Novel 3′-ribonuclease and 3′-phosphatase activities of the bacterial non-homologous end-joining protein, DNA ligase D Journal Article
| Authors: | Zhu, H.; Shuman, S. |
| Article Title: | Novel 3′-ribonuclease and 3′-phosphatase activities of the bacterial non-homologous end-joining protein, DNA ligase D |
| Abstract: | Pseudomonas aeruginosa DNA ligase D (PaeLigD) exemplifies a family of bacterial DNA end-joining proteins that consist of a ligase domain fused to a polymerase domain and a putative nuclease module. The LigD polymerase preferentially adds single ribonucleotides at blunt DNA ends and, as we show here, is also capable of adding up to 4 ribonucleotides to a DNA primer-template. We report that PaeLigD has an intrinsic ability to resect the short tract of 3′-ribonucleotides of a primer-template substrate to the point at which the primer strand has a single 3′-ribonucleotide remaining. The failure to digest beyond this point reflects a requirement for a 2′-OH group on the penultimate nucleoside of the primer strand. Replacing the 2′-OH by a 2′-F, 2′-NH 2, 2′-OCH 3, or 2′-H abolishes the resection reaction. The ribonucleotide resection activity resides within a 187-amino acid N-terminal nuclease domain and is the result of at least two component steps: (i) the 3′-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3′-PO 4 terminus, and (ii) the 3′-PO 4 is hydrolyzed to a 3′-OH. The 3′-ribonuclease and 3′-phosphatase activities are both dependent on a divalent cation, specifically manganese. PaeLigD preferentially remodels the 3′-ends of a duplex primer-template substrate rather than a single strand of identical composition, and it prefers DNA primer strands containing a short 3′-ribonucleotide tract to an all-RNA primer. The nuclease domain of PaeLigD and its bacterial homologs has no apparent structural or mechanistic similarity to previously characterized nucleases. Thus, we surmise that it exemplifies a novel phosphoesterase family, defined in part by conserved residues Asp-50, Arg-52, and His-84, which are essential for the 3′-ribonuclease and 3′-phosphatase reactions. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. American Cancer Society Research. |
| Keywords: | unclassified drug; mutation; dose response; nonhuman; molecular genetics; protein domain; phosphatase; enzymology; dose-response relationship, drug; enzyme activity; physiology; time; time factors; rna; amine; bacterial protein; chemistry; bacterial proteins; dna; double stranded dna; dna modification; amino acid sequence; molecular sequence data; sequence homology, amino acid; amino terminal sequence; kinetics; nucleotide sequence; polymerization; nuclease; base sequence; protein structure, tertiary; dna mutational analysis; alanine; dna primers; primer dna; sequence homology; polydeoxyribonucleotide synthase; enzyme structure; bacterial dna; hydrolysis; phosphoric monoester hydrolases; protein tertiary structure; aspartic acid; centrifugation, density gradient; enzymes; arginine; histidine; ribonucleases; nucleoside; polyacrylamide gel electrophoresis; electrophoresis, polyacrylamide gel; archaeal proteins; dna ligase; dna ligases; dna directed rna polymerase; dna-directed rna polymerases; functional group; ribonuclease; hydrogen; ribonucleotide; pseudomonas aeruginosa; bacteria; glycerol; hydroxyl group; fluorine; archaeal protein; manganese; ribonucleotides; reaction kinetics; polydeoxyribonucleotide synthase (atp); density gradient centrifugation; nucleotide metabolism; positive ions; polydeoxyribonucleotide synthase d; nucleoside 3' phosphatase; primer rna; end-joining proteins; putative nuclease module; 3' ribonuclease; methoxy group |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 280 |
| Issue: | 28 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2005-07-15 |
| Start Page: | 25973 |
| End Page: | 25981 |
| Language: | English |
| DOI: | 10.1074/jbc.M504002200 |
| PROVIDER: | scopus |
| PUBMED: | 15897197 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 20" - "Export Date: 24 October 2012" - "CODEN: JBCHA" - "Source: Scopus" |
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