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
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-77951187826&partnerID=40&md5=e668c7fb45895f308db6cbab7ab0626d
Notes:	--- - "Cited By (since 1996): 2" - "Export Date: 20 April 2011" - "CODEN: JBCHA" - "Source: Scopus"

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