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

Dahl, Joseph M.; Wang, Hongyun; Lázaro, José Portolés; Salas, Margarita; Lieberman, Kate R.

doi:10.1074/jbc.m113.535666

Cited by 6 publications

(35 citation statements)

References 23 publications

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“…For D12A/D66A complexes formed with DNA1-H_H in Mg 2ϩ , the dNTP binding affinity is relative to Mg 2ϩ is primarily due to an ϳ18-fold decrease in the dNTP dissociation rate, in good accord with an earlier comparison between dNTP binding parameters for the D12A/D66A mutant captured in 10 mM Mg 2ϩ or in 2 mM Mn 2ϩ (33). The more substantial enhancement in dNTP binding affinity for complexes captured in Ca 2ϩ occurs due to a combination of an ϳ9-fold increase in the dNTP association rate constant and an ϳ23-fold decrease in the dNTP dissociation rate, relative to the binding rates for complexes captured in Mg 2ϩ .…”

Section: Resultssupporting

confidence: 65%

“…The Influence of Me 2ϩ on Complementary dNTP BindingMe 2ϩ ions are essential for both dNTP binding and catalysis in the polymerase active site. The identity of the Me 2ϩ ions bound in the polymerase active site can strongly influence dNTP binding affinity and binding rates (33), as well as the fidelity of dNTP selection and the ability to catalyze phosphodiester bond formation (see Refs. 10, 42, and 43 and references therein).…”

Section: Resultsmentioning

confidence: 99%

“…Based on earlier findings that both r 1 and k off are slower for complexes captured in 2 mM Mn 2ϩ than for complexes captured in 10 mM Mg 2ϩ , we had hypothesized that Mn 2ϩ may exert its effects on both r 1 and k off by decreasing the rate of fingers opening from the closed complex (33). This hypothesis is based upon the structural model proposed for translocation (3), which predicts that decreasing the rate of fingers opening in the pre-translocation state would lead to a decrease in the rate of the forward translocation (r 1 ).…”

Section: Me 2ϩ Ions Alter the Equilibrium Across The Translocationmentioning

confidence: 99%

“…In prior studies (23,24,26,32,33), we applied these capabilities to examine the kinetic mechanisms by which mutations of highly conserved DNAP residues, alterations in DNA substrate Complexes were formed between the D12A/D66A mutant of ⌽29 DNAP and (i) DNA1-H_OH; or (ii) and (iii) DNA1-H_H. In (iii) 4 M dGTP was present in the cis chamber.…”

mentioning

confidence: 99%

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Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Dahl¹,

Lieberman²,

Wang

2016

Journal of Biological Chemistry

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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...

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Section: Resultssupporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Section: Me 2ϩ Ions Alter the Equilibrium Across The Translocationmentioning

confidence: 99%

mentioning

confidence: 99%

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Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Dahl¹,

Lieberman²,

Wang

2016

Journal of Biological Chemistry

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“…These nanopore-based experiments have also recognized enzyme-P/T binary complex and enzyme-P/T-dNTP ternary complex discrimination [43], incorrect dNTP binding [2], and pre-equilibrium kinetics [46]. Nanopore devices based on ϕ29 DNA polymerase have facilitated understanding of deoxyribose discrimination [8], substrate binding, and dynamics of translocation [7,47]. …”

Section: Current and Potential Single-molecule Sequencing Techniques mentioning

confidence: 99%

Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates

Pugliese

Weiss

2017

Current Opinion in Chemical Biology

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DNA polymerases must discriminate the correct Watson-Crick base pair-forming deoxynucleoside triphosphate (dNTP) substrate from three other dNTPs and additional triphosphates found in the cell. The rarity of misincorporations in vivo, then, belies the high tolerance for dNTP analogs observed in vitro. Advances over the last 10 years in single-molecule fluorescence and electronic detection of dNTP analog incorporation enable exploration of the mechanism and limits to base discrimination by DNA polymerases. Such studies reveal transient motions of DNA polymerase during substrate recognition and mutagenesis in the context of erroneous dNTP incorporation that can lead to evolution and genetic disease. Further improvements in time resolution and noise reduction of single-molecule studies will uncover deeper mechanistic understanding of this critical, first step in evolution.

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Kinetic Mechanisms Governing Stable Ribonucleotide Incorporation in Individual DNA Polymerase Complexes

et al. 2014

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Ribonucleoside triphosphates (rNTPs) are frequently incorporated during DNA synthesis by replicative DNA polymerases (DNAPs), and once incorporated are not efficiently edited by the DNAP exonucleolytic function. We examined the kinetic mechanisms that govern selection of complementary deoxyribonucleoside triphosphates (dNTPs) over complementary rNTPs and that govern the probability of a complementary ribonucleotide at the primer terminus escaping exonucleolytic editing and becoming stably incorporated. We studied the quantitative responses of individual Φ29 DNAP complexes to ribonucleotides using a kinetic framework, based on our prior work, in which transfer of the primer strand from the polymerase to exonuclease site occurs prior to translocation, and translocation precedes dNTP binding. We determined transition rates between the pre-translocation and post-translocation states, between the polymerase and exonuclease sites, and for dNTP or rNTP binding, with single-nucleotide spatial precision and submillisecond temporal resolution, from ionic current time traces recorded when individual DNAP complexes are held atop a nanopore in an electric field. The predominant response to the presence of a ribonucleotide in Φ29 DNAP complexes before and after covalent incorporation is significant destabilization, relative to the presence of a deoxyribonucleotide. This destabilization is manifested in the post-translocation state prior to incorporation as a substantially higher rNTP dissociation rate and manifested in the pre-translocation state after incorporation as rate increases for both primer strand transfer to the exonuclease site and the forward translocation, with the probability of editing not directly increased. In the post-translocation state, the primer terminal 2′-OH group also destabilizes dNTP binding.

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

Cited by 6 publications

References 23 publications

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates

Kinetic Mechanisms Governing Stable Ribonucleotide Incorporation in Individual DNA Polymerase Complexes

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