ATR-Chk1 activation mitigates replication stress caused by mismatch repair-dependent processing of DNA damage

Abstract

The mismatch repair pathway (MMR) is essential for removing DNA polymerase errors, thereby maintaining genomic stability. Loss of MMR function increases mutation frequency and is associated with tumorigenesis. However, how MMR is executed at active DNA replication forks is unclear. This has important implications for understanding how MMR repairs -methylguanine/thymidine (G/T) mismatches created upon exposure to DNA alkylating agents. If G/T lesion recognition by MMR initiates mismatch excision, the reinsertion of a mismatched thymidine during resynthesis could initiate futile repair cycles. One consequence of futile repair cycles might be a disruption of overall DNA replication in the affected cell. Herein, we show that in MMR-proficient HeLa cancer cells, treatment with a DNA alkylating agent slows S phase progression, yet cells still progress into the next cell cycle. In the first S phase following treatment, they activate ataxia telangiectasia and Rad3-related (ATR)-Checkpoint Kinase 1 (Chk1) signaling, which limits DNA damage, while inhibition of ATR kinase activity accelerates DNA damage accumulation and sensitivity to the DNA alkylating agent. We also observed that exposure of human embryonic stem cells to alkylation damage severely compromised DNA replication in a MMR-dependent manner. These cells fail to activate the ATR-Chk1 signaling axis, which may limit their ability to handle replication stress. Accordingly, they accumulate double-strand breaks and undergo immediate apoptosis. Our findings implicate the MMR-directed response to alkylation damage as a replication stress inducer, suggesting that repeated MMR processing of mismatches may occur that can disrupt S phase progression.

Functional Interrogation of Lynch Syndrome Associated MSH2 Missense Variants Using CRISPR-Cas9 Gene Editing in Human Embryonic Stem Cells

“…We also selected two variants previously determined to be benign polymorphisms and two others previously deemed pathogenic mutations as positive and negative controls, respectively. We also used the parental H1 hESCs and an H1 hESC clone in which we used CRISPR-Cas9 to disrupt the MSH2 loci in a homozygous fashion as described previously (16) as additional positive and negative controls, respectively. To assess the functional effects of MSH2 VUS on MMR function in a human cell culture model, we used CRISPR-Cas9 gene editing to introduce each variant directly into the endogenous MSH2 locus of H1 hESCs.…”

“…Human cancer cells undergo a permanent cell cycle arrest in response to various DNA damaging agents including S N 1 alkylating agents like MNNG, however, this response is dependent on a functional MMR pathway (5,6). More recently, we have demonstrated that human pluripotent stem cells instigate a rapid apoptotic response to MNNG, also in a MMR-dependent manner (16,30). To assess the ability of the MSH2 VUS lines to induce cell death in response to MNNG, we used an MTT assay to measure cell survival following treatment.…”

XAB2 prevents abortive recombinational repair of replication-associated DNA double-strand breaks and its loss is synthetic lethal with RAD52 inhibition

“…The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) removes the most cytotoxic lesion induced by TMZ, O 6 -methylguanine (O 6 -meG) [3], providing a major TMZ resistance mechanism [4]. Epigenetic silencing of MGMT is a predictive and prognostic biomarker in TMZ-treated GBM patients [5].…”

“…O 6 -meG lesions left unrepaired by MGMT generate O 6 -meG/thymidine mismatches during S phase. These mismatches are recognized, but not resolved, by the mismatch repair (MMR) pathway, resulting in futile repair cycles and persistent single-stranded DNA (ssDNA) gaps that cause replication fork collapse and DNA double-stranded DNA breaks (DSBs) during a subsequent round of replication [6,7].…”

“…Bidirectional resection then takes place, mediated by MRE11 exonuclease activity in the 3'-5' direction and EXO1/BLM/DNA2 in the opposite direction, generating ssDNA that recruits RPA. Although the mechanisms leading 6 to the subsequent release of Ku remain obscure, for about 40% of seDSBs induced by the topoisomerase I poison camptothecin (CPT), they involve the coordinated nuclease activities of MRE11 and CtIP, and activation by ATM [33]. Regulation of end resection also involves p53 binding protein 1 (53BP1), effector molecules [34] and the helicase HELB [35].…”