Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+ dependent DNA ligase (LigA)
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 Nz) 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.
Nucleic acids research