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J. Biol. Chem., Vol. 276, Issue 39, 36100-36109, September 28, 2001

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NAD⁺-dependent DNA Ligase Encoded by a Eukaryotic Virus^*

Verl Sriskanda, Richard W. Moyer^§, and Stewart Shuman^¶

From the  Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the ^§ Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610

We report the production, purification, and characterization of an NAD⁺-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD⁺ ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD⁺ ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD⁺. Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD⁺. Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD⁺ ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO₄ nicks and the reaction with NAD⁺ to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr³⁹, Tyr⁴⁰, Asp⁴⁸, and Asp⁵²) or abolished (Tyr⁵¹) sealing of a 5'-PO₄ nick and adenylyl transfer from NAD⁺ without affecting ligation of DNA-adenylate. We conclude that: (i) NAD⁺-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD⁺.

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