Gap filling activities of Pseudomonas DNA ligase D (LigD) polymerase and functional interactions of LigD with the DNA end-binding Ku protein Journal Article

Authors: Zhu, H.; Shuman, S.
Article Title: Gap filling activities of Pseudomonas DNA ligase D (LigD) polymerase and functional interactions of LigD with the DNA end-binding Ku protein
Abstract: Many bacterial pathogens, including Pseudomonas aeruginosa, have a nonhomologous end joining (NHEJ) system of DNA double strand break (DSB) repair driven by Ku and DNA ligase D(LigD). LigD is a multifunctional enzyme composed of a ligase domain fused to an autonomous polymerase module (POL) that adds ribonucleotides or deoxyribonucleotides to DSB ends and primer-templates. LigD POL and the eukaryal NHEJ polymerase λ are thought to bridge broken DNA ends via contacts with a duplex DNA segment downstream of the primer terminus, a scenario analogous to gap repair. Here, we characterized the gap repair activity of Pseudomonas LigD POL, which is more efficient than simple templated primer extension and relies on a 5′-phosphate group on the distal gap strand end to confer apparent processivity in filling gaps of 3 or 4 nucleotides. Mutations of the His-553, Arg-556, and Lys-566 side chains implicated in DNA 5′-phosphate binding eliminate the preferential filling of 5′-phosphate gaps. Mutating Phe-603, which is imputed to stack on the nucleobase of the template strand that includes the 1st bp of the downstream gap duplex segment, selectively affects incorporation of the final gap-closing nucleotide. We find that Pseudomonas Ku stimulates POL-catalyzed ribonucleotide addition to a plasmid DSB end and promotes plasmid end joining by full-length Pseudomonas LigD. A series of incremental truncations from the C terminus of the 293-amino acid Ku polypeptide identifies Ku-(1-229) as sufficient for homodimerization and LigD stimulation. The slightly longer Ku-(1-253) homodimer forms stable complexes at both ends of linear plasmid DNA that protect the DSBs from digestion by 5′- and 3′-exonucleases. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords: controlled study; unclassified drug; mutation; dna-binding proteins; dna polymerase; protein function; dna repair; genes; protein protein interaction; enzyme activity; bacteria (microorganisms); bacterial proteins; dna; amino acid sequence; eukaryota; dna breaks, double-stranded; double stranded dna break; dimerization; catalysis; amino acids; organic acids; ku antigen; microbiology; dna binding; protein structure, quaternary; repair; side chains; complexation; dna ligases; ligase; plasmid dna; pseudomonas aeruginosa; dna-directed dna polymerase; nucleotides; polymers; c terminus; functional interaction; dna double strand breaks; phosphate group; dna ligase d; dna protein complex; pseudomonas; duplex dna; nonhomologous end joining; bacterial pathogens; filling gaps; gap filling; homodimerization; homodimers; nucleobases; phosphate binding; primer extension; primer terminus; processivity; ribonucleotides; stable complexes; template strand; templated; functional polymers; joining; deoxyribonucleotide
Journal Title: Journal of Biological Chemistry
Volume: 285
Issue: 7
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 2010-02-12
Start Page: 4815
End Page: 4825
Language: English
DOI: 10.1074/jbc.M109.073874
PUBMED: 20018881
PROVIDER: scopus
PMCID: PMC2836087
Notes: --- - "Cited By (since 1996): 2" - "Export Date: 20 April 2011" - "CODEN: JBCHA" - "Source: Scopus"
Citation Impact
MSK Authors
  1. Stewart H Shuman
    504 Shuman
  2. Hui P Zhu
    14 Zhu