Replication across O⁶-methylguanine activates futile cycling of DNA mismatch repair attempts assisted by the chromatin-remodelling enzyme Smarcad1

S_N1-type alkylating reagents generate O⁶-methylguanine (^meG) lesions that activate the mismatch repair (MMR) response. Since post-replicative MMR specifically targets the nascent strand, ^meG on the template strand is refractory to rectification by MMR and, therefore, can induce non-productive MMR reactions. The cycling of futile MMR attempts is proposed to cause DNA double-strand breaks in the subsequent S phase, leading to ATR-checkpoint-mediated G2 arrest and apoptosis. However, the mechanistic details of futile MMR cycling, especially how this reaction is maintained in chromatin, remain unclear. Using replication-competent Xenopus egg extracts, we herein establish an in vitro system that recapitulates futile MMR cycling in the chromatin context. The ^meG–T mispair, but not the ^meG–C pair, is efficiently targeted by MMR in our system. MMR attempts on the ^meG-strand result in the ^meG-to-A correction, whilst those on the T-strand induce iterative cycles of strand excision and resynthesis. Likewise, replication across ^meG generates persistent single-strand breaks on the daughter DNA containing ^meG. Moreover, the depletion of Smarcad1, a chromatin remodeller previously reported to facilitate MMR, impairs the retention of single-strand breaks. Our study thus provides experimental evidence that chromatin replication across ^meG induces futile MMR cycling that is assisted by Smarcad1.