Mechanism of RNA 20,30-cyclic phosphate end healing by T4 polynucleotide kinase–phosphatase

T4 polynucleotide kinase–phosphatase (Pnkp) exemplifies a family of enzymes with 50-kinase and 30-phosphatase activities that function in nucleic acid repair. The polynucleotide 30-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acyl-phosphatase superfamily. The 30-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid–base catalyst Asp167. We report that Pnkp also has RNA 20-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 20,30-cyclic phosphate end (RNA>p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA>p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA>p to RNAOH, mutant D167N converts RNA>p to RNA 30-phosphate, which it sequesters in the phosphatase active site. In support of the intermedi-acy of an RNA phosphomonoester, the reaction of mutant S211A with RNA>p results in transient ac-cumulation of RNAp en route to RNAOH. Our results suggest that healing of 20,30-cyclic phosphate ends is a four-step processive reaction: RNA>p+Pnk