Direct Observation of Translocation in Individual DNA Polymerase Complexes

Dahl, Joseph M.; H., Ai; Cherf, Gerald Maxwell; Jetha, Nahid N.; Garalde, Daniel R.; Marziali, Andre; Akeson, Mark; Wang, Hongyun; Lieberman, Kate R.

doi:10.1074/jbc.m111.338418

Cited by 28 publications

(115 citation statements)

References 30 publications

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“…It has recently been shown that the translocation dynamics of DNA polymerases may significantly vary between different DNA sequences (41). Two distinct patterns have been identified: (i) prolonged dwelling in either the pretranslocated or posttranslocated state (e.g., TEC C 12 and TEC A 11 , respectively) or (ii) rapid exchange between these states, accompanied by a short duration in each state (41).…”

Section: Discussionmentioning

confidence: 99%

“…Two distinct patterns have been identified: (i) prolonged dwelling in either the pretranslocated or posttranslocated state (e.g., TEC C 12 and TEC A 11 , respectively) or (ii) rapid exchange between these states, accompanied by a short duration in each state (41). Comparatively Nun-resistant TECs, such as G 9 , may belong to the latter category of rapidly translocating complexes, which have very short-lived and poorly defined translocation states, presenting Nun with a challenge to interact with either of these states.…”

Section: Discussionmentioning

confidence: 99%

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Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.T ranscription elongation is highly processive, yet the rate of nucleotide addition varies significantly for different ternary elongation complexes (TECs). In the normal elongation pathway, each nucleotide addition is followed by translocation of RNA polymerase (RNAP) along DNA in single-nucleotide increments. This process transfers the RNA 3′ end from the i + 1 site to the i site of the active center, thus allowing binding of the next NTP and subsequent phosphodiester bond formation. Translocation is thought to be a stochastic, rapid, and fully reversible process and not rate-limiting for elongation (1, 2). Instead, NTP sequestration, phosphodiester bond formation, or pyrophosphate release has been suggested to be rate-limiting for transcription elongation (3, 4). However, several reports strongly suggest that translocation may be at least partially rate-limiting for elongation (5-9).Immediately following bond formation, a catalytically inactive and highly pyrophosphorolytic (pretranslocated) state is formed. In this state, the 3′ RNA end remains in the i + 1 site of the active center. The 3′ RNA thus prevents NTP binding and generates a substrate for pyrophosphorolysis. To bind the next NTP, RNAP must translocate 1-bp forward along the DNA register to form a catalytically active and pyrophosphate-resistant (posttranslocated) state. Stationary RNAP has been suggested to "ratchet" between the two states via Brownian motion (5). NTP bound to the transient posttranslocated state acts as a pawl that interferes with backward translocation, thereby favoring the formation of a phosphodiester bond. Retention of this NTP in the posttranslocated TEC is facilitated by isomerization of the active site that blocks substrate exit, and aligns it for catalysis (10, 11)...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Addition of dNTP complementary to the templating base at n ϭ 0 (dGTP) stabilizes the lower amplitude post-translocation state ( Fig. 2A, ii) (23,25). In contrast to complexes formed with DNA1-H_H ( Fig.…”

mentioning

confidence: 98%

“…1, A and B) that permits quantification of the rates of translocation fluctuations, rates of the primer strand transfer between the polymerase and exonuclease sites, and rates of dNTP binding, in individual DNAP-DNA complexes (23)(24)(25)(26). We used the B-family replicative ⌽29 DNAP, which serves as an excellent model system for leading strand DNA synthesis catalyzed in more complex B family replisomes.…”

mentioning

confidence: 99%

“…Individual complexes reside atop the nanopore for tens of seconds, during which the measured ionic current fluctuates on the millisecond time scale between two amplitude levels (inset). Transition between the two amplitudes corresponds to movement of the DNA substrate relative to the enzyme and the nanopore by the distance of a single nucleotide (23,35,39). This spatial displacement of the DNA substrate that occurs during the translocation is detected in the time traces of ionic current by the use of a reporter group of five consecutive abasic (1Ј-H, 2Ј-H) residues in the template strand (red circles in the schematic).…”

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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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Protein-Primed Replication of Bacteriophage Φ29 DNA

Salas

Vega

2016

DNA Replication Across Taxa

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Direct Observation of Translocation in Individual DNA Polymerase Complexes

Cited by 28 publications

References 30 publications

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

Protein-Primed Replication of Bacteriophage Φ29 DNA

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