NAD+-dependent DNA ligase encoded by a eukaryotic virus

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• NAD(+)-dependent DNA ligases are crucial for DNA repair and replication.
• Previously, these enzymes were exclusively identified in eubacteria.
• Eubacterial NAD(+) ligases share conserved domains, including a zinc-binding tetracysteine and a BRCT domain.

Purpose of the Study:

• To characterize a novel NAD(+)-dependent DNA ligase from the Amsacta moorei entomopoxvirus (AmEPV).
• To investigate the structural and functional differences between the viral ligase and its eubacterial counterparts.
• To determine the role of specific domains in the viral ligase's cofactor specificity.

Main Methods:

• Production, purification, and enzymatic characterization of AmEPV DNA ligase.
• Limited proteolysis to probe enzyme structure.
• Site-directed mutagenesis to assess the function of specific domains and residues.
• Analysis of DNA nick sealing and NAD(+) adenylylation reactions.

Main Results:

• The AmEPV ligase functions as an NAD(+)-dependent DNA ligase, independent of ATP.
• It lacks the zinc-binding tetracysteine and BRCT domains found in eubacterial ligases.
• Domain Ia, unique to NAD(+) ligases, was identified as crucial for NAD(+) cofactor specificity and adenylyl transfer.
• Specific residues within domain Ia were shown to be essential for nick sealing and NAD(+) adenylylation.

Conclusions:

• NAD(+)-dependent DNA ligases are not exclusive to eubacteria and exist in eukaryotic viral systems.
• The N-terminal domain Ia of AmEPV ligase is a key determinant of cofactor specificity.
• Domain Ia likely interacts directly with the nicotinamide mononucleotide portion of NAD(+)