Processing of O6-methylguanine by mismatch correction in human cell extracts

Abstract

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.

Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase

“…Furthermore, these experiments demonstrated that the repair synthesis triggered by the presence of 6Me G residues in the DNA is RPA independent and that it gives rise to open circular DNA, in contrast to the in vitro replication reaction of the same, but unmodified, plasmid, which yielded almost exclusively supercoiled DNA molecules. These results could be taken as further evidence of the persistence of unligatable singlestranded regions or breaks in the plasmid DNA incubated with MMR-proficient cell extracts (Ceccotti et al 1996). Direct support for the persistence of incompletely replicated DNA comes from our experiments, in which the genomic DNA of MNNG-treated MMR-proficient cells after the first S phase was shown to contain numerous single-strand breaks, as witnessed by the appearance of DNA tails in alkaline comet assays (Fig.…”

“…However, as the 6Me G residues persist in the template strand, resynthesis of this region would again generate 6Me G/T or 6Me G/C mispairs. The repeated processing of these mispairs by the MMR system (Karran and Bignami 1996) will likely lead to stalling of the replication fork. One might pose the question of why these structures would fail to activate the S-phase checkpoint, when other polymerase-arresting agents such as HU or aphidicolin do so extremely efficiently (Abraham 2001;Osborn et al 2002;Shiloh 2003).…”

“…An alternative explanation is that unlike HU, which brings about a depletion of the purine nucleotide pool, or aphidicolin, which directly inhibits the replica-tive polymerases, 6Me G residues in the template strand do not prevent a replication restart downstream from the modified base. Indirect support for the replication restart hypothesis comes from in vitro experiments carried out with MNU-modified plasmid DNA; the observed DNA repair synthesis required nucleoside triphosphates (NTPs), which would be required by a primase (Ceccotti et al 1993(Ceccotti et al , 1996. Furthermore, these experiments demonstrated that the repair synthesis triggered by the presence of 6Me G residues in the DNA is RPA independent and that it gives rise to open circular DNA, in contrast to the in vitro replication reaction of the same, but unmodified, plasmid, which yielded almost exclusively supercoiled DNA molecules.…”

Genomic predictors of interindividual differences in response to DNA damaging agents

“…1A). Interestingly, although not known to repair damage induced by MNNG, MYH is known to interact with the MutS␣ heterodimer (Gu et al 2002) that binds O 6 MeG mispairs in DNA to initiate the triggering of apoptotic cell death (Ceccotti et al 1996;Hickman and Samson 2004). It remains to be determined whether MYH influences alkylation sensitivity via its interaction with the MMR machinery.…”

Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O6-methylguanine due to high E2F1 regulated mismatch repair

“…14,15 In this process, the heterodimer MutSa, comprised of MSH2 and MSH6, is critically involved because of its binding to O 6 MeG adducts mispaired with thymine. 13,26 It has been proposed that unsuccessful removal of thymine by MutSa leads to a futile DNA repair cycle that either directly or indirectly, via the formation of DNA double-stranded breaks (DSBs), gives rise to apoptosis. 24,27 If this does not occur, cells will survive and O 6 MeG adducts will give rise to mutations.…”