Treatment of T7 DNA ligase with a range of proteases generates two maj
or fragments which are resistant to further digestion, These fragments
, of molecular weight 16 and 26 kDa, are derived from the N- and C-ter
mini of the protein, respectively, The presence of ATP or a non-hydrol
ysable analogue, ADPNP, during limited proteolysis greatly reduces the
level of digestion, The N-terminal 16 kDa region of the intact T7 lig
ase is labelled selectively in the presence of [alpha-P-32]ATP, confir
ming that it contains the active site lysine residue, In common with t
he intact enzyme, the C-terminal portion of the protein retains the ab
ility to band shift DNA fragments of various lengths, implicating it i
n DNA binding, It can also inhibit ligation by the intact protein, app
arently by competing for target sites on DNA, We conclude that the N-t
erminal region, which contains the putative active site lysine, plays
a role in the transfer of AMP from the enzyme-adenylate complex to the
5' phosphate at the nick site, while the C-terminal 26 kDa fragment a
ppears to position the enzyme at the target site on DNA.