Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA) Journal Article

Authors: Chauleau, M.; Shuman, S.
Article 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. © 2016 The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Journal Title: Nucleic Acids Research
Volume: 44
Issue: 5
ISSN: 0305-1048
Publisher: Oxford University Press  
Date Published: 2016-03-18
Start Page: 2298
End Page: 2309
Language: English
DOI: 10.1093/nar/gkw049
PROVIDER: scopus
PMCID: PMC4797296
PUBMED: 26857547
Notes: Article -- Export Date: 2 May 2016 -- Source: Scopus
Citation Impact
MSK Authors
  1. Stewart H Shuman
    514 Shuman