Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus.

ABSTRACT: An NAD(+)-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg(2+)or Mn(2+)as the metal cofactor. Ca(2+)and Ni(2+)mainly support formation of DNA-adenylate intermediates. The catalytic cycle is characterized by a low k (cat)value of 2 min(-1)with concomitant accumulation of the DNA - adenylate intermediate when Mg(2+)is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A < or = G/C < C/G < A/T on both the 3'- and 5'-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mism

SUBMITTER: Tong J 

PROVIDER: S-EPMC111035 | biostudies-literature | 2000 Mar

REPOSITORIES: biostudies-literature