Role of the first aspartate residue of the "YxDTDS" motif of phi29 DNA polymerase as a metal ligand during both TP-primed and DNA-primed DNA synthesis.

Abstract:

Almost all known nucleic acid polymerases require three acidic residues to bind the metal ion during catalysis of nucleotide incorporation. Nevertheless, recent crystallographic data on bacteriophage RB69 DNA polymerase indicate that the first aspartate residue belonging to the conserved motif "YxDTDS" could have a merely structural role. To address this question, a mutant protein at the homologous aspartate residue (Asp456) in phi29 DNA polymerase was made 3'-5' exonuclease deficient. This allowed us to analyse the functional importance of this residue in different metal-dependent reactions that can be performed using either terminal protein (TP) or DNA primers. When Mg2+ was used as the metal activator, the synthetic activities of the mutant phi29 DNA polymerase, TP-primed initiation and DNA-primed polymerisation, were about 50-fold less efficient than those of the wild-type enzyme. Interestingly, the use of Mn2+ as the metal activator partially restored the wild-type phenotype. When polymerisation required an efficient translocation along the template, mutation of Asp456 strongly affected the catalytic efficiency of phi29 DNA polymerase. The results presented here indicate that Asp456 has a catalytic role as a metal-activator ligand, but also contributes to enzyme translocation along the DNA, required during consecutive nucleotide incorporation cycles. Moreover, Asp456 appears to be critical to remodel the active site during transition from TP priming to DNA priming. The results are discussed in the light of structural information corresponding to distantly related polymerases.

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