Distinct Double- and Single-Stranded DNA Binding of <i>E. coli</i> Replicative DNA Polymerase III α Subunit

McCauley, Micah J.; Shokri, Leila; Sefcikova, Jana; Venclovas, C̆eslovas; Beuning, Penny J.; Williams, Mark C.

doi:10.1021/cb8001107

Cited by 32 publications

(51 citation statements)

References 49 publications

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“…Because OB folds commonly bind to ssDNA, a proposal was made that the OB fold could be part of the sensing network 25, 26 . Consistent with this hypothesis, the ssDNA-binding portion of Pol III was localized to a C-terminal region of α that contains the OB-fold element 34 . A test of the importance of the OB-fold motif was made using a mutant in which three basic residues located in the β1-β2 loop were changed to serine 11 .…”

Section: Discussionmentioning

confidence: 53%

The Rate of Polymerase Release upon Filling the Gap between Okazaki Fragments Is Inadequate to Support Cycling during Lagging Strand Synthesis

Dohrmann

Manhart

Downey

et al. 2011

Journal of Molecular Biology

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Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 second to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5′-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 minutes. Reduction of gap size to one nucleotide increased the rate of dissociation 2.5-fold and complete filling of the gap increased the off rate an additional three-fold (t½ ∼ 2 min). An exogenous primed template and ATP accelerated dissociation an additional four-fold in a reaction that required complete filling of the gap. Neither a 5′-triphosphate nor 5′-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nucleotide) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-crosslinking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating it serves as the sensor.

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Section: Discussionmentioning

confidence: 53%

The Rate of Polymerase Release upon Filling the Gap between Okazaki Fragments Is Inadequate to Support Cycling during Lagging Strand Synthesis

Dohrmann

Manhart

Downey

et al. 2011

Journal of Molecular Biology

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“…In contrast, ε by itself has been shown to be an ssDNA exonuclease, and the catalytic activity of ε is similar on ssDNA and mispaired primer termini. The ε subunit preferentially binds ssDNA, whereas α binds both ssDNA and dsDNA and prefers a primer‐template junction 30, 54. In addition, a recent NMR study showed that a primer destabilized due to mismatches increases the propensity of the mismatch to reach the ε subunit, enabling ε to correct for the mismatches in a passive manner 55.…”

Section: Discussionmentioning

confidence: 99%

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

et al. 2017

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Pol III core is the three‐subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two‐step reaction scheme in which pol III core binds bimolecularly to the primer‐template junction, then converts at rate k 2 into an exo‐competent conformation. Fits to the force dependence of k init show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature‐ and mismatch‐dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.

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“…The full-length crystal structure of Thermus aquaticus Pol III reveals similarities to E. coli Pol III [286], and a recent structure of the T. aquaticus α subunit shows the OB fold bound to DNA [290]. Single molecule DNA stretching experiments characterized the dsDNA and ssDNA binding activity of E. coli Pol III α, quantifying binding to both forms of DNA by distinct regions of the protein [291]. …”

Section: Proteins That Bind Both Double and Single-stranded Dnamentioning

confidence: 99%

Biophysical characterization of DNA binding from single molecule force measurements

Chaurasiya

Paramanathan

McCauley

et al. 2010

Physics of Life Reviews

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166

149

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Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

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Distinct Double- and Single-Stranded DNA Binding of E. coli Replicative DNA Polymerase III α Subunit

Cited by 32 publications

References 49 publications

The Rate of Polymerase Release upon Filling the Gap between Okazaki Fragments Is Inadequate to Support Cycling during Lagging Strand Synthesis

The Rate of Polymerase Release upon Filling the Gap between Okazaki Fragments Is Inadequate to Support Cycling during Lagging Strand Synthesis

Single‐molecule mechanochemical characterization of E. coli pol III core catalytic activity

Biophysical characterization of DNA binding from single molecule force measurements

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