Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

Abstract

5=-and 3=-end-healing reactions are key steps in nucleic acid break repair in which 5=-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3=-PO 4 or 2=,3=-cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5=-PO 4 and 3=-OH termini required for sealing by classic polynucleotide ligases. Endhealing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2=,3=-phosphoesterase HD domain and a C-terminal 5=-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5=-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2=,3=-cyclic phosphate, RNA 3=-phosphate, RNA 2=-phosphate, and DNA 3=-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCEThe present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5=-and 3=-end-healing enzymes that phosphorylate 5=-OH termini and dephosphorylate 2=,3=-cyclic-PO 4 , 3=-PO 4 , and 2=-PO 4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. KEYWORDS 3= phosphatase, nucleic acid repair, polynucleotide kinase P olynucleotide kinases (Pnks) are a widely distributed class of cellular and virusencoded nucleic acid repair enzymes that convert 5=-OH termini into 5=-PO 4 ends that can be sealed by RNA or DNA ligases. Pnks are members of the P-loop phosphotransferase superfamily; they catalyze metal-dependent transfer of the ␥ phosphate of a nucleoside triphosphate (NTP) donor to a 5=-OH polynucleotide acceptor. In many repair systems, a Pnk enzyme is fused in a modular fashion to one or more other repair enzymes within a single multifunctional polypeptide.For example, bacteriophage T4 encodes a bifunctional 5=-OH polynucleotide kinase-3=-phosphatase (Pnkp) consisting of an N-terminal Pnk domain fused to a C-terminal

Structure-Function Analysis of the Phosphoesterase Component of the Nucleic Acid End-Healing Enzyme Runella slithyformis HD-Pnk

“…HD-Pnk dephosphorylates RNA 2=,3=-cyclic-PO 4 , RNA 3=-PO 4 , RNA 2=-PO 4 , and DNA 3=-PO 4 ends in the presence of a transition metal, which can be either nickel, copper, or cobalt. Copper was the most effective of the three metals in supporting DNA 3= phosphatase activity (8). The 3=-and 5=-end-healing activities are functionally separable, i.e., mutation of Asp254 or Arg257 of the DXXR motif effaced kinase activity without affecting the phosphatase and mutation of His73 of the HD motif abolished 3= phosphatase activity without affecting the kinase (8).…”

“…We recently identified and characterized Runella slithyformis and Deinococcus radiodurans HD-Pnk enzymes as the founders of a new dual 3=-and 5=-end-healing enzyme family (8,9). HD-Pnk consists of an N-terminal module of the HD phosphoesterase domain clade fused to a C-terminal Pnk domain ( Fig.…”

“…The invocation of a DNA or RNA repair function for HD-Pnk was underscored by its gene context in the R. slithyformis and D. radiodurans chromosomes (8,9). The HD-Pnk open reading frame (ORF) is located immediately downstream of a cooriented ORF encoding a polynucleotide ligase (LIG), in an operon-like fashion in which the 3= end of the LIG ORF overlaps the 5= end of the HD-Pnk ORF (Fig.…”

“…Whereas HD-Pnk is inessential for D. radiodurans growth, deletion of the gene encoding HD-Pnk sensitizes D. radiodurans to ionizing radiation and mitomycin C (9). Because HD-Pnk homologs are present in many bacterial taxa (8), often in an operon with LIG, we envision that the HD-Pnk and LIG activities are functionally related.…”

“…Previously, we showed that recombinant Runella slithyformis HD-Pnk has 3=-and 5=-end-healing activities (8). The kinase activity of HD-Pnk phosphorylates 5=-OH polynucleotides (Ն9 nucleotides in length) in the presence of magnesium using any standard NTP as the phosphate donor.…”