Characterization of a thermostable archaeal polynucleotide kinase homologous to human Clp1

Clp1 proteins are essential components of the eukaryal mRNA 3′ cleavage-polyadenylation machinery. Human Clp1 has an additional function as an RNA-specific 5′-OH polynucleotide kinase, which is implicated in RNA end healing. Yeast Clp1 has no kinase activity, although it binds ATP. Here we report that Clp1-like proteins are extant in archaea. Purification and characterization of Pyrococcus horikoshii Clp1 ( Pho Clp1) reveals it to be a thermostable 5′-OH polynucleotide kinase optimally active at 55°C to 85°C. Pho Clp1 catalyzes transfer of the gamma phosphate from ATP ( K m 16 μM) to either 5′-OH RNA or DNA ends, although it prefers RNA in a competitive situation. Increasing the monovalent salt concentration to 250 mM suppresses the DNA kinase without affecting RNA phosphorylation, suggesting that RNA is a likely substrate for this enzyme in vivo. Indeed, we show that expression of Pho Clp1 in budding yeast can complement a lethal mutation in the 5′-OH RNA kinase module of tRNA ligase. Pho Clp1 is a member of the P-loop phosphotransferase superfamily. Alanine mutations at the P-loop lysine (Lys49) and a conserved aspartate (Asp73) inactivate the kinase. Our studies fortify emerging evidence for an enzymatic RNA repair capacity in archaea and provide a new reagent for polynucleotide phosphorylation at high temperatures.