Repair of O6-G-Alkyl-O6-G Interstrand Cross-Links by Human O6-Alkylguanine-DNA Alkyltransferase

O (6)-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our u...

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Bibliographic Details
Published in:Biochemistry (Easton) 2008-10, Vol.47 (41), p.10892-10903
Main Authors: FANG, Qingming, NORONHA, Anne M., MURPHY, Sebastian P., WILDS, Christopher J., TUBBS, Julie L., TAINER, John A., CHOWDHURY, Goutam, GUENGERICH, F. Peter, PEGG, Anthony E.
Format: Article
Language:English
Subjects:
Blotting, Western
Chromatography, High Pressure Liquid
DNA Damage
DNA Modification Methylases - chemistry
DNA Modification Methylases - metabolism
DNA Repair
DNA Repair Enzymes - chemistry
DNA Repair Enzymes - metabolism
Electrophoresis, Polyacrylamide Gel
Humans
Models, Molecular
Tumor Suppressor Proteins - chemistry
Tumor Suppressor Proteins - metabolism
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Summary:O (6)-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine- O (6) in each strand. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi8008664