DNA Rereplication Is Susceptible to Nucleotide-Level Mutagenesis

Abstract

Abstract The initiation of eukaryotic DNA replication at replication origins is tightly regulated to prevent re-initiation and re-replication within each cell cycle. This regulation is critical for genome stability as re-replication is an extremely potent inducer... The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA rereplication, which arises when the mechanisms that prevent the reinitiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae, we previously showed that DNA rereplication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of rereplication forks to break. Here, we examine the ability of DNA rereplication to induce nucleotide-level mutations. During normal replication these mutations are restricted by three overlapping error-avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsEA14, a frameshift reporter that is poorly proofread, we show that rereplication induces up to a 30× higher rate of frameshift mutations and that this mutagenesis is due to passage of the rereplication fork, not secondary to rereplication fork breakage. Rereplication can also induce comparable rates of frameshift and base-substitution mutations in a more general mutagenesis reporter CAN1, when the proofreading activity of DNA polymerase ε is inactivated. Finally, we show that the rereplication-induced mutagenesis of both lys2InsEA14 and CAN1 disappears in the absence of mismatch repair. These results suggest that mismatch repair is attenuated during rereplication, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, rereplication can facilitate nucleotide-level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability.