A primer-dependent polymerase function of Pseudomonas aeruginosa ATP-dependent DNA ligase (LigD) Journal Article


Authors: Zhu, H.; Shuman, S.
Article Title: A primer-dependent polymerase function of Pseudomonas aeruginosa ATP-dependent DNA ligase (LigD)
Abstract: Pseudomonas aeruginosa encodes two putative DNA ligases: a classical NAD +-dependent DNA ligase (LigA) plus an ATP-dependent DNA ligase (LigD). LigD exemplifies a family of bacterial proteins that consist of a ligase domain fused to flanking domains that resemble nucleases and/or polymerases. Here we purify LigD and show that it possesses an intrinsic polymerase function resident within an autonomous C-terminal polymerase domain, LigD-(533-840), that flanks an autonomous DNA ligase domain, LigD-(188-527). Native LigD and the polymerase domain are both monomeric proteins. The polymerase activity is manifest in three ways: (i) non-templated nucleotide addition to a blunt-ended duplex DNA primer; (ii) non-templated addition to a single-stranded DNA primer; and (iii) templated extension of a 5′-tailed duplex DNA primer-template. The divalent cation cofactor requirement for non-templated and templated polymerase activity is satisfied by manganese or cobalt. rNTPs are preferred over dNTPs as substrates for non-templated blunt-end addition, which typically entails the incorporation of only 1 or 2 nucleotides at the primer terminus. Templated dNMP addition to a 5′-tailed substrate is efficient with respect to dNTP utilization; the primer is elongated to the end of the template strand and is then further extended with a non-templated nucleotide. The polymerase activity is abolished by alanine substitution for two aspartates (Asp-669 and Asp-671) within the putative metal-binding site. We speculate that polymerase activity is relevant to LigD function in nonhomologous end-joining.
Keywords: controlled study; unclassified drug; sequence analysis; nonhuman; dna polymerase; dna replication; protein domain; protein function; proteins; protein analysis; amino acid substitution; carboxy terminal sequence; enzyme activation; enzyme activity; structure-activity relationship; mutational analysis; bacteria (microorganisms); bacterial proteins; dna; amino acid sequence; molecular sequence data; protein purification; sequence alignment; substrate specificity; nuclease; protein structure, tertiary; binding energy; alanine; dna primers; primer dna; cobalt; protein structure; polydeoxyribonucleotide synthase; aspartic acid; enzymes; substrates; protein isolation; dna ligases; ribonucleotide; pseudomonas aeruginosa; dna-directed dna polymerase; dna template; bacteria; bacterial enzyme; manganese; pseudomonas; deoxyribonucleotide; positive ions; negibacteria; species comparison; atp dependent dna ligase; flanking domains; polymerase activity
Journal Title: Journal of Biological Chemistry
Volume: 280
Issue: 1
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 2005-01-07
Start Page: 418
End Page: 427
Language: English
DOI: 10.1074/jbc.M410110200
PUBMED: 15520014
PROVIDER: scopus
DOI/URL:
Notes: --- - "Cited By (since 1996): 28" - "Export Date: 24 October 2012" - "CODEN: JBCHA" - "Source: Scopus"
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MSK Authors
  1. Stewart H Shuman
    504 Shuman
  2. Hui P Zhu
    14 Zhu