The Hna Phage Defence Protein: A DNA ‐Stimulated Helicase With Cryptic Nuclease Activity

The Hna phage defence system consists of a single protein containing helicase and nuclease motifs. Hna provides defence through abortive infection, meaning that it prevents production of new phages without saving the infected cell, but its specific mechanism of action is currently unknown. We previously observed that Hna causes cell death when phage 5A single-stranded DNA binding protein (SSB) is expressed in Sinorhizobium meliloti. Here, we recreated this response by expressing Hna and phage SSB in the heterologous host Escherichia coli, indicating that no other host-specific proteins are required for Hna-mediated cell death. We also find that increased expression of Hna is lethal in E. coli even in the absence of phage SSB. We tagged and purified the Hna protein from S. meliloti, as well as helicase and nuclease motif mutants, and show that wildtype Hna binds ssDNA, performs DNA-stimulated ATP hydrolysis and unwinds short DNA duplexes. The nuclease mutant exhibits increased DNA-dependent stimulation of ATPase activity. Hna demonstrates cryptic nuclease activity: we do not observe nuclease activity in vivo, but the purified protein showed inefficient degradation of phage and bacterial genomic DNA and a ssDNA oligonucleotide in vitro. Further research should elucidate how these activities contribute to cell death.