文摘
The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD+)-dependent DNA ligases [J. Virol. 73 (1999) 533]. This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD+ ligases. Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD+-dependent fashion, while adenosine 5′-monophosphate (ATP) cannot serve as an adenylation cofactor. The ligation activity of MsEPV ligase requires Mg2+ or Mn2+. MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol. In comparison, bacterial NAD+-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol. MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A). MsEPV NAD+-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD+ DNA ligases. T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3′ side of the nick but substantial 5′ T/G mismatch ligation on an RNA template. In contrast, MsEPV ligase joins mismatches at the 3′ side of the nick more frequently than at the 5′ side of the nick on an RNA template. The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts.
