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Synthesis of Modified Thymidine and Intrastrand Cross-Linked DNA Probes to Investigate Repair by O6-Alkylguanine DNA Alkyltransferases and Bypass by Human DNA Polymerase η

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Synthesis of Modified Thymidine and Intrastrand Cross-Linked DNA Probes to Investigate Repair by O6-Alkylguanine DNA Alkyltransferases and Bypass by Human DNA Polymerase η

O'Flaherty, Derek Kyle (2016) Synthesis of Modified Thymidine and Intrastrand Cross-Linked DNA Probes to Investigate Repair by O6-Alkylguanine DNA Alkyltransferases and Bypass by Human DNA Polymerase η. PhD thesis, Concordia University.

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Abstract

O6-Alkylguanine-DNA alkyltransferases (AGTs) are responsible for genomic maintenance by repairing O6-alkyl-2’-deoxyguanosine (O6-alkyl-dG) and O4-alkyl-thymidine (O4-alkyl-dT) adducts. AGT-mediated repair was investigated against DNA intrastrand cross-links (IaCL). A variety of cross-linked dimers linking the O6-atom of dG or O4-atom of dT were prepared synthetically to produce precursors for IaCL DNA that either lack or containing an intradimer phosphodiester group in the oligonucleotide backbone. Studies with AGTs demonstrated that: 1) O6-dG-alkylene-O6-dG flexible IaCL DNA (lacking the phosphodiester linkage) were efficiently repaired by hAGT. Repair of the model IaCL DNA occurred more efficiently in comparison to similar ICL DNA; 2) O6-dG-alkylene-O6-dG IaCL DNA containing the intradimer phosphodiester were moderately repaired by hAGT. Efficiency of the hAGT-mediated repair was contingent on the presence of the intradimer phosphate, which suggest conformational flexibility may be a requirement for repair by AGTs; 3) Flexible O4-dT-alkylene-O4-dT IaCL DNA evaded repair from all AGTs tested, whereas the flexible IaCL 5'-O4-dT-alkylene-O6-dG were efficiently repaired by hAGT. Interestingly, the 5'-O6-dG-alkylene-O4-dT was not proficiently repaired by hAGT supporting the importance of the 3'-phosphate group of the target dG nucleotide. 4) Flexible IaCL can be employed to generate DNA-protein cross-links (DPCs), with good conversions, as observed with repair of O6-dG-alkylene-O6-dG and 5'-O4-dT-alkylene-O6-dG by hAGT. The use of such cross-linking experiments may be useful for elucidating substrate discrimination across AGTs by X-ray crystallography.
Translesion synthesis (TLS) may be activated by the cell as a coping mechanism when DNA damage evades repair or remains otherwise irreparable by repair mechanisms. Human DNA polymerase η (hPol η) is a key TLS Pol involved in the bypass of certain UV-induced DNA damage, and lesions formed by platinum-containing chemotherapeutics. Bypass experiments were conducted to determine if conformational freedom of the lesion impacted hPol η processivity. Towards this end, bicyclic pyrimidines linking the C5-atom to the O4-atom, by an ethylene or a propylene bridge, were synthesized as conformationally locked mimics of the biologically relevant DNA damage O4-methyl thymidine (O4-MedT) and O4-ethyl thymidine (O4-EtdT), respectively. Bypass studies revealed that: 1) The conformationally locked pyrimidyl analogues described above were bypassed by hPol η with different profiles, relative to O4-MedT and O4-EtdT. All thymidinyl modifications evoked an error-prone behavior from hPol η, with insertion of dGMP being incorporated most-frequently in the growing strand. 2) IaCL bypass profiles of O6-dG-alkylene-O6-dG containing the intradimer phosphodiester group behaved significantly different relative to those IaCL lacking it. hPol η inserted the correct nucleotide (dCMP) across the 3'-dG residue for all IaCL studied, whereas an error-prone behavior was observed across the 5'-dG residue. While the lack of the intradimer phosphodiester caused frameshift adduct formation across the 5'-dG, hPol η inserted the incorrect dTMP across the 5'-dG of the canonical IaCL DNA. More studies are required to elucidate whether this dependence is shared for other types of lesions.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:O'Flaherty, Derek Kyle
Institution:Concordia University
Degree Name:Ph. D.
Program:Chemistry
Date:11 March 2016
Thesis Supervisor(s):Wilds, Christopher J.
ID Code:980938
Deposited By: DEREK O'FLAHERTY
Deposited On:16 Jun 2016 15:08
Last Modified:01 Apr 2018 00:00
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