Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2002
Publication Date: N/A
Citation: N/A Interpretive Summary: Melanoplus sanguinipes entomopoxvirus (MsEPV) is an ancestor of modern poxviruses and a potential grasshopper bio-control agent. Understanding MsEPV survival strategies and interactions with the host are important to develop new grasshopper bio-control strategies and they provide insight on other poxviral-host interaction mechanisms. Here we demonstrate that an open reading frame contained in the MsEPV genome is a functional NAD-dependent DNA ligase and suggest that it may be part of DNA repair pathway. The presence of a functional NAD ligase in eukaryotic system has not previously been described and this gene represent a new class of NAD ligase which is structurally different from all known eubacterial NAD ligases.
Technical Abstract: Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial NAD dependent DNA ligases (Afonso et al: J. of Virology, 73: 533). This 522 amino acid open reading frame from the eukaryotic virus contains all conserved nucleotidyl transferase motif but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD ligases. This putative DNA ligase, combined with a uracil glycosylase and an AP endonuclease DNA polymerase beta fusion protein, may constitute a uracil base excision repair pathway in MsEPV. In this work, the ligation properties of this new class of ligase are investigated. The cloned MsEPV ligase seals DNA nicks in an NAD dependent fashion. ATP can not serve as adenylation cofactor. The metal cofactor requirement can be satisfied by Mg2+ or Mn2+. MsEPV ligase seals sticky ends efficiently, but has little activity on I nucleotide gap or blunt ended DNA substrates even in the presence of polyethylene glycol. The ligation fidelity discriminating against mismatched DNA substrates at either 3' or 5' side of the nick is low. However, A templated mismatches (A/A, G/A and C/A) are not ligated. Like T4 DNA ligase, MsEPV DNA ligase is able to ligate DNA probes on RNA template. T4 DNA ligase shows no detectable mismatch ligation at the 3' side of the nick but substantial 5' T/G mismatch ligation on RNA template. On the contrary, MsEPV ligase is quite tolerant of mismatches located at the 3' side of the nick. but is severely inhibited by mismatches located at the 5' side of the nick. These RNA-templated ligation properties have implications in allele specific detection in RNA transcripts.