Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase Journal Article
| Authors: | Wang, L. K.; Nair, P. A.; Shuman, S. |
| Article Title: | Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase |
| Abstract: | NAD+-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg333); penetrate the minor grove and distort the nick (Val383 and Ile384); or stabilize the OB fold (Arg379). The essential constituents of the HhH domain include: four glycines (Gly455, Gly489, Gly 521, Gly553), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg487, which penetrates the minor groove at the outer margin on the 3′-OH side of the nick; and Arg446, which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | unclassified drug; gene mutation; gene deletion; genetics; missense mutation; mutation; mutation, missense; nonhuman; molecular genetics; protein domain; genes; protein dna binding; protein stability; enzymology; mutational analysis; bacterial protein; chemistry; amines; dna; double stranded dna; amino acid sequence; molecular sequence data; sequence homology, amino acid; nucleotide sequence; escherichia coli; nucleic acids; base sequence; crystal structure; models, molecular; crystallography, x-ray; isoleucine; protein structure, tertiary; chemical structure; 3' untranslated region; dna mutational analysis; alanine; amino acids; organic acids; conformation; nucleic acid conformation; protein folding; sequence homology; zinc; x ray crystallography; polydeoxyribonucleotide synthase; bacterial dna; zinc finger protein; protein tertiary structure; genetic conservation; molecular conformation; enzymes; glycine; arginine; valine; oligonucleotides; dna ligase; dna ligases; catalytic cores; protein clamps; functional groups; functional group; modular structures; phosphodiester; nucleotidyltransferase; liga; minor grooves; zinc fingers; dna supercoiling; modular construction; brct domains; do-mains; function analysis; phosphate backbones; protein brct; protein hhh; protein liga; protein ob; polydeoxyribonucleotide synthase (atp) |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 283 |
| Issue: | 34 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2008-08-22 |
| Start Page: | 23343 |
| End Page: | 23352 |
| Language: | English |
| DOI: | 10.1074/jbc.M802945200 |
| PUBMED: | 18515356 |
| PROVIDER: | scopus |
| PMCID: | PMC2516987 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 2" - "Export Date: 17 November 2011" - "CODEN: JBCHA" - "Source: Scopus" |
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