Unveiling Replication Timing-Dependent Mutational Biases: Mechanistic Insights from Gene Knockouts and Genotoxins Exposures.

Replication timing (RT), the temporal order of DNA replication during S phase, influences regional mutation rates, yet the mechanistic basis for RT-associated mutagenesis remains incompletely defined. To identify drivers of RT-dependent mutation biases, we analyzed whole-genome sequencing data from cells with disruptions in DNA replication/repair genes or exposed to mutagenic compounds. Mutation distributions between early- and late-replicating regions were compared using bootstrapping and statistical modeling. We identified 14 genes that exhibit differential effects in early- or late-replicating regions, encompassing multiple DNA repair pathways, including mismatch repair (MLH1, MSH2, MSH6, PMS1, and PMS2), trans-lesion DNA synthesis (REV1) and double-strand break repair (DCLRE1A and PRKDC), DNA polymerases (POLB, POLE3, and POLE4), and other genes central to genomic instability (PARP1 and TP53). Similar analyses of mutagenic compounds revealed 19 compounds with differential effects on replication timing. These results establish replication timing as a critical modulator of mutagenesis, with distinct DNA repair pathways and exogenous agents exhibiting replication timing-specific effects on genomic instability. Our systematic bioinformatics approach identifies new DNA repair genes and mutagens that exhibit differential activity during the S phase. These findings pave the way for further investigation of factors that contribute to genome instability during cancer transformation.