DNA replication initiation timing is important for maintaining genome integrity

Abstract

ABSTRACTDNA replication is regulated by factors that promote or inhibit initiation. InBacillus subtilis,YabA is a negative regulator of DNA replication initiation while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of DNA replication to genome stability remain unclear. In this work, we measure origin to terminus ratios as a proxy for replication initiation activity. We show that ΔccrZand severalccrZalleles under-initiate DNA replication while ablation ofyabAor 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 low formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating DNA 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 were both sensitive to mitomycin C and that changes in replication initiation frequency cause increased sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to a shortage of DNA for repair through homologous recombination. For cells over-initiating DNA 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 as 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. For 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. Further, cells over-initiating DNA replication experience replication fork stress at levels that phenocopy cells encountering DNA damage from the crosslinking agent 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.