Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.
To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.
Jaiswal
M
M
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Lipinski
L J
LJ
Bohr
V A
VA
Mazur
S J
SJ
D000080242:8-Hydroxy-2'-Deoxyguanosine; D002460:Cell Line; D002474:Cell-Free System; D003057:Cockayne Syndrome / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects; D003843:Deoxycytidine Monophosphate / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000528:radiation effects; D006801:Humans; D008027:Light; D008214:Lymphocytes / Q000166:cytology / Q000528:radiation effects; D008751:Methylene Blue; D011838:Radiation-Sensitizing Agents; D013347:Subcellular Fractions / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000378:metabolism
D003848:Deoxyguanine Nucleotides; D011838:Radiation-Sensitizing Agents; D003843:Deoxycytidine Monophosphate; C007257:2'-deoxyguanosine 5'-phosphate; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine; D008751:Methylene Blue