed by the Ku heterodimer and DNA-PKcs, is responsible for the initial protection and synapses of the ends and recruits other NHEJ factors to the DSB. These factors include the nuclease Artemis and polymerases Polμ and Polλ that will remove and add nucleotides to replace possible damaged bases generated during the breaking process. The final ligation step is performed by DNA ligase IV (LigIV), whose recruit‐ ment to DSBs depends on its close association with XRCC4, a process aided by XLF. The regulation of these steps is still not clear but it is known that DNA-PKcs phosphor‐ ylates several NHEJ factors and can induce its own removal from DSBs, and that ubiq‐ uitination also plays a role in disassembling complexes once the damage has been repaired [11-14]. Furthermore, ATM can phosphorylate NHEJ factors, although their role

During evolution, the end protection properties of some NHEJ factors were recycled into protecting the natural ends of chromosomes. How telomeres manage to harbor NHEJ factors to protect their ends while preventing them from triggering end-to-end fusions is still an open question [16]. Later on, NHEJ was again recycled into joining physiologically program‐ med DSBs that occur during V(D)J recombination and class switch recombination (CSR) in B- and T- lymphocytes. These pathways ensure antigen-binding diversity in antibodies as well as the presence of different antibody isotypes capable of binding different downstream effectors. Consistent with a role of NHEJ in these pathways, mutations in several NHEJ fac‐ tors are associated with diseases in which the immune system is compromised. For instance, mutations in XLF, DNA-PKcs and Artemis are present in patients suffering from severe

For the last two decades, research in NHEJ has mostly focused on the identification of genes involved in this pathway and the dissection of their enzymatic activities [9]. The structures of most NHEJ factors have been solved and this is starting to unravel how NHEJ is regulated throughout the cell cycle [18]. Despite these advances, we are still lacking a clear model of how all these factors assemble at DSBs and whether different

during NHEJ remains to be elucidated [15].

42 New Research Directions in DNA Repair

combined immunodeficiency or SCID [17].

**2. Multimerization of NHEJ factors**

Once a break forms, one of the first responders is the Ku heterodimer, an abundant protein (~400,000 molecules per cell) formed by the Ku70 and Ku80 subunits [24]. Ku is an obligate dimer as in the absence of one subunit the other subunit disappears from cell extracts, pre‐ sumably due to lack of proper folding [25, 26]. Despite showing low sequence similarities, both Ku70 and Ku80 contain nearly identical domains and have very similar secondary and tertiary structures. Both subunits share a vonWillebrand domain (vWA, also referred to as the a/b domain) in their N-termini followed by a central dimerization domain that can also bind DNA [27]. The only divergence between both subunits is the presence of a C-terminus SAP domain (SAF-A/B, Acinus and PIAS) exclusively in Ku70, which is replaced in KU80 by a domain that is involved in recruiting DNA-PKcs to DSBs [24, 27, 28].

The structure of Ku shows a quasi-symmetrical configuration with both central domains dimerizing and forming a DNA binding ring flanked by the two vWA domains on oppo‐ site sides of the ring [27]. This creates a toroidal structure with a basket-like shape that can thread onto DNA (Figure 1A). Ku binds duplex DNA ends with great affinity (Kd~10-9 M) and in a sequence independent manner, hence its role as the first DSB recog‐ nition factor [29-31]. Ku needs at least 14bp to bind DNA and since the DNA binding ring is preformed, Ku requires a free end to associate with DNA [27]. Consistent with this, the affinity of Ku to circular DNA is orders of magnitude lower compared with linear DNA [32]. Similarly, Ku's affinity to single-stranded DNA (ssDNA) is lower than to doublestranded DNA (dsDNA), which presumably favors HR over NHEJ once resection of ends becomes too extensive to hold Ku [7].

Resolution of the X-ray structure of DNA bound Ku indicated that binding occurs with a pre‐ ferred orientation that places the Ku70 vWA domain closest to the end and the Ku80 vWA fur‐ thest from the end [27]. In the budding yeast *S. cerevisae*, mutations in α-helix5 (α5), the most outer structure in the vWA domain had opposite effects in each subunit [33]. Whereas Yku80 a5 mutations disrupt Ku's telomeric silencing function without perturbing DNA repair abili‐ ties, mutations in Yku70-α5 impair NHEJ while preserving Ku's telomeric functions [33]. This suggests that Ku is spatially organized in two faces with distinct roles in NHEJ and telomeric functions, a hypothesis termed the two-face model. In essence, this model states that the in‐ ward face, composed mostly of the Ku80 vWA and Ku70 C-terminal domain (CTD), is oriented towards internal tracks of DNA and has telomeric roles. In contrast, the outward face of Ku is the closest to the DNA end and its main role is to engage the nearby DNA end in NHEJ. Consis‐ tent with this, mutations in both the Ku70 vWA domain and the Ku80 CTD, the two major com‐ ponents of the outer face, impair NHEJ [33, 34]. The most recent discovery that mutations in human Ku70-α5 also diminish NHEJ suggests that the two-face model may also be conserved in mammalian cells [35].

chromatography showed that while recombinant Ku exists as a single heterodimer in solution, the addition of 24bp DNA fragments promotes the appearance of Ku multi‐ mers whose sizes correspond to that of a heterotetramer [40]. More importantly, using a combination of electron microscopy (EM) and atomic force microscopy (AFM), they vi‐ sualized Ku-dependent end-to-end bridging events involving either ends of two DNA fragments or loops within a single DNA fragment. Ku was found forming higher order multimers in the junction of those events, which suggests that Ku multimerization is re‐ sponsible for its end bridging properties. To note, this was not the result of non-specific aggregation of Ku as multimers could only be detected with DNA-bound Ku and not with free Ku molecules. The ability of Ku to synapse two ends was later confirmed by Ramsden *et al* using a mix of biotinylated DNA fragments with 32P-radiolabeled dsDNA [41]. When streptavidin beads were used to pull down biotinylated DNA, researchers could recover radiolabeled DNA only when recombinant Ku was also present in the mix. This co-precipitation could not be explained by a single Ku molecule binding and stabilizing the junction of two DNA fragments with complementary ends as similar re‐ sults were obtained when DNA fragments with non-compatible ends were used. This result suggests that synapses were achieved through the interaction of at least two Ku molecules each bound to a differently labeled DNA. More recently, DNA-bound Ku het‐ erotetramers have been demonstrated as supershifts in electromobility shift assays (EM‐ SA) and EM studies have visualized formation of end bridges using recombinant yeast Ku protein, indicating that multimerization-dependent synapses may be evolutionarily

The Role of Multimerization During Non-Homologous End Joining

http://dx.doi.org/10.5772/53982

45

Recent work with the Ku80 CTD suggests that Ku heterotetramerization may occur through the interaction of two outward faces [42]. This long and flexible domain interacts with the Ku core domain and upon binding to DNA it undergoes a conformational change that relocates it to the outward face [43]. Researchers have now shown that the Ku80 CTD can dimerize and thus, can putatively mediate Ku-Ku interactions across a DSB enabling the tethering of two DNA ends [42]. In fact, Ku proteins bearing Ku80 CTD truncations have reduced ability to form heterotetramers as shown by reduced supershift EMSA signals compared with wild type [42]. Ku80 CTD truncations impair NHEJ, but this result cannot be fully attributed to reduced heterotetramer formation as the Ku80 CTD is also involved in recruiting DNA-PKcs to DSBs. Intriguingly, a mutation in the out‐ ward face located Ku70-α5 also impairs NHEJ, although the effect of this mutation on Ku

While the presence of Ku multimers of higher order than heterotetramers has been detected in EM and AFM using recombinant Ku proteins, its functional significance remains to be elucidated and evidence for its existence in living cells is lacking [40, 44-46]. A single hetero‐ tetramer is sufficient to create a synaptic complex across DSB and it is difficult to envision how higher order Ku complexes may aid in this process. Since Ku can slide towards internal tracks of DNA, one possibility is that multiple Ku molecules threaded into a single DNA end could form filaments held by interactions between inward and outward faces. Howev‐

heterotetramerization remains to be investigated [35].

er, fluorescence anisotropy studies do not support this model [47].

conserved [42].

**Figure 1.** Molecular modeling of Ku and DNA-PKcs dimerization. A Possible Ku tetramer formation through outward face interactions. Each Ku dimer is represented in red and blue, whereas DNA is depicted as black line (Adapted from 1JEY). B Possible Head-to-Head mediated dimerization of two DNA-PKcs molecules (Adapted from 3KGV).

Ku plays multiple roles during NHEJ. Initially, Ku not only binds and detects DSB but also protects the ends from nucleolytic degradation and tilts the choice of DSB repair towards NHEJ and away from HR [7, 36]. Perhaps the most extensive role for Ku is to recruit NHEJ factors to DSB sites. The Ku80 CTD recruits DNA-PKcs to DSBs and binds a long list of NHEJ factors including XRCC4, LigIV, XLF, Polμ, and Polλ [9, 10]. Moreover, Ku's require‐ ment for the recruitment of DNA-PKcs, XLF and XRCC4 has been demonstrated *in vivo* [37, 38]. In addition, Ku possesses some catalytic activities and can function as a deubiquitylat‐ ing enzyme or as a 5'-dRP/AP lyase which suggests that Ku may aid in processing DNA ends before the final ligation [39].

The ability of the Ku heterodimer to self-associate and create higher order multimers was already apparent in early studies. Seminal work from Cary *et al* using gel filtration chromatography showed that while recombinant Ku exists as a single heterodimer in solution, the addition of 24bp DNA fragments promotes the appearance of Ku multi‐ mers whose sizes correspond to that of a heterotetramer [40]. More importantly, using a combination of electron microscopy (EM) and atomic force microscopy (AFM), they vi‐ sualized Ku-dependent end-to-end bridging events involving either ends of two DNA fragments or loops within a single DNA fragment. Ku was found forming higher order multimers in the junction of those events, which suggests that Ku multimerization is re‐ sponsible for its end bridging properties. To note, this was not the result of non-specific aggregation of Ku as multimers could only be detected with DNA-bound Ku and not with free Ku molecules. The ability of Ku to synapse two ends was later confirmed by Ramsden *et al* using a mix of biotinylated DNA fragments with 32P-radiolabeled dsDNA [41]. When streptavidin beads were used to pull down biotinylated DNA, researchers could recover radiolabeled DNA only when recombinant Ku was also present in the mix. This co-precipitation could not be explained by a single Ku molecule binding and stabilizing the junction of two DNA fragments with complementary ends as similar re‐ sults were obtained when DNA fragments with non-compatible ends were used. This result suggests that synapses were achieved through the interaction of at least two Ku molecules each bound to a differently labeled DNA. More recently, DNA-bound Ku het‐ erotetramers have been demonstrated as supershifts in electromobility shift assays (EM‐ SA) and EM studies have visualized formation of end bridges using recombinant yeast Ku protein, indicating that multimerization-dependent synapses may be evolutionarily conserved [42].

Resolution of the X-ray structure of DNA bound Ku indicated that binding occurs with a pre‐ ferred orientation that places the Ku70 vWA domain closest to the end and the Ku80 vWA fur‐ thest from the end [27]. In the budding yeast *S. cerevisae*, mutations in α-helix5 (α5), the most outer structure in the vWA domain had opposite effects in each subunit [33]. Whereas Yku80 a5 mutations disrupt Ku's telomeric silencing function without perturbing DNA repair abili‐ ties, mutations in Yku70-α5 impair NHEJ while preserving Ku's telomeric functions [33]. This suggests that Ku is spatially organized in two faces with distinct roles in NHEJ and telomeric functions, a hypothesis termed the two-face model. In essence, this model states that the in‐ ward face, composed mostly of the Ku80 vWA and Ku70 C-terminal domain (CTD), is oriented towards internal tracks of DNA and has telomeric roles. In contrast, the outward face of Ku is the closest to the DNA end and its main role is to engage the nearby DNA end in NHEJ. Consis‐ tent with this, mutations in both the Ku70 vWA domain and the Ku80 CTD, the two major com‐ ponents of the outer face, impair NHEJ [33, 34]. The most recent discovery that mutations in human Ku70-α5 also diminish NHEJ suggests that the two-face model may also be conserved

**Figure 1.** Molecular modeling of Ku and DNA-PKcs dimerization. A Possible Ku tetramer formation through outward face interactions. Each Ku dimer is represented in red and blue, whereas DNA is depicted as black line (Adapted from

Ku plays multiple roles during NHEJ. Initially, Ku not only binds and detects DSB but also protects the ends from nucleolytic degradation and tilts the choice of DSB repair towards NHEJ and away from HR [7, 36]. Perhaps the most extensive role for Ku is to recruit NHEJ factors to DSB sites. The Ku80 CTD recruits DNA-PKcs to DSBs and binds a long list of NHEJ factors including XRCC4, LigIV, XLF, Polμ, and Polλ [9, 10]. Moreover, Ku's require‐ ment for the recruitment of DNA-PKcs, XLF and XRCC4 has been demonstrated *in vivo* [37, 38]. In addition, Ku possesses some catalytic activities and can function as a deubiquitylat‐ ing enzyme or as a 5'-dRP/AP lyase which suggests that Ku may aid in processing DNA

The ability of the Ku heterodimer to self-associate and create higher order multimers was already apparent in early studies. Seminal work from Cary *et al* using gel filtration

1JEY). B Possible Head-to-Head mediated dimerization of two DNA-PKcs molecules (Adapted from 3KGV).

in mammalian cells [35].

44 New Research Directions in DNA Repair

ends before the final ligation [39].

Recent work with the Ku80 CTD suggests that Ku heterotetramerization may occur through the interaction of two outward faces [42]. This long and flexible domain interacts with the Ku core domain and upon binding to DNA it undergoes a conformational change that relocates it to the outward face [43]. Researchers have now shown that the Ku80 CTD can dimerize and thus, can putatively mediate Ku-Ku interactions across a DSB enabling the tethering of two DNA ends [42]. In fact, Ku proteins bearing Ku80 CTD truncations have reduced ability to form heterotetramers as shown by reduced supershift EMSA signals compared with wild type [42]. Ku80 CTD truncations impair NHEJ, but this result cannot be fully attributed to reduced heterotetramer formation as the Ku80 CTD is also involved in recruiting DNA-PKcs to DSBs. Intriguingly, a mutation in the out‐ ward face located Ku70-α5 also impairs NHEJ, although the effect of this mutation on Ku heterotetramerization remains to be investigated [35].

While the presence of Ku multimers of higher order than heterotetramers has been detected in EM and AFM using recombinant Ku proteins, its functional significance remains to be elucidated and evidence for its existence in living cells is lacking [40, 44-46]. A single hetero‐ tetramer is sufficient to create a synaptic complex across DSB and it is difficult to envision how higher order Ku complexes may aid in this process. Since Ku can slide towards internal tracks of DNA, one possibility is that multiple Ku molecules threaded into a single DNA end could form filaments held by interactions between inward and outward faces. Howev‐ er, fluorescence anisotropy studies do not support this model [47].

#### **2.2. The DNA-PKcs**

With over 400 kDa, the DNA-PKcs is one of the largest kinases in mammalian cells. Along with ATM and ATR, it belongs to the phosphatidylinositol-3-OH kinase (PI3K)-related kin‐ ase (PIKK) family that preferentially phosphorylates serines and threonines followed by a glutamine [48]. Although DNA-PKcs can bind directly to DNA, during NHEJ it is recruited to DSBs by the Ku80 CTD flexible domain, which increases the affinity of DNA-PKcs to DNA by 100 fold [49-51]. Therefore, assembly of the DNA-PK complex only occurs at DSBs where it induces Ku displacement one helix turn away from the end and positions DNA-PKcs at the very tip of the break [44]. The DNA-PK complex performs two major roles dur‐ ing NHEJ: it forms a synaptic complex across DSBs and serves as a scaffold for the recruitment of all other NHEJ factors [52]. DNA-PKcs is responsible for the recruitment of Artemis to DSBs, which provides the NHEJ machinery with a variety of end processing ac‐ tivities including 5' endonuclease, 3' endonuclease and hairpin opening [53]. In addition, DNA-PKcs directly binds XRCC4 and stimulates the ligase activity of XRCC4/Ligase IV complex [52, 54, 55]. Both dimerization and XRCC4 interaction induces DNA-PKcs kinase activity which is known to phosphorylate several NHEJ factors such as Ku, Artemis, XRCC4, LigIV and XLF, although the role of these phosphorylation events in NHEJ remains to be elucidated [56, 57]. More importantly, DNA-PKcs possesses over 15 autophosphoryla‐ tion sites that become phosphorylated after formation of the synaptic complex and that are involved in releasing DNA-PKcs from Ku and DSB [11]. Consistent with this, non-autophos‐ phorylatable mutations or kinase-dead DNA-PKcs mutants still localize to DSBs but are re‐ tained longer at sites of DNA damage [43].

whose size was consistent with that of a DNA-PKcs dimer. While DNA-PKcs was sufficient to create a synaptic complex, these were significantly more abundant when Ku was present [64]. Importantly, end-to-end bridges still occurred in the presence of kinase inhibitors, indi‐ cating that autophosphorylation events were not required for synaptic complex formation [64]. Further single particle EM, cryo-EM and SAXS studies have visualized dimers of DNA-PKcs molecules that form in a concentration dependent manner and in a process that is highly enhanced in the presence of Ku [43, 60, 63]. These techniques detected two types of DNA-PKcs dimers with different orientations depending on the DNA molecules used. In the presence of 40bp Y-shaped DNA fragments, DNA-PKcs dimers formed in a palm-topalm fashion whereas, in the presence of a 40bp hairpin DNA, DNA-PKcs dimers had the opposite orientation and formed through head-to-head interactions [43, 60, 63]. These two kinds of DNA-PKcs dimers were observed in the presence or absence of Ku. The reason for the existence of two DNA-PKcs dimer subspecies is not clear but authors speculate that the Y-shape DNA induced orientation may be caused by the binding of two DNA-PKcs mole‐ cules to the same DNA fragment, suggesting that dimers generated by head-to-head interac‐ tions may be the only ones capable of bridging two DNA fragments [43]. Corroborating this hypothesis, DNA-PKcs dimers with a head-to-head orientation were the only type of dimers observed in the absence of DNA [43]. Overall, the current model proposes that Ku recruits DNA-PKcs to sites of DNA damage where it dimerizes through head-to-head interactions, creating synaptic complexes across DSBs [10]. DNA-PKcs dimerization at breaks stimulates auto-transphosphorylation, which in turn induces conformational changes that disassemble

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the DNA-PK complex and promotes its timely release from DNA ends [11].

the Rad50 ABC-ATPase domain in the MRN complex [74, 77, 78].

The association of the conserved Mre11 and Rad50 subunits along with Nbs1 (a protein whose functional homolog in *S. cerevisiae* is Xrs2), makes up the mammalian MRN complex, also known as the MRX complex in yeast [65-68]. This complex plays vital roles in multiple DNA repair pathways, including HR and NHEJ, and is responsible for the co-activation of the DDR in the presence of DNA injury [68-70]. Analysis of the domain composition and en‐ zymatic activities of each subunit suggest possible mechanistic roles for the MRN complex during DNA repair. Rad50 is a member of the SMC protein family whose members play roles in chromosome condensation and cohesion [71, 72]. A key feature of these proteins is the presence of long coiled-coil domains that can fold on themselves via an antiparallel man‐ ner, bringing the N- and C-terminus in close proximity [73]. In Rad50, folding of the coiledcoil domain permits the reconstitution of a bipartite ATP-binding cassette (ABC)-type ATPase globular domain made of N-terminal Walker A and C-terminal Walker B nucleotide binding motifs [74, 75]. In other complexes, binding and hydrolysis of ATP by similar ABC-ATPase domains, mediate large conformational changes that can be transmitted to other members of the complex [71, 76]. Crystallography and SAXS data support a similar role for

As is the case for Rad50, Mre11 can bind DNA and possesses a specific region capable of capping DNA ends [20, 79]. Mre11 contains a phosphoesterase domain in its N-terminus

**2.3. The MRN complex**

Given its large size, the complete structure of DNA-PKcs has been elusive at the atomic lev‐ el. Single particle cryo-EM, small-angle X-ray scattering (SAXS) experiments and more re‐ cently, the crystal structure at 6.6 angstroms resolution, have shown that multiple Nterminus HEAT repeats, encompassing ~66 helices, form a ring-like structure with a gap at one end (Figure 1B) [58-60]. This structure is usually referred to as the `palm' region and it also encloses a globular DNA binding domain, although a clear picture of how DNA-PKcs interacts with DNA is missing. The top of the palm houses the so-called `crown' or `head' that includes the globular C-terminus kinase domain, along with FAT and FATC domains. Also missing is the exact location of the Ku80 CTD interaction and the details of how the whole Ku heterodimer is accommodated by the DNA-PKcs structure to create the DNA-PK complex [61]. Several pieces of evidence indicate that DNA-PKcs undergoes conformational changes as a result of autophosphorylation [43, 62, 63]. SAXS analysis has detected a phos‐ phorylation-driven conformational change that repositions the head with respect to the palm [43], whereas the crystal structure suggests that auto-transphosphorylation widens the gap at the end of the palm and facilitates disassembly of DNA-PKcs from Ku and DSBs [58].

During NHEJ, DNA-PKcs multimerization is limited to the dimerization of the two DNA-PKcs located at opposite ends of a DSB to create a synaptic complex. Early studies demon‐ strated the ability of DNA-PK to mediate co-immunoprecipitation of biotinylated DNA fragments with radiolabeled probes [64]. In agreement, initial EM experiments visualized the ability of DNA-PKcs to circularize DNA fragments and detected synaptic complexes whose size was consistent with that of a DNA-PKcs dimer. While DNA-PKcs was sufficient to create a synaptic complex, these were significantly more abundant when Ku was present [64]. Importantly, end-to-end bridges still occurred in the presence of kinase inhibitors, indi‐ cating that autophosphorylation events were not required for synaptic complex formation [64]. Further single particle EM, cryo-EM and SAXS studies have visualized dimers of DNA-PKcs molecules that form in a concentration dependent manner and in a process that is highly enhanced in the presence of Ku [43, 60, 63]. These techniques detected two types of DNA-PKcs dimers with different orientations depending on the DNA molecules used. In the presence of 40bp Y-shaped DNA fragments, DNA-PKcs dimers formed in a palm-topalm fashion whereas, in the presence of a 40bp hairpin DNA, DNA-PKcs dimers had the opposite orientation and formed through head-to-head interactions [43, 60, 63]. These two kinds of DNA-PKcs dimers were observed in the presence or absence of Ku. The reason for the existence of two DNA-PKcs dimer subspecies is not clear but authors speculate that the Y-shape DNA induced orientation may be caused by the binding of two DNA-PKcs mole‐ cules to the same DNA fragment, suggesting that dimers generated by head-to-head interac‐ tions may be the only ones capable of bridging two DNA fragments [43]. Corroborating this hypothesis, DNA-PKcs dimers with a head-to-head orientation were the only type of dimers observed in the absence of DNA [43]. Overall, the current model proposes that Ku recruits DNA-PKcs to sites of DNA damage where it dimerizes through head-to-head interactions, creating synaptic complexes across DSBs [10]. DNA-PKcs dimerization at breaks stimulates auto-transphosphorylation, which in turn induces conformational changes that disassemble the DNA-PK complex and promotes its timely release from DNA ends [11].

#### **2.3. The MRN complex**

**2.2. The DNA-PKcs**

46 New Research Directions in DNA Repair

tained longer at sites of DNA damage [43].

With over 400 kDa, the DNA-PKcs is one of the largest kinases in mammalian cells. Along with ATM and ATR, it belongs to the phosphatidylinositol-3-OH kinase (PI3K)-related kin‐ ase (PIKK) family that preferentially phosphorylates serines and threonines followed by a glutamine [48]. Although DNA-PKcs can bind directly to DNA, during NHEJ it is recruited to DSBs by the Ku80 CTD flexible domain, which increases the affinity of DNA-PKcs to DNA by 100 fold [49-51]. Therefore, assembly of the DNA-PK complex only occurs at DSBs where it induces Ku displacement one helix turn away from the end and positions DNA-PKcs at the very tip of the break [44]. The DNA-PK complex performs two major roles dur‐ ing NHEJ: it forms a synaptic complex across DSBs and serves as a scaffold for the recruitment of all other NHEJ factors [52]. DNA-PKcs is responsible for the recruitment of Artemis to DSBs, which provides the NHEJ machinery with a variety of end processing ac‐ tivities including 5' endonuclease, 3' endonuclease and hairpin opening [53]. In addition, DNA-PKcs directly binds XRCC4 and stimulates the ligase activity of XRCC4/Ligase IV complex [52, 54, 55]. Both dimerization and XRCC4 interaction induces DNA-PKcs kinase activity which is known to phosphorylate several NHEJ factors such as Ku, Artemis, XRCC4, LigIV and XLF, although the role of these phosphorylation events in NHEJ remains to be elucidated [56, 57]. More importantly, DNA-PKcs possesses over 15 autophosphoryla‐ tion sites that become phosphorylated after formation of the synaptic complex and that are involved in releasing DNA-PKcs from Ku and DSB [11]. Consistent with this, non-autophos‐ phorylatable mutations or kinase-dead DNA-PKcs mutants still localize to DSBs but are re‐

Given its large size, the complete structure of DNA-PKcs has been elusive at the atomic lev‐ el. Single particle cryo-EM, small-angle X-ray scattering (SAXS) experiments and more re‐ cently, the crystal structure at 6.6 angstroms resolution, have shown that multiple Nterminus HEAT repeats, encompassing ~66 helices, form a ring-like structure with a gap at one end (Figure 1B) [58-60]. This structure is usually referred to as the `palm' region and it also encloses a globular DNA binding domain, although a clear picture of how DNA-PKcs interacts with DNA is missing. The top of the palm houses the so-called `crown' or `head' that includes the globular C-terminus kinase domain, along with FAT and FATC domains. Also missing is the exact location of the Ku80 CTD interaction and the details of how the whole Ku heterodimer is accommodated by the DNA-PKcs structure to create the DNA-PK complex [61]. Several pieces of evidence indicate that DNA-PKcs undergoes conformational changes as a result of autophosphorylation [43, 62, 63]. SAXS analysis has detected a phos‐ phorylation-driven conformational change that repositions the head with respect to the palm [43], whereas the crystal structure suggests that auto-transphosphorylation widens the gap at the end of the palm and facilitates disassembly of DNA-PKcs from Ku and DSBs [58].

During NHEJ, DNA-PKcs multimerization is limited to the dimerization of the two DNA-PKcs located at opposite ends of a DSB to create a synaptic complex. Early studies demon‐ strated the ability of DNA-PK to mediate co-immunoprecipitation of biotinylated DNA fragments with radiolabeled probes [64]. In agreement, initial EM experiments visualized the ability of DNA-PKcs to circularize DNA fragments and detected synaptic complexes

The association of the conserved Mre11 and Rad50 subunits along with Nbs1 (a protein whose functional homolog in *S. cerevisiae* is Xrs2), makes up the mammalian MRN complex, also known as the MRX complex in yeast [65-68]. This complex plays vital roles in multiple DNA repair pathways, including HR and NHEJ, and is responsible for the co-activation of the DDR in the presence of DNA injury [68-70]. Analysis of the domain composition and en‐ zymatic activities of each subunit suggest possible mechanistic roles for the MRN complex during DNA repair. Rad50 is a member of the SMC protein family whose members play roles in chromosome condensation and cohesion [71, 72]. A key feature of these proteins is the presence of long coiled-coil domains that can fold on themselves via an antiparallel man‐ ner, bringing the N- and C-terminus in close proximity [73]. In Rad50, folding of the coiledcoil domain permits the reconstitution of a bipartite ATP-binding cassette (ABC)-type ATPase globular domain made of N-terminal Walker A and C-terminal Walker B nucleotide binding motifs [74, 75]. In other complexes, binding and hydrolysis of ATP by similar ABC-ATPase domains, mediate large conformational changes that can be transmitted to other members of the complex [71, 76]. Crystallography and SAXS data support a similar role for the Rad50 ABC-ATPase domain in the MRN complex [74, 77, 78].

As is the case for Rad50, Mre11 can bind DNA and possesses a specific region capable of capping DNA ends [20, 79]. Mre11 contains a phosphoesterase domain in its N-terminus that endows the MRN complex with ssDNA endonuclease and 3' to 5' dsDNA exonuclease enzymatic activities. Mre11 is the only subunit that interacts with all components of the complex as it also binds Nbs1 whose main function is to recruit DNA repair factors to DSB. For instance, the Nbs1 N-terminus is responsible for bringing ATM and ATR to DNA dam‐ age locations and hence, acts as a DSB sensor during the initiation of the DDR [80, 81]. Simi‐ larly, the Nbs1 N-terminus contains BRCT and FHA domains that bind and recruit CtIP (Sae2 in *S. cerevisiae*), an important nuclease during DNA repair, to DSBs [28, 82] as well as MDC1 [83, 84] and ATR[85], and the WRN helicase [86].

ABC-ATPase domains bound to ATP can dimerize in head-to-tail fashion between N- and Cterminal domains, trapping two ATP molecules in the process [74]. Moreover, Mre11 dimers bind ABC-ATPase dimers forming the so-called M2R2 head region. In this disposition, a heter‐ ohexamer has a circular shape formed by two coiled-coil regions as semicircles are united at one end by the Zn-hook domain and by interactions within the M2R2 head at the other end (Figure 2C). Mre11 also contains a helix-loop-helix domain in its C-terminus that extends away from the N-terminal globular region and binds the base of the Rad50 coiled-coil region in the vicinity of the ABC-ATPase domain, further reinforcing the interaction between Mre11 and Rad50 dimers (Figure 2A). In contrast, Nbs1 does not form dimers nor does it contribute to het‐ erohexamer-ization. Recent studies indicate that the MRN heterohexamer presents two dis‐ tinct configurations [77, 78, 105, 106]. In the absence of ATP binding, the MRN complex adopts an `open' conformation where Mre11 dimers localize in between the two Rad50 ABC-ATPase domains, preventing their dimerization (Figure 2A). In this `open' configuration, Rad50 can only dimerize through the Zn-hook domain. Upon ATP binding, a conformational change al‐ lows displacement of the Mre11 dimers and dimerization of two Rad50 ABC-ATPase domains (Figure 2B). This `close' configuration is substantially more rigid and may promote DNA bind‐ ing by Rad50 [77, 78, 105, 106]. Subsequent ATP hydrolysis disrupts ABC-ATPase mediated

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**Figure 2.** The MRN complex undergoes ATP-Driven conformational changes. A Molecular structure of the open state where Mre11 dimer is depicted in red and Rad50 in blue (Adapted from 3QG5). B Closed conformation of the MRN com‐ plex. (Adapted from 3QF7 and 3THO). C Tethering of DSB ends by different multimerization states of the MRN complex.

Rad50 dimerization and stimulates Mre11 nuclease activity [107].

The MRN complex is at the center of the decision process that governs whether a DSB is re‐ paired by NHEJ or HR [87, 88]. The current model indicates that, in the presence of DNA injury, recruitment of CtIP to DSB provides the MRN complex with the 5' to 3' exonuclease activity necessary to chew away part of the DNA ends and create an initial ~50-100nt ssDNA 3' overhang [18, 82, 89-92]. This overhang is a poor binding substrate for Ku but an ideal substrate for the HR initiation factor RPA and thus, it favors DSB repair by HR over NHEJ [32, 93]. Since CtIP activity and recruitment to DSBs is dependent upon CDK phosphoryla‐ tion of both CtIP and Nbs1 during S and G2 phases [93-95], the lack of these post-transla‐ tional modifications during G1 prevents overhang formation at DSB ends in this phase and tilts the choice of DNA repair pathway towards NHEJ. Therefore, and according to this model, NHEJ remains active throughout the cell cycle but is overpowered by HR during S and G2 due to CDK-dependent 3' overhang formation by the MRN/CtIP complex. This sys‐ tem ensures that HR is only active when an identical copy of the damaged DNA is available.

Despite favoring HR, the MRN complex also plays essential roles during NHEJ when HR is inhibited. Yeast defective for any MRX subunit are inviable in the presence of a single HO– induced break that can only be repaired by NHEJ [96]. Similarly, mammalian cells depleted of Mre11 display reduced end-joining activities [97], impaired NHEJ [69, 98] and, in the case of B-lymphocytes, markedly reduced CSR [99]. Intriguingly, yeast nuclease-dead Mre11 mu‐ tants can carry end-joining activity near wild type levels [100] and B lymphocytes only show mild defects in CSR in the presence of a Mre11 mutation lacking nuclease activity [99]. These results indicate that the MRN complex mostly plays a signaling and structural role during NHEJ, and that its end processing capabilities are dispensable or can be compensated by other nucleases. Scanning force microscopy (SFM) and AFM have demonstrated the ability of the MRN complex to create long range bridges across DNA molecules [101, 102] implicat‐ ing DNA end tethering as the most likely structural role for MRN during NHEJ.

Formation of higher order multimers is essential to our understanding of the mechanistic roles of MRN during NHEJ. The MRX complex assembles as a heterohexamer where two Mre11 molecules bind simultaneously to two Rad50 and two Nbs1 subunits (Figure2A) [70, 103]. A combination of SAXS with X-ray crystallography has shown that, through interactions of the N-terminus globular domain, Mre11 form very stable dimers capable of forming bridges be‐ tween two DNA molecules [20, 104]. Rad50 can also form dimers through two different dimeri‐ zation regions located at opposite poles of the molecule [74]. At the end of the coiled-coil region, Rad50 contains a CXXC motif that can dimerize through the formation of two zinc-hook (Zn-hook) domains that lock in a single Zn(2+) ion [103]. At the opposite pole, two globular ABC-ATPase domains bound to ATP can dimerize in head-to-tail fashion between N- and Cterminal domains, trapping two ATP molecules in the process [74]. Moreover, Mre11 dimers bind ABC-ATPase dimers forming the so-called M2R2 head region. In this disposition, a heter‐ ohexamer has a circular shape formed by two coiled-coil regions as semicircles are united at one end by the Zn-hook domain and by interactions within the M2R2 head at the other end (Figure 2C). Mre11 also contains a helix-loop-helix domain in its C-terminus that extends away from the N-terminal globular region and binds the base of the Rad50 coiled-coil region in the vicinity of the ABC-ATPase domain, further reinforcing the interaction between Mre11 and Rad50 dimers (Figure 2A). In contrast, Nbs1 does not form dimers nor does it contribute to het‐ erohexamer-ization. Recent studies indicate that the MRN heterohexamer presents two dis‐ tinct configurations [77, 78, 105, 106]. In the absence of ATP binding, the MRN complex adopts an `open' conformation where Mre11 dimers localize in between the two Rad50 ABC-ATPase domains, preventing their dimerization (Figure 2A). In this `open' configuration, Rad50 can only dimerize through the Zn-hook domain. Upon ATP binding, a conformational change al‐ lows displacement of the Mre11 dimers and dimerization of two Rad50 ABC-ATPase domains (Figure 2B). This `close' configuration is substantially more rigid and may promote DNA bind‐ ing by Rad50 [77, 78, 105, 106]. Subsequent ATP hydrolysis disrupts ABC-ATPase mediated Rad50 dimerization and stimulates Mre11 nuclease activity [107].

that endows the MRN complex with ssDNA endonuclease and 3' to 5' dsDNA exonuclease enzymatic activities. Mre11 is the only subunit that interacts with all components of the complex as it also binds Nbs1 whose main function is to recruit DNA repair factors to DSB. For instance, the Nbs1 N-terminus is responsible for bringing ATM and ATR to DNA dam‐ age locations and hence, acts as a DSB sensor during the initiation of the DDR [80, 81]. Simi‐ larly, the Nbs1 N-terminus contains BRCT and FHA domains that bind and recruit CtIP (Sae2 in *S. cerevisiae*), an important nuclease during DNA repair, to DSBs [28, 82] as well as

The MRN complex is at the center of the decision process that governs whether a DSB is re‐ paired by NHEJ or HR [87, 88]. The current model indicates that, in the presence of DNA injury, recruitment of CtIP to DSB provides the MRN complex with the 5' to 3' exonuclease activity necessary to chew away part of the DNA ends and create an initial ~50-100nt ssDNA 3' overhang [18, 82, 89-92]. This overhang is a poor binding substrate for Ku but an ideal substrate for the HR initiation factor RPA and thus, it favors DSB repair by HR over NHEJ [32, 93]. Since CtIP activity and recruitment to DSBs is dependent upon CDK phosphoryla‐ tion of both CtIP and Nbs1 during S and G2 phases [93-95], the lack of these post-transla‐ tional modifications during G1 prevents overhang formation at DSB ends in this phase and tilts the choice of DNA repair pathway towards NHEJ. Therefore, and according to this model, NHEJ remains active throughout the cell cycle but is overpowered by HR during S and G2 due to CDK-dependent 3' overhang formation by the MRN/CtIP complex. This sys‐ tem ensures that HR is only active when an identical copy of the damaged DNA is available.

Despite favoring HR, the MRN complex also plays essential roles during NHEJ when HR is inhibited. Yeast defective for any MRX subunit are inviable in the presence of a single HO– induced break that can only be repaired by NHEJ [96]. Similarly, mammalian cells depleted of Mre11 display reduced end-joining activities [97], impaired NHEJ [69, 98] and, in the case of B-lymphocytes, markedly reduced CSR [99]. Intriguingly, yeast nuclease-dead Mre11 mu‐ tants can carry end-joining activity near wild type levels [100] and B lymphocytes only show mild defects in CSR in the presence of a Mre11 mutation lacking nuclease activity [99]. These results indicate that the MRN complex mostly plays a signaling and structural role during NHEJ, and that its end processing capabilities are dispensable or can be compensated by other nucleases. Scanning force microscopy (SFM) and AFM have demonstrated the ability of the MRN complex to create long range bridges across DNA molecules [101, 102] implicat‐

ing DNA end tethering as the most likely structural role for MRN during NHEJ.

Formation of higher order multimers is essential to our understanding of the mechanistic roles of MRN during NHEJ. The MRX complex assembles as a heterohexamer where two Mre11 molecules bind simultaneously to two Rad50 and two Nbs1 subunits (Figure2A) [70, 103]. A combination of SAXS with X-ray crystallography has shown that, through interactions of the N-terminus globular domain, Mre11 form very stable dimers capable of forming bridges be‐ tween two DNA molecules [20, 104]. Rad50 can also form dimers through two different dimeri‐ zation regions located at opposite poles of the molecule [74]. At the end of the coiled-coil region, Rad50 contains a CXXC motif that can dimerize through the formation of two zinc-hook (Zn-hook) domains that lock in a single Zn(2+) ion [103]. At the opposite pole, two globular

MDC1 [83, 84] and ATR[85], and the WRN helicase [86].

48 New Research Directions in DNA Repair

**Figure 2.** The MRN complex undergoes ATP-Driven conformational changes. A Molecular structure of the open state where Mre11 dimer is depicted in red and Rad50 in blue (Adapted from 3QG5). B Closed conformation of the MRN com‐ plex. (Adapted from 3QF7 and 3THO). C Tethering of DSB ends by different multimerization states of the MRN complex.

In the MRN complex, multimerization regulates formation of DNA bridges, with increasing order of multimers providing longer range tethering capabilities. Short range bridges can be achieved by single heterohexamers, where each Mre11 subunit is bound to a different DNA end (Figure 2C) [103]. In contrast with Ku heterotetramers and DNA-PKcs dimers, where multimerization occurs after the assembly of subunits located at each end of a DSB, short range bridges mediated by single heterohexamers must be achieved without disruption of the MRN complex as heterohexamers are predicted to be pre-assembled before binding DNA ends. Therefore, single heterohexamers may only bridge ends that are in close proxim‐ ity (~100 angstroms) as to allow simultaneous binding of each end to the MRN complex without disrupting the M2R2 head [20, 103]. The role that DNA binding activity of the Rad50 dimers may play in synapse formation by heterohexamers remains to be elucidated but SFM has shown that Rad50 is also able to bind and tether DNA molecules in the absence of Mre11 [108].

to DSBs, Ku is capable of bringing both XRCC4-LigIV and XLF to sites of DNA damage in

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Both XLF and XRCC4 are obligated homodimers that have very similar structures where the presence of an N-terminal globular domain, or `head', is followed by a coiled-coil re‐ gion that mediates homodimerization [112, 117, 118]. In this disposition, the two head do‐ mains on each homodimer face each other with opposite orientation. Given its heterogeneity and flexible nature, the structure of the C-terminal domain for both XRCC4 and XLF remain to be resolved. Nevertheless, XRCC4 SAXS analysis is consistent with the C-terminal domain folding backwards and interacting with the head domain [119]. Like‐ wise, XLF's structure resolution revealed a fold back in the coiled-coil region that shortens the helix and creates a kink that likely positions the C-terminal domain in close proximity to the head domain [118]. The presence of discrete regions in both XRCC4 and XLF corre‐ lates with the spatial organization of their interactions with NHEJ factors. While their Nterminal domains mediate XRCC4-XLF interaction, the XRCC4-LigIV interaction maps to the XRCC4 coiled-coil region and the C-terminal domain of XLF interacts with Ku [22, 38, 116, 119-122]. In addition, both XRCC4 and XLF can bind DNA. EMSA analysis has iden‐ tified the upper part of the XRCC4 coiled-coil domain and the XLF C-terminus as their re‐

Besides forming homodimers, XRCC4 also exists as tetramers and higher order multimers in solution [119, 123]. Although coiled-coil mediation of tetramerization was initially proposed, SAXS analyses have demonstrated that tetramerization is mostly mediated by the interac‐ tion of two N-terminal domains in a way that leaves the stalks of each dimer pointing in op‐ posite directions [119, 124, 125]. These head-to-head interactions can also drive formation of XRCC4 filaments, as detected by SAXS [119]. Interestingly, while full length XRCC4 mostly exists as tetramers and filaments in solution, truncation of the C-terminus makes homodim‐ ers the predominant XRCC4 form, suggesting that the C-terminus also contributes to tetra‐ merization and filament formation. The presence of a LigIV BRCT region responsible for binding the XRCC4 coiled-coil region also made XRCC4 filaments unstable, indicating that the XRCC4-LigIV complex does not exist as part of a filament and suggesting that under physiological conditions, XRCC4 remains in multiple configurations [119]. Given the fact that mammalian cells contain six times more XRCC4 molecules than LigIV, XRCC4 fila‐ ments may constitute a protein reservoir that can readily be mobilized in the presence of DNA damage. The ability of XLF to form higher order assemblies in solution suggests that

In addition, XRCC4 can also form filaments through its interaction with XLF. SAXS analysis, EM, SFM and crystallography have all detected long filaments of alternating XRCC4 and XLF molecules bound through head-to-head interactions (Figure 3) [21-23, 119, 126]. In this conformation, XRCC4 and XLF stalks are both oriented towards the same direction, albeit with a 30-degree offset from each other. Furthermore, two filaments can intertwine through XRCC4-XRCC4 interactions to form a left-handed helix with a ~220 angstrom diameter where head domains reside in the interior while coiled-coil regions stick out to the exterior. Higher order multimers where several filaments constitute a thicker fiber have also been

the absence of DNA-PKcs [37, 38, 54, 115, 116].

spective DNA binding regions [22, 23, 118].

XLF filaments may also exist in cells [118].

Longer range bridges can be achieved through the formation of higher order multimers where two heterohexamers, each bound to a different end, combine to form a structure ca‐ pable of tethering DNA molecules as far as 1200 angstroms apart [20, 103]. This configura‐ tion has been confirmed by AFM and EM and consists of two M2R2 heads separated by two long coiled-coil regions held together by two Zn-hooks (Figure 2C) [102, 103]. Consistent with this, AFM has demonstrated that upon DNA binding, heterohexamers extend their two coiled-coil regions in a parallel fashion that disrupts Zn-hook mediated dimerization within the heterohexamer and favors formation of Zn-hook interactions with other heterohexamers [102]. These assemblies have been proposed to mediate long distance tethering of homolo‐ gous sequences during HR and to hold DNA ends in close proximity during NHEJ, prevent‐ ing them from going astray and facilitating DSB repair by the rest of the NHEJ machinery [70, 109]. Consistent with this, in yeast, loss of the Rad50 CxxC Zn-hook motif abolishes DNA repair while replacing it with a *FKBP* homodimerization domain has no major effect [110]. Similarly, truncations of the coiled-coil region in Rad50 impairs DNA repair [109]. Since heterohexamers could potentially form Zn-hook interactions with more than one het‐ erohexamer at a time, further higher order arrangements have been proposed to form multi‐ ple interactions to secure bridges across DNA molecules, a possibility whose biological significance remains to be investigated.

#### **2.4. XLF and XRCC4**

Although neither of them have any intrinsic enzymatic activity, XLF (also known as Cernnu‐ nos) and XRCC4 are involved in the final ligation step catalyzed by LigIV. XRCC4 binds tightly to LigIV and drives its localization to sites of DNA damage, whereas XLF stimulates the ability of the XRCC4-LigIV complex to ligate DSBs 20-200 fold, especially in the presence of non-cohesive ends [111-114]. XRCC4 is also capable of binding several other NHEJ factors including XLF, DNA-PKcs and the Ku heterodimer, while known interactions for XLF in‐ clude XRCC4 and the Ku heterodimer. In addition, both XLF and XRCC4 can bind DNA, although their localization to sites of DNA damage is dependent on their interaction with members of the DNA-PK complex. While DNA-PKcs can recruit the XRCC4-LigIV complex to DSBs, Ku is capable of bringing both XRCC4-LigIV and XLF to sites of DNA damage in the absence of DNA-PKcs [37, 38, 54, 115, 116].

In the MRN complex, multimerization regulates formation of DNA bridges, with increasing order of multimers providing longer range tethering capabilities. Short range bridges can be achieved by single heterohexamers, where each Mre11 subunit is bound to a different DNA end (Figure 2C) [103]. In contrast with Ku heterotetramers and DNA-PKcs dimers, where multimerization occurs after the assembly of subunits located at each end of a DSB, short range bridges mediated by single heterohexamers must be achieved without disruption of the MRN complex as heterohexamers are predicted to be pre-assembled before binding DNA ends. Therefore, single heterohexamers may only bridge ends that are in close proxim‐ ity (~100 angstroms) as to allow simultaneous binding of each end to the MRN complex without disrupting the M2R2 head [20, 103]. The role that DNA binding activity of the Rad50 dimers may play in synapse formation by heterohexamers remains to be elucidated but SFM has shown that Rad50 is also able to bind and tether DNA molecules in the absence