ABSTRACT5′- and 3′-end healing are key steps in nucleic acid break repair in which 5′-OH and 3′-PO4or 2′,3′-cyclic-PO4ends are converted to 5′-PO4and 3′-OH termini suitable for sealing by polynucleotide ligases. Here, we characterizeDeinococcus radioduransHD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5′-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3′-phosphatase and 2′,3′-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis ofp-nitrophenylphosphate. HD-Pnk is encoded by the LIG–PARG–HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential forDeinococcusgrowth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ΔHD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitizeDeinococcusto killing by mitomycin C.IMPORTANCEEnd healing is a process whereby nucleic acid breaks with “dirty” 3′-PO4or 2′,3′-cyclic-PO4and 5′-OH ends are converted to 3′-OH and 5′-PO4termini that are amenable to downstream repair reactions.Deinococcus radioduransis resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlightsDeinococcusHD-Pnk as a bifunctional 3′- and 5′-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.
Holliday intermediates and reaction by-products in FLP protein-promoted site-specific recombination.
