Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells.
Human cell-free extracts were used to detect activities specifically incising O6-methylguanine (m6G) paired with C or T in DNA. A 45-bp double-stranded DNA containing one m6G across from a T (m6G:T) was the test substrate. Extracts from glioblastoma cell lines A172 and A1235 (lacking the m6G-specific repair protein m6G-DNA methyltransferase, MGMT) and colon carcinoma cell line HT29, containing MGMT, showed incision activities specific for the T strand of m6G:T [and G:T, as reported previously by Wiebauer and Jiricny (1989)] substrates, but did not cleave m6G:C (or G:C) substrates. Competition experiments showed that the activity was similar to, if not identical with, the activity in human cells that incises G:T mismatches. The incision sites were similar to those recognized by human G:T- or G:A-specific mismatch enzymes, i.e., the phosphodiester bonds both 3' and 5' to the poorly matched T, suggesting the glycolytic removal of the poorly matched T followed by backbone incisions by class I or II AP endonucleases. Three experiments in which MGMT was inactivated showed that the m6G:T incision activity was not simply due to a two-step mechanisms in which MGMT would first mediate conversion of the m6G:T substrate to a G:T substrate which would serve as a substrate for G:T incision. Extracts from HT29 contained a DNA-binding factor, possibly DNA sequence-specific, that inhibited incision of the m6G:T (but not the G:T) substrate, that was removed by the addition of synthetic DNA to the reaction.
Sibghat-Ullah
Department of Medicine, Cross Cancer Institute, Edmonton, Alberta, Canada.
Day
R S
RS
D001482:Base Composition; D001483:Base Sequence; D001667:Binding, Competitive; D003110:Colonic Neoplasms / Q000201:enzymology; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000378:metabolism; D005910:Glioma / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013941:Thymine / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D008769:Methylnitronitrosoguanidine; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003850:Deoxyribonuclease I; D013941:Thymine