A phage mechanism for selective nicking of dUMP-containing DNA

Significance Studying the interactions of bacterial viruses (phages) with their bacterial hosts may lead to better understanding of bacterial mechanisms and consequently enable better manipulation of bacterial pathogens. In this study, we characterized the activity of a protein from phage T5, called T5.015. This protein binds to another protein, Ung, and uses its activity to selectively cleave dUMP-containing DNA. Such cleavage of the bacterial DNA stops bacterial DNA replication and also prevents bacterial division. Presumably, the phage DNA is protected from this activity as Ung does not act on the phage DNA, probably due to lower incorporation of the Ung substrate, dUMP. We believe that the findings are general to many phages and reveal a mechanism of self-versus-foreign DNA discrimination.
Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.