Increased replication-associated single-stranded DNA promotes formaldehyde-induced mutagenesis.

Formaldehyde (FA) is an environmentally abundant and endogenously produced aldehyde that has been shown to cause DNA damage, mutagenesis, and carcinogenesis. Several studies have demonstrated that FA induces guanine mutations resulting in a mutation signature like SBS40. In this work, we demonstrate that replication defects generating single-stranded DNA (ssDNA) caused by the downregulation of the major replicative polymerases results in elevated FA mutagenesis. We found that loss of Mrc1 (CLASPIN) resulted in a high accumulation of ssDNA and FA mutagenesis, and that these phenotypes were not dependent on Mrc1's checkpoint activity. Loss of DNA-protein crosslink repair results in elevated FA sensitivity with no alteration to mutagenesis, likely due to the inability of the fork to bypass unprocessed protein adducts. Finally, we show that FA-induced mutagenesis is dependent on Pol ζ-mediated translesion synthesis, while deficiencies in the template switching pathway do not alter error-free bypass of FA adducts. Overall, our work points towards replication-associated ssDNA as a major substrate for FA-induced damage and elucidates the pathways that function to prevent FA mutagenesis at replication forks.