title: Mechanism of RNA 2',3'-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase.
abstract: T4 polynucleotide kinase-phosphatase (Pnkp) exemplifies a family of enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The polynucleotide 3'-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3'-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 2'-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2',3'-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 RNA(OH), mutant D167N converts RNA > p to RNA 3'-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNA(OH). Our results suggest that healing of 2',3'-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3'-phosphoaspartyl)-Pnkp → RNA(3')p + Pnkp → RNA(OH) + phosphoaspartyl-Pnkp → P(i) + Pnkp.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors.forename: Ushati
authors.initials: U
authors.lastname: Das
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology*; D020134:Catalytic Domain; D009154:Mutation; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010710:Phosphates; D001224:Aspartic Acid; D012313:RNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D010744:Phosphoric Monoester Hydrolases; D010727:Phosphoric Diester Hydrolases