A primer-dependent polymerase function of pseudomonas aeruginosa ATP-dependent DNA ligase (LigD).
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.
Zhu
Hui
H
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
Stewart
S
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C120640:DNA ligase (NAD); D000409:Alanine