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

2000; Oxford University Press; Volume: 28; Issue: 6 Linguagem: Inglês

10.1093/nar/28.6.1447

1362-4962

Janelle Tong,

DNA and Nucleic Acid Chemistry

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 Mg2+ or Mn2+ as the metal cofactor. Ca2+ and Ni2+ mainly support formation of DNA–adenylate intermediates. The catalytic cycle is characterized by a low kcat value of 2 min–1 with concomitant accumulation of the DNA–adenylate intermediate when Mg2+ 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 ≤ 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 mismatched base pair on the 3′-side of the nick junction. The requirement of 3′ complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3′- or 5′-side of the nick. Furthermore, most of the unligatable 3′ mismatched base pairs prohibit formation of the DNA–adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complemen­tarity on one side of the nick affects substrate adenylation on the 5′-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.