Biomaterial Database

Mechanism of RNA 2',3'-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase. 2013

Ushati Das, and Stewart Shuman

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

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.

UI	MeSH Term	Description	Entries
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D010710	Phosphates	Inorganic salts of phosphoric acid.	Inorganic Phosphate,Phosphates, Inorganic,Inorganic Phosphates,Orthophosphate,Phosphate,Phosphate, Inorganic
D010727	Phosphoric Diester Hydrolases	A class of enzymes that catalyze the hydrolysis of one of the two ester bonds in a phosphodiester compound. EC 3.1.4.	Phosphodiesterase,Phosphodiesterases,Hydrolases, Phosphoric Diester
D010744	Phosphoric Monoester Hydrolases	A group of hydrolases which catalyze the hydrolysis of monophosphoric esters with the production of one mole of orthophosphate.	Phosphatase,Phosphatases,Phosphohydrolase,Phosphohydrolases,Phosphomonoesterase,Phosphomonoesterases,Phosphoric Monoester Hydrolase,Hydrolase, Phosphoric Monoester,Hydrolases, Phosphoric Monoester,Monoester Hydrolase, Phosphoric
D011116	Polynucleotide 5'-Hydroxyl-Kinase	An enzyme that catalyzes the transfer of a phosphate group to the 5'-terminal hydroxyl groups of DNA and RNA. EC 2.7.1.78.	Polynucleotide Hydroxylkinase,Polynucleotide Kinase,5'-Hydroxylpolynucleotide Kinase,DNA 5'-Hydroxylkinase,DNA Kinase,Polynucleotide 5'-Hydroxyl Kinase,Polynucleotide Hydroxykinase,5' Hydroxylpolynucleotide Kinase,5'-Hydroxyl Kinase, Polynucleotide,5'-Hydroxyl-Kinase, Polynucleotide,5'-Hydroxylkinase, DNA,DNA 5' Hydroxylkinase,Hydroxykinase, Polynucleotide,Hydroxylkinase, Polynucleotide,Kinase, 5'-Hydroxylpolynucleotide,Kinase, DNA,Kinase, Polynucleotide,Kinase, Polynucleotide 5'-Hydroxyl,Polynucleotide 5' Hydroxyl Kinase
D001224	Aspartic Acid	One of the non-essential amino acids commonly occurring in the L-form. It is found in animals and plants, especially in sugar cane and sugar beets. It may be a neurotransmitter.	(+-)-Aspartic Acid,(R,S)-Aspartic Acid,Ammonium Aspartate,Aspartate,Aspartate Magnesium Hydrochloride,Aspartic Acid, Ammonium Salt,Aspartic Acid, Calcium Salt,Aspartic Acid, Dipotassium Salt,Aspartic Acid, Disodium Salt,Aspartic Acid, Hydrobromide,Aspartic Acid, Hydrochloride,Aspartic Acid, Magnesium (1:1) Salt, Hydrochloride, Trihydrate,Aspartic Acid, Magnesium (2:1) Salt,Aspartic Acid, Magnesium-Potassium (2:1:2) Salt,Aspartic Acid, Monopotassium Salt,Aspartic Acid, Monosodium Salt,Aspartic Acid, Potassium Salt,Aspartic Acid, Sodium Salt,Calcium Aspartate,Dipotassium Aspartate,Disodium Aspartate,L-Aspartate,L-Aspartic Acid,Magnesiocard,Magnesium Aspartate,Mg-5-Longoral,Monopotassium Aspartate,Monosodium Aspartate,Potassium Aspartate,Sodium Aspartate,Aspartate, Ammonium,Aspartate, Calcium,Aspartate, Dipotassium,Aspartate, Disodium,Aspartate, Magnesium,Aspartate, Monopotassium,Aspartate, Monosodium,Aspartate, Potassium,Aspartate, Sodium,L Aspartate,L Aspartic Acid
D012313	RNA	A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed)	RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D017122	Bacteriophage T4	Virulent bacteriophage and type species of the genus T4-like phages, in the family MYOVIRIDAE. It infects E. coli and is the best known of the T-even phages. Its virion contains linear double-stranded DNA, terminally redundant and circularly permuted.	Bacteriophage T2,Coliphage T2,Coliphage T4,Enterobacteria phage T2,Enterobacteria phage T4,Phage T2,Phage T4,T2 Phage,T4 Phage,Phage, T2,Phage, T4,Phages, T2,Phages, T4,T2 Phages,T2, Enterobacteria phage,T4 Phages
D020134	Catalytic Domain	The region of an enzyme that interacts with its substrate to cause the enzymatic reaction.	Active Site,Catalytic Core,Catalytic Region,Catalytic Site,Catalytic Subunit,Reactive Site,Active Sites,Catalytic Cores,Catalytic Domains,Catalytic Regions,Catalytic Sites,Catalytic Subunits,Core, Catalytic,Cores, Catalytic,Domain, Catalytic,Domains, Catalytic,Reactive Sites,Region, Catalytic,Regions, Catalytic,Site, Active,Site, Catalytic,Site, Reactive,Sites, Active,Sites, Catalytic,Sites, Reactive,Subunit, Catalytic,Subunits, Catalytic