Strand- and replication timing-dependent functions of DNA polymerase η in human DNA replication

Efficient DNA replication requires coordinated use of replicative and error-prone polymerases, which provide flexibility at the cost of mutagenesis. DNA polymerase η (Pol η) tolerates UV-induced lesions via translesion synthesis (TLS), but may contribute more broadly to DNA synthesis. Using polymerase usage sequencing (Pu-seq), we map Pol η activity genome-wide during unperturbed replication in human cells. Pol η preferentially participates in lagging strand replication, as in budding yeast. This bias requires direct PCNA interaction, suggesting stochastic engagement of PCNA-bound intermediates at replication forks. Pol η usage also varies across S phase, with PCNA ubiquitylation-dependent enrichment in late-replicating domains. In cancer genomes, characteristic Pol η mutations are enriched in late-replicating regions and correlate with RAD18 expression, linking PCNA ubiquitylation to Pol η-mediated mutagenesis. Thus, Pol η usage is biased by strand and replication timing during unperturbed replication and may contribute to mutational burden in human genomes.