The bacteriophage ϕ29 DNA polymerase

Replicative DNA polymerases (DNAPs) require divalent metal cations for phosphodiester bond formation in the polymerase site and for hydrolytic editing in the exonuclease site. Me 2؉ ions are intimate architectural components of each active site, where they are coordinated by a conserved set of amino acids and functional groups of the reaction substrates. DNA polymerases (DNAPs) 4 are responsible for accurate replication of DNA genomes. Replicative DNAPs achieve this by catalyzing template-directed polymerization of deoxyribonucleoside triphosphates (dNTPs) with extremely high fidelity (with error rates of ϳ10 Ϫ6 to ϳ10 Ϫ8 ) (1, 2). Phosphodiester bond formation, the chemical transformation during polymerization, is catalyzed in the polymerase active site, in the 5Ј to 3Ј direction. Many replicative DNAPs also catalyze a second nucleotidyl transfer reaction, the exonucleolytic cleavage of the primer strand in the 3Ј to 5Ј direction. This editing reaction allows for removal of incorrect nucleotides inserted during polymerization, contributing ϳ1-2 orders of magnitude to replication fidelity (1). Exonucleolytic editing occurs in a separate active site that is typically ϳ30 -40 Å from the polymerase site (3-9), and it requires that ϳ3 base pairs of the nascent primertemplate duplex be melted to allow the primer strand to be transferred from the polymerase site to the exonuclease site. An essential role for two divalent metal cations (Me 2ϩ ions) in the mechanism of numerous enzyme-catalyzed nucleotidyl transfer reactions has been characterized (10), in which one Me 2ϩ ion (termed metal A) serves primarily to activate the nucleophile, whereas the other (termed metal B) mitigates the negative charge that builds in the transition state (10, 11). For replicative DNAPs, this two Me 2ϩ ion mechanism applies to both phosphodiester bond formation in the polymerase site, and to hydrolysis of the primer terminal dNMP residue in the exonuclease site (12, 13). In accord with their roles in the chemical transformations, the Me 2ϩ ions are intimate architectural components of each of the active sites. They are coordinated by a highly conserved set of acidic amino acid side chains in each site, as well as by specific functional groups of the reaction substrates. Therefore, in addition to their essential role in the chemical reactions, Me 2ϩ ions may also influence the reversible noncovalent transitions that govern the fate of DNAP-DNA complexes after each covalent nucleotide addition. These transitions include (i) the primer strand transfer between the polymerase and exonuclease sites, (ii) the translocation fluctuations, in which the DNA substrate moves between the pretranslocation and post-translocation states in the DNAP polymerase site, a spatial displacement of the distance of a single nucleotide, and (iii) dNTP binding in the polymerase site. In contrast to the roles of the Me 2ϩ ions in the chemical steps of dNTP polymerization and exonucleolysis, much less is known about the effects of Me 2ϩ ions on these noncovalent trans...

mentioning

confidence: 99%

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Dahl¹,

Lieberman²,

Wang

2016

“…The B-family DNA polymerase from bacteriophage phi29 contains 5Ј-3Ј-polymerase and 3Ј-5Ј-exonuclease functions within a single ϳ66.5-kDa protein chain (17,18). This polymerase catalyzes the processive replication of tens of kilobases of DNA in vitro without the need for accessory proteins such as sliding clamps or helicases.…”

mentioning

confidence: 99%

Direct Observation of Translocation in Individual DNA Polymerase Complexes

Dahl

Cherf

et al. 2012

110

Background: DNA polymerases translocate along DNA by one nucleotide in each catalytic cycle. Results: The DNA polymerase translocation step is observed with single nucleotide and submillisecond precision. Conclusion: DNA polymerase complexes fluctuate between pre-and post-translocation states and are rectified to the posttranslocation state by dNTP. Significance: These results provide insight into the translocation mechanism and its integration into the DNA polymerase catalytic pathway.

“…The DNAP from the bacteriophage ⌽29 is a B-family polymerase that catalyzes highly processive DNA synthesis (1)(2)(3), without the need for accessory proteins, such as sliding clamps or helicases, because it remains tightly associated with its DNA substrate and promotes downstream strand displacement during replication (1,4,5). In addition to its 5Ј-3Ј polymerase active site, ⌽29 DNAP has a 3Ј-5Ј exonuclease active site, located in a separate domain of the protein, ϳ30 Å from the polymerase active site (2)(3)(4)6).…”

mentioning

confidence: 99%

Dynamics of Translocation and Substrate Binding in Individual Complexes Formed with Active Site Mutants of Φ29 DNA Polymerase

Dahl

Wang

Lázaro

et al. 2014

Self Cite

Background: Tyr-226 and Tyr-390 in the ⌽29 DNA polymerase active site are implicated in the mechanism of translocation. Results: Y226F and Y390F differ in their effects on translocation and on dNTP and pyrophosphate binding. Conclusion: Mutations in the ⌽29 DNA polymerase and exonuclease active sites perturb dNTP or pyrophosphate binding rates. Significance: DNA polymerase architecture is finely tuned to integrate translocation and substrate binding.