Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Abstract

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...

Key features of magnesium that underpin its role as the major ion for electrophilic biocatalysis

“…Our results are, consistent with previous data testing a number of Me 2+ ions with DNAPs [38][39][40][52][53][54][55][56][57]. However, in many cases we observed significant differences.…”

“…5C-F, enhanced stimulation by a Me 2+ in the absence of template was due to a significant loss of inhibition by that Me 2+ at elevated concentrations, apparently by raising the optimal Me 2+ concentration. The effect of the DNA template strand on Me 2+ -mediated cleavage might also involve an equilibrium between the 3 0 primer terminus occupying either the polymerizing or the hydrolytic site [40]. Such an equilibrium (which could depend on the identity of the Me 2+ ) is absent in complexes lacking the DNA template strand.…”

Two-Metal-Ion Catalysis: Inhibition of DNA Polymerase Activity by a Third Divalent Metal Ion

“…The fact that these metal ions bind makes it very challenging to correlate structures with kinetics, particularly in the chemical step of the pol reaction. In addition to catalysis, divalent metal ions can also modulate noncovalent kinetic behaviors ( Dahl et al, 2016 ).…”

Single-molecule nanopore enzymology

“…DNA polymerization above the nanopore revealed different states in the polymerisation reaction as the position of the abasic insert moved inside the αHL pore. Further work showed that this system can be used to reveal the details of enzymes reactions, such as the dynamics of the pre- and post-translocation steps of binary and ternary complex formation [64, 65] and the primer strand transfer from the polymerase to the exonuclease site within the enzyme [66]. Using an identical approach but with MspA, a nanopore with a narrower constriction than αHL, the Gundlach group revealed DNA polymerisation at a higher resolution [67].…”