DNA replication initiation timing is important for maintaining genome integrity.

Abstract

DNA replication is regulated by factors that promote or inhibit initiation. In Bacillus subtilis, YabA is a negative regulator of replication initiation, while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of replication on genome stability remain unclear. In this work, we measure the origin-to-terminus ratio as a proxy for replication initiation activity. We show that ΔccrZ and several ccrZ alleles under-initiate DNA replication, while ablation of yabA or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by the low frequency formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA-damaging agents. We show that cells under-initiating and over-initiating DNA replication are both sensitive to mitomycin C, demonstrating that changes in replication initiation frequency cause an increase in sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to asynchronous DNA replication, leading to inefficient homologous recombination. In cells over-initiating replication, we propose that an increase in the number of replication forks leads to replication fork stress, which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled because changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.IMPORTANCEThe regulation of DNA replication is fundamental to cell growth and cell cycle control. In eukaryotes, under-initiation or over-initiation leads to genome instability. In bacteria, it is unclear how changes in replication initiation frequency impact DNA replication status and genome integrity. We show that tight regulation of DNA replication initiation is critical for maintaining genome integrity. Cells over-initiating or under-initiating DNA replication are sensitive to DNA damage. Furthermore, cells over-initiating DNA replication experience replication fork stress at levels that phenocopy those observed in cells challenged with DNA damage from mitomycin C. Our results establish the critical importance of properly regulating DNA replication initiation frequency because an imbalance in initiation results in replication fork perturbations, deficiencies in DNA repair, and genome instability.