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 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 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 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.

Keywords: Bacillus subtilis; CcrZ; DNA replication; DnaA; RecA.

Figure 1.. Establishing oriC/ter ratio of CcrZ expression under the amyE locus.

Shown are qPCR-generated oriC/ter ratios for the B. subtilis strains indicated grown in LB medium at 37°C as described in Materials and Methods. Data points plotted with means represented by a horizontal bar and error bars representing standard error of the mean for at least six independent replicates. Statistical significance of increased or decreased ratios compared to WT assessed by two-tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The data shown here are also summarized in Table 1.

Figure 2.. ccrZ and ccrZ mutant D166A under xylose-inducible promoters lead to increased oriC/ter ratios.

Strains were grown in LB medium and 0.25% xylose in LB medium used for all strains with a xylose-inducible promoter. The oriC/ter ratios generated by qPCR are shown as individual data points. The crossbar represents the mean and error bars shown indicate standard error of the mean for at least six independent replicates. Statistical significance assessed by two-tailed t-test compared to ΔccrZ, amyE::P_ccrZ-ccrZ, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The data shown here are also summarized in Table 1.

Figure 3.. Mutations that disrupt CcrZ function have lowered oriC/ter ratios.

Various strains carrying ccrZ mutants were grown in LB media with oriC/ter ratios assessed by qPCR. The individual data points are shown. The horizontal bars indicate the mean while the error bars represent standard error of the mean for each strain. The data shown are from six independent replicates. Statistical significance compared to ΔccrZ, amyE::P_ccrZ-ccrZ assessed by two-tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The data shown here are also summarized in Table 1.

Figure 4.. ccrZ alleles defective in replication initiation confer sensitivity to DNA damage.

Shown is a spot titer assay with the indicated strains spotted onto LB or LB with 25ng/ml mitomycin C (MMC). Each ccrZ allele is expressed from the amyE locus undercontrol of the native ccrZ promoter as described (35).

Figure 5.. RecA-GFP foci are elevated in cells with increased DNA replication initiation.

Shown are representative images of cells with the indicated genotypes. The left most column is RecA-GFP, followed by the middle column membrane stained with FM 4–64 and the merge of the GFP and FM 4–64 channels (composite) to the right.