Efficient repair of O6-ethylguanine, but not O4-ethylthymine or O2-ethylthymine, is dependent upon O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair activities in human cells.
The formation and persistence of O6-ethylguanine, O4-ethylthymine, and O2-ethylthymine were quantitated in the genomic DNA of human lymphoblasts exposed to 1.0 mM N-ethyl-N-nitrosourea using immunoslot-blot. The three cell lines used included one which lacks O6-alkylguanine-DNA alkyltransferase, one deficient in nucleotide excision repair, and a third which is competent in both of these repair pathways. The activity of O6-alkylguanine-DNA alkyltransferase was further modulated with O6-benzylguanine, a specific inhibitor of this protein. Repair of the O-ethylated thymines was slow and not related to either DNA repair phenotype. O6-Ethylguanine was repaired with a half-life of about 8 h in cells which expressed both O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair functions. Cells expressing O6-alkylguanine-DNA alkyltransferase activity but lacking nucleotide excision repair showed only slow repair of O6-ethylguanine (half-life of O6-ethylguanine, 43 h), while cells lacking the alkyltransferase showed little or no repair of O6-ethylguanine regardless of nucleotide excision repair activity (half-lives of O6-ethylguanine, 53 to greater than 100 h). We conclude that O6-alkylguanine-DNA alkyltransferase and nucleotide excision repair cooperate in the repair of O6-ethylguanine in human cells.
Bronstein
S M
SM
Department of Pathology, Duke University, Durham, North Carolina 27710.
Skopek
T R
TR
Swenberg
J A
JA
D001402:B-Lymphocytes; D002460:Cell Line; D004260:DNA Repair; D005038:Ethylnitrosourea / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D008954:Models, Biological; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011740:Pyrimidine Dimers; D013379:Substrate Specificity; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism; D013997:Time Factors
D011740:Pyrimidine Dimers; C040384:4-ethylthymine; C052666:2-ethylthymine; C007673:6-ethylguanine; D006147:Guanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005038:Ethylnitrosourea; D013941:Thymine