Untersuchungen zur Schädigung von DNA

Köhler, Anne-Kathrin. Untersuchungen zur Schädigung von DNA. 2004, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: http://edoc.unibas.ch/diss/DissB_6721

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The aim of this work was to investigate DNA damage. One area of interest was the mechanism of electron transfer which is important for repair processes, while the second topic of investigation was the breakage of DNA strands caused by a new class of photocleaving reagents (acridicinium salts) in the presence and absence of oxygen. The mechanism of the aerobic and anaerobic cleavage was to be examined. The mechanism of electron transfer in DNA has been widely debated in recent years. It was possible to explain the contradictory results regarding the range and rate of the electron transfer by a new model: the hopping model. In this model it is assumed that a charge can travel great distances in DNA by hopping between guanine bases (figure A). This is only possible if the distance between the guanine bases is less than four AT base pairs. The total charge transport is considered to be a sequence of single, reversible transfer steps between guanine bases, and these steps are highly distance dependent since the charge is tunnelling between donor and acceptor. The bridge (AT base pairs) is not oxidized or reduced in this process. It is characterized as a super exchange mechanism (figure A). With the hopping model that has been proposed, it was not possible to explain the electron
transfer via ten or more AT base pairs with no guanine bases inbetween. Therefore a new model
was developed which takes into consideration the oxidation of adenine. In this A-hopping model
it is assumed that adenine contributes directly to the charge transport process (figure B). To prove the participation of adenine in the charge transport process, the electron transfer via
two, three and seven AT base pairs was investigated. Over a distance of seven AT base pairs
(A-hopping) adenine participation could be detected. Therefore it was concluded that over long
distances, adenine is oxidized and contributes to the charge tranport process. For short distances
(two of three AT base pairs) no such oxidation or participation could be detected due to the
tunnelling of the charge (super exchange mechanism) and a high distance dependence was
Therefore it was shown that the charge transport can occur via different mechanisms, depending
on distance and base sequence.
Acridicinium salts (figure C) are a new class of DNA intercalating dyes which are able to cleave
DNA strands upon photolytic excitation in the presence and absence of oxygen. The aim of this
part of the work was to investigate the mechanism of cleavage under aerobic and anaerobic
conditions. By comparing the rate of strand breakage of long and short DNA strands, it was found that the
length of the DNA strand does not affect the degree to which DNA is damaged by acridicinium
salts. Furthermore, it was observed that single strands are also cleaved upon irradiation in the
presence of acridicinium salts. Under anaerobic conditions the cleavage of single strands was
even more effective than that of double strands. Hence, intercalation is not required for DNA
strand cleavage.
When the DNA was treated with ammonia or piperidine after irradiation, additional strand
breakage was observed under aerobic conditions which was due to alkali labile modifications.
Under anaerobic conditions none of these modifications could be detected. Consequently, it was
proposed that two different mechanisms were involved.
Irradiation in the presence of suitable additives (NaN3, D2O, tert-butyl alcohol) led to the
assumption that singlet oxygen and hydroxyl radicals are the reactive species under aerobic and
anaerobic conditions respectively. For further confirmation, the influence of the nucleotides or the DNA sequence was investigated. It was found that in the presence of oxygen damage occured
exclusively at the guanine bases. The more guanines the DNA strand contained the more
effective was the damage. On the other hand the damage under anaerobic conditions occured at
all nucleotides without any preference. Since singlet oxygen is known for its G selective reactions
and hydroxyl radicals are known for their high reactivity and lack of selectivity, this result added
proof to the theory of these two species being responsible for the DNA damage.
Thus, it could be shown that acridicinium salts do not react with the DNA directly but generate
reactive oxygen species upon photolytic excitation. Under aerobic conditions singlet oxygen is
generated from oxygen dissolved in the aqueous solution. Under anaerobic conditions the
reaction of activated acridicinium salt results in hydroxyl radicals.
These two reactive oxygen species are responsible for the observed strand breaks and oxidative
damage (scheme A).
Advisors:Giese, Bernd
Committee Members:Bickle, Thomas A.
Faculties and Departments:05 Faculty of Science > Departement Chemie > Former Organization Units Chemistry > Bioorganische Chemie (Giese)
UniBasel Contributors:Giese, Bernd
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6721
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:110
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 14:47

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