Essential constituents of the 3′-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme Journal Article
| Authors: | Zhu, H.; Wang, L. K.; Shuman, S. |
| Article Title: | Essential constituents of the 3′-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme |
| Abstract: | DNA ligase D (LigD) catalyzes end-healing and end-sealing steps during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal 3′-phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at a duplex primer-template with a short 3′-ribonucleotide tract. The phosphodiesterase, which cleaves a 3′-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3′-PO 4 terminus, requires the vicinal 2′-OH of the penultimate ribose. The phosphomonoesterase converts the terminal ribonucleoside 3′-PO 4 to a 3′-OH. Here we show that the PE domain has a 3′-phosphatase activity on an all-DNA primer-template, signifying that the phosphomonoesterase reaction does not depend on a 2′-OH. The distinctions between the phosphodiesterase and phosphomonoesterase activities are underscored by the results of alanine-scanning, limited proteolysis, and deletion analysis, which show that the two reactions depend on overlapping but nonidentical ensembles of protein functional groups, including: (i) side chains essential for both ribonuclease and phosphatase activity (His-42, His-48, Asp-50, Arg-52, His-84, and Tyr-88); (ii) side chains important for 3′-phosphatase activity but not for 3′ ribonucleoside removal (Arg-14, Asp-15, Glu-21, Gin-40, and Glu-82); and (iii) side chains required selectively for the 3′-ribonuclease (Lys-66 and Arg-76). These constellations of critical residues are unique to LigD-like proteins, which we propose comprise a new bifunctional phosphoesterase family. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | unclassified drug; sequence analysis; gene deletion; scanning; nonhuman; dna polymerase; protein domain; protein function; dna damage; dna repair; phosphatase; protein degradation; enzyme activity; tyrosine; bacteria (microorganisms); rna; bacterial proteins; dna; amino acid sequence; amino acid; alanine; catalysis; dna primers; primer dna; deletion analysis; adenosine triphosphate; enzyme kinetics; molecular biology; polydeoxyribonucleotide synthase; biochemistry; glutamic acid; catalytic domain; phosphate; phosphoric monoester hydrolases; aspartic acid; dna, bacterial; reaction analysis; lysine; glycine; arginine; histidine; ribonucleases; dna ligases; dna-directed rna polymerases; functional groups; ribonuclease; pseudomonas aeruginosa; bacteria; dna ligase d; manganese; ribose; templates, genetic; proteolysis; phosphodiesterase; ribonucleoside; adenosinetriphosphate |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 280 |
| Issue: | 40 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2005-10-07 |
| Start Page: | 33707 |
| End Page: | 33715 |
| Language: | English |
| DOI: | 10.1074/jbc.M506838200 |
| PUBMED: | 16046407 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 17" - "Export Date: 24 October 2012" - "CODEN: JBCHA" - "Source: Scopus" |
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