title: Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA).
abstract: Escherichia coli DNA ligase (EcoLigA) repairs 3'-OH/5'-PO4 nicks in duplex DNA via reaction of LigA with NAD(+) to form a covalent LigA-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the nick 5'-PO4 to form an AppDNA intermediate (step 2); and attack of the nick 3'-OH on AppDNA to form a 3'-5' phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA-AMP. For substrates with correctly base-paired 3'-OH/5'-PO4 nicks, kstep2 was fast (6.8-27 s(-1)) and similar to kstep3 (8.3-42 s(-1)). Absent ammonium, kstep2 and kstep3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3'-OH base mispairs and 3' N:abasic lesions, which elicited 1000- to >20000-fold decrements in kstep2. The exception was the non-canonical 3' A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3' nucleoside for catalysis of 5' adenylylation; and (ii) EcoLigA's potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5'-phosphate base mispairs and 5' N:abasic lesions.
authors: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
authors: Mathieu
authors: M
authors: Chauleau
authors: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA s-shuman@ski.mskcc.org.
authors: Stewart
authors: S
authors: Shuman
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry* / Q000378:metabolism; D000645:Ammonium Sulfate / Q000737:chemistry / Q000494:pharmacology; D020137:Base Pair Mismatch; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics; D004260:DNA Repair; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D000249:Adenosine Monophosphate; D004247:DNA; D011088:DNA Ligases; C497118:LigA protein, E coli; D000645:Ammonium Sulfate