Processing of O6-methylguanine by mismatch correction in human cell extracts.
Human cell extracts perform an aberrant form of DNA synthesis on methylated plasmids [1], which represents processing of O6-methylguanine (O6-meG). Here, we show that extracts of colorectal carcinoma cells with defects in the mismatch repair proteins that normally correct replication errors do not carry out this synthesis. hMSH2-defective LoVo cell extracts (hMSH for human MutS homologue) performed O6-meG-dependent DNA synthesis only after the addition of the purified hMutS alpha mismatch recognition complex. Processing of O6-meG by mismatch correction requires PCNA and therefore probably DNA polymerase delta and/or epsilon. Mismatch repair-defective cells withstand O6-meG in their DNA [2], making them tolerant to methylating agents. Methylation-tolerant HeLaMR clones, with a mutator phenotype and a defect in either mismatch recognition or correction in vitro, also performed little O6-meG-dependent DNA synthesis. Assays of pairwise combinations of tolerant and colorectal carcinoma cell extracts identified hMLH1 as the missing mismatch repair function in a group of tolerant clones. The absence of processing by extracts of methylation-tolerant cells provides the first biochemical evidence that lethality of DNA O6-meG derives from its interaction with mismatch repair.
Ceccotti
S
S
Istituto Superiore di Sanita, Rome, Italy.
Aquilina
G
G
Macpherson
P
P
Yamada
M
M
Karran
P
P
Bignami
M
M
D002457:Cell Extracts; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000096:biosynthesis*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D004273:DNA, Neoplasm; D006147:Guanine; C008449:O-(6)-methylguanine