Activation of Intra‐S Phase Checkpoint Facilitates Tolerance of Replication Stress Caused by Mismatch Repair Processing of DNA Damage
Authors: Gupta Dipika , Lin Bo , Heinen Christopher D 
Published: November 2023

The DNA mismatch repair (MMR) pathway maintains genomic integrity by increasing DNA replication fidelity. This critical role in maintenance of genomic stability is underscored by the association between inherited mutations in key MMR genes and the colorectal cancer predisposition disorder Lynch syndrome. Furthermore, 10–40% of spontaneously arising colorectal and other tumor types lose MMR function. In addition to repairing mistakes made by the replicative polymerases, the MMR pathway also elicits a cell cycle arrest and apoptotic response to DNA damaging agents, particularly a class of DNA methylating agents commonly used in chemotherapy. The MMR‐dependent cytotoxicity to SN1 type alkylating agents results from the generation of O6‐methylguanine (MeG) lesions in DNA which commonly mispair with thymidine during DNA replication. These MeG/T lesions invoke a MMR‐dependent, permanent G2 arrest in transformed cell models, however only after cells have undergone two rounds of DNA replication. The “futile cycle model” posits that MeG/T mismatch recognition by MMR initiates excision of the mismatch containing daughter strand followed by DNA resynthesis. However, persistence of MeG on the parental DNA strands results in MeG/T lesion resynthesis, driving iterative cycles of futile repair ultimately leaving unreplicated gaps after the first S phase. Although this is accompanied by the activation of the ataxia telangiectasia and Rad3‐related (ATR) ‐ Checkpoint Kinase 1 (Chk1) signaling axis, its significance in the MMR‐dependent DNA damage response is unknown. During the second S phase, replication forks encountering the single strand gaps collapse, forming double strand breaks (DSB), leading to a permanent G2 arrest from which the cells never recover. In sharp contrast to this, our studies in human pluripotent stem cells (hPSCs) demonstrate the activation of a MMR‐dependent apoptotic response in the first replication cycle after exposure to DNA alkylating agents. We therefore wished to investigate the effects of MMR processing of MeG/T lesions on active replication forks to explain the differential outcomes observed in these two cell types. We found that hPSCs treated with an alkylating agent accumulate single‐stranded DNA stretches without activating Chk1 that is accompanied by severe hindrance to replication fork progression, extensive double strand break formation and immediate apoptosis. In HeLa cancer cells treated with the drug, progression through the first S‐phase is delayed with activation of the ATR‐Chk1 signaling axis limiting DNA damage accumulation and allowing cells to progress into the second cell cycle. We propose that MMR‐dependent processing of alkylation damage during the first S‐phase leads to replication stress and that cancer cells with active MMR may be particularly dependent on a functional ATR‐Chk1 dependent intra‐S phase checkpoint. By understanding how cancers respond to chemotherapies, this study could ultimately lead to the development of more effective drug regimens to selectively sensitize cancers to chemotherapeutic DNA alkylating agents.Support or Funding Information1. Scholarship from the Kenneth & Paula Munson Charitable Lead Annuity Trust2. State of Connecticut Regenerative Medicine Research Fund 13SCB‐UCHC‐06