Characterization of Escherichia coli Translesion Synthesis Polymerases and Their Accessory Factors

Beuning, Penny J.; Simon, Sharotka M.; Godoy, Veronica G.; Jarosz, Daniel F.; Walker, Graham C.

doi:10.1016/s0076-6879(06)08020-7

Cited by 49 publications

(99 citation statements)

References 75 publications

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“…Primer extension assays were carried out as described previously58 using 32P‐labeled primers annealed to 61‐mer template. Reactions contained a final concentration of 25 nM DNA polymerase, 100 nM primer/template DNA, 100 µM dNTPs, 7.5 mM MgSO 4 , 30 mM HEPES (pH 7.5), 20 mM NaCl, 2 mM DTT, 1% (w/v) bovine serum albumin, and 4% glycerol.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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“…Purification of UmuDЈ 2, UmuD2, and UmuD(F94C)2 (49) and cross-linking (50) were performed as previously described. A plasmid encoding UmuD(F94C) was produced from pSG5 by using the Stratagene QuikChange site-directed mutagenesis kit (49). Protein concentration was determined by using the Bio-Rad protein assay.…”

Section: Methodsmentioning

confidence: 99%

“…Protein concentration was determined by using the Bio-Rad protein assay. DinB was a kind gift from Daniel Jarosz (49). ClpXP and the ␤ subunit of Pol III were generously provided by the Baker Laboratory at Massachusetts Institute of Technology (11) and the Beuning Laboratory at Northeastern University (15), respectively.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Simon

Sousa²,

Mohana‐Borges³

et al. 2008

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

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Products of the umuD gene in Escherichia coli play key roles in coordinating the switch from accurate DNA repair to mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD 2 is up-regulated 10-fold immediately after damage, after which slow autocleavage removes the Nterminal 24 amino acids of each UmuD. The remaining fragment, UmuD 2, is required for mutagenic TLS. The small proteins UmuD2 and UmuD 2 make a large number of specific protein-protein contacts, including three of the five known E. coli DNA polymerases, parts of the replication machinery, and RecA recombinase. We show that, despite forming stable homodimers, UmuD 2 and UmuD 2 have circular dichroism (CD) spectra with almost no ␣-helix or ␤-sheet signal at physiological concentrations in vitro. High protein concentrations, osmolytic crowding agents, and specific interactions with a partner protein can produce CD spectra that resemble the expected ␤-sheet signature. A lack of secondary structure in vitro is characteristic of intrinsically disordered proteins (IDPs), many of which act as regulators. A stable homodimer that lacks significant secondary structure is unusual but not unprecedented. Furthermore, previous single-cysteine cross-linking studies of UmuD 2 and UmuD 2 show that they have a nonrandom structure at physiologically relevant concentrations in vitro. Our results offer insights into structural characteristics of relatively poorly understood IDPs and provide a model for how the umuD gene products can regulate diverse aspects of the bacterial SOS response.natively unfolded ͉ SOS response ͉ unstructured ͉ denatured ͉ DNA repair

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“…DinB was purified as described previously by Beuning et al [Beuning et al, 2006] and stored in single-use aliquots at 2808C. The DNA template containing a single N 2 -furfuryl-dG adduct was prepared as described [DeCorte et al, 1996;Jarosz et al, 2006].…”

Section: Experimental Methodsmentioning

confidence: 99%

“…DNA primer (standing start or MatchC) and the template containing N 2 -furfuryl-dG were combined to a final ratio of 1:1 (100 nM) and annealed in annealing buffer [20 mM Hepes (pH 7.5) and 5 mM Mg(OAc) 2 ] by heating for 2 min at 958C, incubating at 508C for 60 min, and then cooling to 378C. The reactions were carried out with 100 nM 32 P-end-labeled primer/template in a reaction buffer containing final concentrations of 30 mM Hepes (pH 7.5), 20 mM NaCl, 7.5 mM MgSO 4 , 2 mM b-mercaptoethanol, 1% bovine serum albumin, and 4% glycerol [Beuning et al, 2006]. Experiments were carried out with dNTP concentrations ranging from 1 lM-5000 lM, unless otherwise noted, and DinB concentration varied based on the extent of activity of each variant.…”

Section: Experimental Methodsmentioning

confidence: 99%

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

Walsh

Parasuram

Rajput

et al. 2012

Environ and Mol Mutagen

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DinB is one of two Y family polymerases in E. coli and is involved in copying damaged DNA. DinB is specialized to bypass deoxyguanosine adducts that occur at the N 2 position, with its cognate lesion being the furfuryl adduct. Active site residues have been identified that make contact with the substrate and carry out deoxynucleotide triphosphate (dNTP) addition to the growing DNA strand. In DNA polymerases, these include negatively charged aspartate and glutamate residues (D8, D103, and E104 in E. coli DNA polymerase IV DinB). These residues position the essential magnesium ions correctly to facilitate nucleophilic attack by the primer hydroxyl group on the a-phosphate group of the incoming dNTP. To study the contribution of DinB residues to lesion bypass, the computational methods THEMATICS and POOL were employed. These methods correctly predict the known active site residues, as well as other residues known to be important for activity. In addition, these methods predict other residues involved in substrate binding as well as more remote residues. DinB variants with mutations at the predicted positions were constructed and assayed for bypass of the N 2 -furfuryl-dG lesion. We find a wide range of effects of predicted residues, including some mutations that abolish damage bypass. Moreover, most of the DinB variants constructed are unable to carry out the extension step of lesion bypass. The use of computational prediction methods represents another tool that will lead to a more complete understanding of translesion DNA synthesis. Environ. Mol. Mutagen. 53:766-776, 2012. V V C 2012 Wiley Periodicals, Inc.

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Characterization of Escherichia coli Translesion Synthesis Polymerases and Their Accessory Factors

Cited by 49 publications

References 75 publications

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

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

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

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