O6-methylguanine residues elicit DNA repair synthesis by human cell extracts

Karran, Peter; Macpherson, Peter; Ceccotti, Sabrina; Dogliotti, Eugenia; Griffin, Shaun; Bignami, Margherita

doi:10.1016/s0021-9258(18)82335-3

Cited by 61 publications

(30 citation statements)

References 40 publications

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“…Interestingly, we detected robust, MMR-dependent, UDS upon incubation of MNU-treated plasmids in NPE. This observation reveals that, not only are O 6 mG:C lesions recognized by MutSα as previously noted ( Duckett et al, 1999 ; Karran et al, 1993 ), but also the whole MMR repair process is engaged and proceeds to completion. We would also like to stress the high sensitivity of the pull-down assay with respect to MMR protein capture.…”

Section: Discussionsupporting

confidence: 76%

“…Response of cells to SN1 methylating agents was shown to be initiated at O 6 mG:T mispairs that form upon DNA replication of O 6 mG-containing DNA template and shown to involve the MMR machinery ( Goldmacher et al, 1986 ; Day et al, 1980 ; Karran et al, 1993 ; Yarosh et al, 1983 ). The O 6 mG:T mispair is efficiently recognized by MutSα, the key MMR initiator protein.…”

Section: Discussionmentioning

confidence: 99%

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Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide

Fuchs

Isogawa

Paulo

et al. 2021

eLife

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Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-studied cell cycle dependent mode of action.

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Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide

Fuchs

Isogawa

Paulo

et al. 2021

eLife

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“…1a and [3]). And third, the aberrant DNA synthesis does not occur to a significant extent on a methyl methanesulfonatetreated substrate that contains essentially no O 6 -meG [1].…”

Section: Resultsmentioning

confidence: 99%

“…HeLa cell extracts perform non-replicative DNA synthesis on plasmids methylated with N-methyl-N-nitrosourea (MNU) and containing approximately one O 6 -meG per molecule [1]. Three lines of evidence indicate that this DNA synthesis represents processing of O 6 -meG.…”

Section: Resultsmentioning

confidence: 99%

“…First, pretreatment of MNU-treated plasmid with a purified recombinant human O 6 -meG-DNA methyltransferase (MGMT), which selectively removes the methyl group from O 6 -meG in DNA, reduces the extent of this type of DNA synthesis (Fig. 1a and [1]). Second, active MGMT in extracts of Mex + HeLaS3 cells (Mex + denotes expression of MGMT) decreases the level of DNA synthesis to approximately half that carried out by Mex -HeLaMR extracts, which do not contain detectable MGMT (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Ceccotti

Aquilina

Macpherson³

et al. 1996

Current Biology

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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.

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2017

Anticancer Therapeutics

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O6-methylguanine residues elicit DNA repair synthesis by human cell extracts

Cited by 61 publications

References 40 publications

Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide

Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide

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

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