Vaccinia DNA ligase and mammalian
DNA ligases II and III comprise a distinct subgroup of structurally homologous
enzymes within the eukaryotic
DNA ligase family. The specificity and fidelity of the viral
enzyme were investigated using purified recombinant
ligase and synthetic duplex
DNA substrates containing a single strand discontinuity.
Vaccinia ligase catalyzed efficient strand joining on nicked DNAs in the presence of
magnesium and
ATP (Km = 95 microM). dATP,
ITP,
AMPPCP, 3'dATP, and
ATP alpha S could not substitute for
ATP; of these, 3'dATP and
ATP alpha S were inhibitors of
ligation. The
vaccinia enzyme was unable to seal strands across a 1 nt (
nucleotide) or 2 nt gap.
Ligase action at a 1 nt gap resulted in accumulation of high levels of the normally undetectable
DNA-adenylate reaction intermediate. In contrast, no
DNA-adenylate was formed at a 2 nt gap. A native gel mobility shift assay showed that
vaccinia DNA ligase was capable of discriminating between nicked and gapped DNAs at the substrate binding step. The
ligase was fairly tolerant of mismatches at a nick involving the 5'
phosphate donor terminus but was inhibited strongly by mismatches at the 3'
OH acceptor terminus, especially by
purine.-
purine mispairs. These findings underscore the importance of a proper 3'
OH terminus in substrate recognition and reaction chemistry but also raise the possibility that
ligase may generate mutations during DNA repair by sealing
DNA molecules with mispaired ends.
Ligase was inhibited by several
DNA binding drugs, including, in order of decreasing potency,
distamycin,
ethidium bromide, and
actinomycin. Strand joining by purified
ligase was not affected by
etoposide, a
drug that inhibits vaccinia virus replication in vivo and which depends on the presence of
vaccinia ligase for its
antiviral action.