Literature DB >> 7645238

Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis.

S Shuman1, X M Ru.   

Abstract

DNA ligation entails AMP transfer from ATP to the 5' end of DNA to form a DNA-adenylate structure, A(5')pp(5')N. A similar reaction involving GMP transfer occurs during 5' capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase-AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.

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Year:  1995        PMID: 7645238     DOI: 10.1006/viro.1995.1380

Source DB:  PubMed          Journal:  Virology        ISSN: 0042-6822            Impact factor:   3.616


  20 in total

Review 1.  Structural and mechanistic conservation in DNA ligases.

Authors:  A J Doherty; S W Suh
Journal:  Nucleic Acids Res       Date:  2000-11-01       Impact factor: 16.971

2.  Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair.

Authors:  C E Huang; J Cruz-Reyes; A G Zhelonkina; S O'Hearn; E Wirtz; B Sollner-Webb
Journal:  EMBO J       Date:  2001-09-03       Impact factor: 11.598

3.  Comparative analysis of editosome proteins in trypanosomatids.

Authors:  Elizabeth A Worthey; Achim Schnaufer; I Saira Mian; Kenneth Stuart; Reza Salavati
Journal:  Nucleic Acids Res       Date:  2003-11-15       Impact factor: 16.971

4.  RNA 3'-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5'-monophosphate ends.

Authors:  Anupam K Chakravarty; Stewart Shuman
Journal:  J Biol Chem       Date:  2010-11-22       Impact factor: 5.157

5.  Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1.

Authors:  C K Ho; J L Van Etten; S Shuman
Journal:  J Virol       Date:  1997-03       Impact factor: 5.103

6.  Identification of essential residues in Thermus thermophilus DNA ligase.

Authors:  J Luo; F Barany
Journal:  Nucleic Acids Res       Date:  1996-08-01       Impact factor: 16.971

7.  Domain structure of vaccinia DNA ligase.

Authors:  J Sekiguchi; S Shuman
Journal:  Nucleic Acids Res       Date:  1997-02-15       Impact factor: 16.971

8.  The two RNA ligases of the Trypanosoma brucei RNA editing complex: cloning the essential band IV gene and identifying the band V gene.

Authors:  L N Rusché; C E Huang; K J Piller; M Hemann; E Wirtz; B Sollner-Webb
Journal:  Mol Cell Biol       Date:  2001-02       Impact factor: 4.272

9.  Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum.

Authors:  V Sriskanda; Z Kelman; J Hurwitz; S Shuman
Journal:  Nucleic Acids Res       Date:  2000-06-01       Impact factor: 16.971

10.  Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Authors:  J Sekiguchi; S Shuman
Journal:  J Virol       Date:  1997-12       Impact factor: 5.103

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