Kinetic analysis of DNA strand joining by Chlorella virus DNA ligase and the role of nucleotidyltransferase motif VI in ligase adenylylation.

ABSTRACT: Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3'-OH and 5'-PO(4) termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PP(i) and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5'-phosphate to form DNA-adenylate; 3) the 3'-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg(2+). Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (k(step3) = 25 s(-1)) exceeds that for DNA adenylylation (k(step2) = 2.4 s(-1)) and that Mg(2+) binds with similar affinity during step 2 (K(d) = 0.77 mM) and step 3 (K(d) = 0.87 mM). The rates of

SUBMITTER: Samai P 

PROVIDER: S-EPMC3436572 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature