Abstract
Bacteriophage ϕ29 DNA polymerase has two activities: DNA polymerization and 3′-5′ exonucleolysis governed by catalytic sites present in two structurally distant domains. These domains must work together to allow the correct replication of the template and to prevent the accumulation of errors in the newly synthesized DNA strand. ϕ29 DNA polymerase is endowed with a high processivity and strand displacement capacity together with a high fidelity. Previous studies of its crystallographic structure suggested possible interactions of residues of the exonuclease domain like the Gln180 with the fingers subdomain, or water mediated and direct hydrogen bond by the polar groups of residues Tyr101 and Thr189 that could stabilize DNA binding. To analyse their functional importance for the exonuclease activity of ϕ29 DNA polymerase we engineered mutations to encode amino acid substitutions. Our results confirm that both residues, Tyr101 and Thr189 are involved in the 3′-5′ exonuclease activity and in binding the dsDNA. In addition, Tyr101 is playing a role in processivity and Thr189 is an important determinant in the fidelity of the DNA polymerase. On the other hand, the biochemical characterization of the mutant derivatives of residue Gln180 showed how the mutations introduced enhanced the 3′-5′ exonuclease activity of the enzyme. A potential structural conformation prone to degrade the substrate is discussed.
Highlights
Φ29 DNA polymerase is the only member of the protein-primed subgroup of DNA polymerases whose structure has been solved
When an incorrect nucleotide is incorporated, the 3′ terminus of the primer must be physically moved from the polymerase to the exonuclease active site for the removal of the misinserted nucleotide, and the corrected primer returns to the polymerization active site[12]
Previous results showed that φ29 DNA polymerase edits the polymerization errors using an intramolecular pathway, so the primer terminus moves from one active site to the other without dissociation from the DNA14
Summary
Φ29 DNA polymerase is the only member of the protein-primed subgroup of DNA polymerases whose structure has been solved. Inspection of the binary and ternary complexes of φ29 DNA polymerase shows how in the open conformation the fingers residue Leu[381] is close to the exonuclease domain residue Gln[180] (see Fig. 1B), allowing the concomitant movement of the exonuclease domain towards the polymerization one, a situation in which the exonuclease active site is catalytically competent Such scenario could be modified once the fingers subdomain rotates into the closed catalytically competent polymerization conformation, promoting the exonuclease domain to adopt a more “opened” and maybe non-catalytically competent position respect to the polymerase domain. After the incorporation of an incorrect nucleotide, the primer terminus has to move from the polymerization to the exonuclease active site to degrade only the misincorporated nucleotide and come back again to the polymerization active site to continue with the elongation of the primer In this process, several interactions between the polymerase and the DNA must be broken and further reestablished to allow the polymerase to continue with the replication after the removal of a wrong nucleotide. The study of three residues of the exonuclease domain (Tyr[101], Gln[180] and Thr189) and their interactions with the DNA and with other residues of the polymerase allow us to improve the understanding of the molecular bases that govern the coordination between the exonuclease and polymerization activities
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