Mechanism of RNA 2′,3′-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase Journal Article
| Authors: | Das, U.; Shuman, S. |
| Article 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 RNAOH, 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 RNAOH. Our results suggest that healing of 2′,3′-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3′- phosphoaspartyl)-Pnkp → RNA3′p + Pnkp → RNA OH + phosphoaspartyl-Pnkp → Pi + Pnkp. © 2012 The Author(s). Published by Oxford University Press. |
| Journal Title: | Nucleic Acids Research |
| Volume: | 41 |
| Issue: | 1 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2013-01-01 |
| Start Page: | 355 |
| End Page: | 365 |
| Language: | English |
| DOI: | 10.1093/nar/gks977 |
| PROVIDER: | scopus |
| PUBMED: | 23118482 |
| PMCID: | PMC3592404 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 1 February 2013" - "CODEN: NARHA" - "Source: Scopus" |
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