Domain structure and mutational analysis of T4 polynucleotide kinase

The Journal of Biological Chemistry
Li Kai Wang, Stewart Shuman

Abstract

T4 polynucleotide kinase (Pnk) is the founding member of a family of 5'-kinase/3'-phosphatase enzymes that heal broken termini in RNA or DNA by converting 3'-PO(4)/5'-OH ends into 3'-OH/5'-PO(4) ends, which are then suitable for sealing by RNA or DNA ligases. Here we employed site-directed mutagenesis and biochemical methods to dissect the domain structure of the homotetrameric T4 Pnk protein and to localize essential constituents of the apparently separate active sites for the 5'-kinase and 3'-phosphatase activities. We characterized deletion mutants Pnk(42-301) and Pnk(1-181), which correspond to domains defined by proteolysis with chymotrypsin. Pnk(1-181) is a monomer with no 3'-phosphatase and low residual 5'-kinase activity. Pnk(42-301) is a dimer with no 5'-kinase and low residual 3'-phosphatase activity. Four classes of missense mutational effects were observed. (i) Mutations K15A, S16A, and D35A inactivated the 5'-kinase but did not affect the 3'-phosphatase or the tetrameric quaternary structure of T4 Pnk. 5'-kinase activity was ablated by the conservative mutations K15R, K15Q, and D35N; however, kinase activity was restored by the S16T change. (ii) Mutation D167A inactivated the 3'-phosphatase without affecting the 5'...Continue Reading

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Citations

Jun 1, 2010·Analytical and Bioanalytical Chemistry·Changbei Ma, Edward S Yeung
Dec 15, 2010·Extremophiles : Life Under Extreme Conditions·Debamitra ChakravortySanjukta Patra
Jul 12, 2002·The EMBO Journal·Li Kai WangStewart Shuman
Mar 28, 2002·Nucleic Acids Research·Vivek AnantharamanL Aravind
Feb 3, 2004·Nucleic Acids Research·Jennifer H EastbergBarry L Stoddard
May 10, 2007·Nucleic Acids Research·Niroshika KeppetipolaStewart Shuman
Nov 3, 2012·Nucleic Acids Research·Ushati Das, Stewart Shuman
Jun 18, 2005·Nucleic Acids Research·Zhiwen TangXiangxian Meng
Oct 19, 2004·Journal of Bacteriology·Małgorzata B ŁobockaFrederick R Blattner
Aug 21, 2013·Archives of Virology·Živilė Strazdaitė-ŽielienėLidija Truncaitė
Feb 20, 2004·Proceedings of the National Academy of Sciences of the United States of America·Beate SchwerStewart Shuman
Nov 11, 2010·The EMBO Journal·Katrin Heindl, Javier Martinez
Jun 20, 2017·PLoS Pathogens·Teja ShidoreLindsay R Triplett

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