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Nucleic Acids Research, 2000, Vol. 28, No. 6 1447-1454
© 2000 Oxford University Press

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

Jie Tong, Francis Barany and Weiguo Cao*

Department of Microbiology and Immunology, Hearst Microbiology Research Center and Strang Cancer Prevention Center, The Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, Box 62, New York, NY 10021, USA

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.

* To whom correspondence should be addressed. Tel: +1 212 746 6517; Fax: +1 212 746 8587; Email: wgc@mail.med.cornell.edu


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