DNA polymerase I is required for the Hachiman-family anti-phage defense system

Abortive infection (Abi) systems protect bacterial populations from bacteriophages by triggering growth arrest or cell death in infected cells; however, how Abi effectors are coupled to changes in host DNA replication and repair remains unclear. The widely conserved Hachiman defense system encodes a nuclease (HamA) and a helicase (HamB) that assemble into the HamAB effector complex, and Escherichia coli carries a related variant, AbpAB. Here, we show that DNA polymerase I (PolA) is required for AbpAB- and HamAB-mediated phage restriction, whereas RNase HI prevents AbpAB-dependent growth inhibition during uninfected growth. In a temperature-sensitive polA mutant (polA12), AbpAB- and HamAB-mediated restriction of multiple phages was lost, as was AbpAB-dependent growth arrest triggered by mitomycin C (MMC)-induced DNA damage or by expression of the T4 single-stranded DNA-binding protein gp32. Truncation analyses suggest that the PolA requirement for AbpAB-dependent growth arrest may involve the PolA-specific N-terminal 5'-3' exonuclease domain, whereas SOS-induced polymerases II, IV, and V were not required. Pull-down assays detected PolA in association with the AbpAB complex in vivo; this co-detection persisted after DNase treatment, increased after MMC treatment, and decreased during T4 infection. In contrast, deletion of rnhA, which encodes RNase HI, rendered AbpAB expression toxic even in the absence of phage. Together, these results identify PolA and RNase HI as host factors that respectively enable Hachiman-family phage defense and prevent AbpAB-dependent growth inhibition during uninfected growth.IMPORTANCEAbortive infection (Abi) systems can stop phage replication by forcing infected cells to arrest growth or die, but this strategy is risky if it is triggered in the absence of phage. Here, we show that two Hachiman-family Abi systems-AbpAB and HamAB-require Escherichia coli DNA polymerase I (PolA) to block phage growth. PolA is also required for AbpAB-dependent growth arrest triggered by DNA damage (mitomycin C) or by expression of the T4 single-stranded DNA-binding protein gp32. In contrast, deleting rnhA (encoding RNase HI) makes AbpAB expression toxic even in the absence of phage. These results show that host replication and repair enzymes can both support anti-phage defense and prevent Abi-associated growth inhibition during uninfected growth.