Hickman, A. B. | bioRxiv Science

bioRxiv2023

Toxic anti-phage defense proteins inhibited by intragenic antitoxin proteins

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic full-length proteins. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. SignificanceHere we document the function of small genes-within-genes, showing they encode antitoxin proteins that block the functions of the toxic DNA endonuclease proteins encoded by the longer rpn genes. Intriguingly, a sequence present in both long and short protein shows extensive variation in the number of four amino acid repeats. Consistent with a strong selection for the variation, we provide evidence that the Rpn proteins represent a phage defense system.

microbiology↗

bioRxiv2022

Formation of the active Hermes transpososome is driven by asymmetric DNA binding of BED domains

The cut-and-paste Hermes DNA transposase stands out among the transposases that have been biochemically or structurally characterized so far. Many transposases function as dimers, but the Hermes transposase forms a tetramer of dimers to achieve its active form in vivo. Intriguingly, the transposition complex, or transpososome, relies on only one dimer to perform the enzymatic reactions necessary to the mobilization of its transposon. Our investigation combining biochemical and structural approaches shows that the Hermes octamer extensively interacts with its transposon left-end (LE) engaging the BED domains of three Hermes protomers belonging to three dimers. By contrast, the right-end (RE) is entirely deprived of such interaction inside the transpososome. Our work suggests that formation of the Hermes synaptic complex is sequential and relies on the considerable difference of affinity of the transposase towards its transposon ends. Thus, we propose that Hermes dimers multimerize to gather enough BED domains to find the LE among the abundant genomic DNA, facilitating the subsequent interaction with the RE, most likely solely based on recognition of its terminal inverted repeat.

biochemistry↗