Substrate specificity and structure-function analysis of the 3′-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D Journal Article


Authors: Zhu, H.; Shuman, S.
Article Title: Substrate specificity and structure-function analysis of the 3′-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D
Abstract: DNA ligase D (LigD) performs end remodeling and end sealing reactions 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 phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3′ end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3′-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3′-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3′-PO4 or deoxyribonucleoside 3′-PO4 of a primer-template to a 3′-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5′ single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2′-OH of the penultimate ribose, it is indifferent to a 2′-OH versus a 2′-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2′-OH is moved by 1 nucleotide in the 5′ direction, suggesting that LigD is an exoribonuclease that cleaves the 3′-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg 52, His84, and Tyr88, which are essential for both the ribonuclease and 3′-phosphatase activities; (ii) Arg 14, Asp15, Glu21, and Glu82, which are critical for 3′-phosphatase activity but not 3′-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3′-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords: controlled study; unclassified drug; gene mutation; mutation; nonhuman; protein function; amino acid substitution; phosphatase; enzyme activity; structure-activity relationship; tyrosine; bacteria (microorganisms); bacterial protein; dna; molecular sequence data; kinetics; enzyme analysis; substrate specificity; base sequence; protein structure, tertiary; dna mutational analysis; dna primers; amino acids; molecular biology; polydeoxyribonucleotide synthase; enzyme specificity; enzyme structure; aspartic acid; enzymes; lysine; substrates; arginine; histidine; dna ligases; ribonuclease; pseudomonas aeruginosa; dna template; bacteria; ribose; phosphodiesterase; phosphoric diester hydrolases; polydeoxyribonucleotide synthase d; exoribonuclease; metal binding; dna ligase d (ligd); phosphoesterase (pe); phosphomonoesterase reactions; protein nonhomologous end joining
Journal Title: Journal of Biological Chemistry
Volume: 281
Issue: 20
ISSN: 0021-9258
Publisher: American Society for Biochemistry and Molecular Biology  
Date Published: 2006-05-19
Start Page: 13873
End Page: 13881
Language: English
DOI: 10.1074/jbc.M600055200
PUBMED: 16540477
PROVIDER: scopus
DOI/URL:
Notes: --- - "Cited By (since 1996): 14" - "Export Date: 4 June 2012" - "CODEN: JBCHA" - "Source: Scopus"
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MSK Authors
  1. Stewart H Shuman
    504 Shuman
  2. Hui P Zhu
    14 Zhu