Structural and functional studies on ø29 DNA polymerase

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Abstract
TheBacillus subtilis phage ø29 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 ø29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of ø29 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 ø29 DNA ends, by means of a “sliding-back” mechanism, could also contribute to increase the fidelity of ø29 DNA replication.

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