| Abstract: | TheBacillus subtilis phage ?29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences
common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance
of a C-terminal conserved region, represented by the Lys-X-Tyr (“K-Y”) motif. Single point mutants have been constructed and
the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated
that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant
Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein
as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates
initiation of ?29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about
5-fold) the insertion fidelity of ?29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a
model in which the special strategy to maintain the integrity of the ?29 DNA ends, by means of a “sliding-back” mechanism,
could also contribute to increase the fidelity of ?29 DNA replication. |
