An Efficient Method for the Preparation of Long Heteroduplex DNA as Substrate for Mismatch Repair by the Escherichia coli MutHLS System

Thomas, Evangelos; Pingoud, Alfred; Friedhoff, Peter

doi:10.1515/bc.2002.166

Cited by 18 publications

(18 citation statements)

References 13 publications

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“…DNA Substrates-Linear DNA substrates (484 bp with or without a GT mismatch) were generated from two 484-bp PCR products amplified by Pfu DNA polymerase with a 5Ј-phosphate at the top or the bottom strand, respectively, using plasmids pET-15b-XhoI (with a GC base pair) and/or pET-15b-HindIII (with an AT base pair), respectively, with primers (BBseq A302/pA302, 5Ј-ATC TTC CCC ATC GGT GAT GTC-3Ј; and BBseq B111/p111, 5Ј-TCA TCC TCG GCA CCG TCA C-3Ј) essentially as described previously (33). The phosphorylated strands were digested by -exonuclease, and the top strand (containing a G at position 385) was annealed to the bottom strand (containing a C or a T at position 385, respectively) to give either a 484-bp homoduplex (GC484) or heteroduplex (GT484), respectively.…”

Section: Methodsmentioning

confidence: 99%

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

Winkler

Marx

Larivière³

et al. 2011

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

Winkler

Marx

Larivière³

et al. 2011

Journal of Biological Chemistry

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“…Run-off transcription yields a 113 nt RNA product. Open templates contained mismatches at position -9, -10 and -11 ("open upstream") or -2, -1 and +1 ("open start") relative to the transcription start site in gdh-C20 and were prepared as reported 20 . Briefly, mutations were introduced by fusion PCR.…”

Section: P Furiosus Transcription Assaysmentioning

confidence: 99%

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

Kostrewa

Zeller

Armache

et al. 2009

Nature

279

423

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Preparation of yeast proteinsEndogenous S. cerevisiae ten-subunit Pol II core enzyme was prepared as described 1 . An E. coli expression vector was derived from pET21b (Novagen) for the coexpression of the translational fusion of S. cerevisiae Rpb4:20 glycine linker:TFIIB and Rpb7:His 6 under the control of separate T7 promoters. Details of the vector design are available on request. Following expression in E. coli, cells were lysed by sonication in buffer A (50 mM Tris, 150 mM NaCl; pH 7.5, 0.3 mg/L leupeptin, 1.4 mg/L pepstatin A, 0.17 g/L PMSF, 0.33 g/L benzamidine and 10 mM β -mercaptoethanol). The lysate was cleared by centrifugation and applied to a Ni-NTA agarose column (Qiagen). The column was washed with buffer A containing 2 M NaCl, and the protein was eluted with a gradient of 10 mM to 200 mM imidazole in buffer A containing 150 mM NaCl. Peak fractions were diluted twofold and loaded onto a Mono-S cation exchange column (Amersham) equilibrated with buffer A containing 100 mM NaCl. The fusion protein was eluted over a total of 15 column volumes with a gradient of 0.1-1 M NaCl in buffer A. Peak fractions were concentrated and applied to a Superose 6 gel filtration column (Amersham) equilibrated with buffer B (5 mM HEPES pH 7.25, 40 mM ammonium sulfate, 10 μM ZnCl 2 , 10 mM DTT). Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. The TBP core domain (S. cerevisiae residues 61-240) expression vector was a generous gift from Dr. Sean Juo. Expression and purification of the yeast TBP core domain was as described 2 except that Superose 12 size exclusion chromatography was performed with buffer B. Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. 10-subunit Pol II was incubated with two molar equivalents of nucleic acid scaffold (Template, 5'-cgacacagcatcaaatgcacgatgtaacttttataggcgcccaacc;Nontemplate, 5'-ggttgggcgcctataaaagttacatcgtgcaaaatcgttatgagaa; RNA, 5'-gctgtgtcg) as described 3 and 2.5 molar equivalents of TBP. After incubation for 20 minutes at 20°C 3-5 molar equivalents of TFIIB-Rpb4/7 fusion protein were added. After incubation for 20 min. at 20°C, the complex was purified on a Superose 6 size exclusion column (Amersham). Fractions corresponding to the complex were pooled and concentrated to 4 mg/ml.Crystallization, data collection, and structure determination Crystals were grown at 20 °C using the hanging drop vapor diffusion method by mixing 1.5 µl of sample solution with 1.5 µl of reservoir solution (800 mM sodium ammonium tartrate, 100 mM HEPES pH 7.5, 5 mM DTT). Crystals were transferred stepwise to mother solution containing additionally 0-22% glycerol over 8 h, slowly cooled down to 8 °C, incubated for another 24 h, and plunged into liquid nitrogen. Diffraction data were collected in 0.75° increments at the protein crystallography beamline ID 29 at ESRF. Diffraction data were processed with XDS and scaled with XSCALE 4 . The structure was solved by molecular replacement with PHASER 5 using the first 12-subunit Pol II ...

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“…DNA Substrates-Linear heteroduplex substrates were generated by annealing two 484-bp PCR products amplified by Taq-DNA polymerase with a single GATC site at position 210 and a G/C or a A/T base pair at position 385 using plasmids and primers as described previously (20). This procedure results in a mixture of 50% homoduplex substrates (G/C and A/T) and 50% heteroduplex substrates (G/T and A/C).…”

mentioning

confidence: 99%

Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking

Giron-Monzon

Manelytė

Ahrends

et al. 2004

Journal of Biological Chemistry

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Strand discrimination in Escherichia coli DNA mismatch repair requires the activation of the endonuclease MutH by MutL. There is evidence that MutH binds to the N-terminal domain of MutL in an ATP-dependent manner; however, the interaction sites and the molecular mechanism of MutH activation have not yet been determined. We used a combination of site-directed mutagenesis and site-specific cross-linking to identify protein interaction sites between the proteins MutH and MutL. Unique cysteine residues were introduced in cysteine-free variants of MutH and MutL. The introduced cysteines were modified with the cross-linking reagent 4-maleimidobenzophenone. Photoactivation resulted in cross-links verified by mass spectrometry of some of the single cysteine variants to their respective Cys-free partner proteins. Moreover, we mapped the site of interaction by cross-linking different combinations of single cysteine MutH and MutL variants with thiol-specific homobifunctional cross-linkers of varying length. These results were used to model the MutH⅐MutL complex and to explain the ATP dependence of this interaction.DNA repair systems are crucial for maintaining a stable genome (1). One of these systems, DNA mismatch repair, is present in most organisms and enhances the fidelity of DNA replication by a factor of up to 1000 by correcting replication errors (2-6). mismatch repair is a complex process that requires the coordination of a number of specialized enzymatic activities involving many different proteins. The paradigm of such a process is the MutHLS system from Escherichia coli (7). Because mismatch repair proteins MutS and MutL are evolutionarily conserved among the three kingdoms of life, it is believed that the basic mechanisms of mismatch repair are similar. In the presence of ATP and a mismatch, MutS recruits MutL and together they activate MutH, which in turn cleaves the newly replicated daughter strand at a transiently unmethylated d(GATC) site. The nick marks the erroneous strand for excision, which is started by the action of DNA helicase II, single-stranded DNA-binding protein, and one of several exonucleases (8). DNA polymerase III, single-stranded DNA-binding protein, and DNA ligase carry out repair synthesis.One of the interesting questions in DNA mismatch repair is how the different Mut proteins interact in time and space. In the E. coli system, the assembly of the active MutS⅐MutL complex will activate the strand discrimination endonuclease MutH. Both MutS and MutL are ATPases, and the roles of ATP binding and hydrolysis are just emerging (9 -13). The mismatch activated form of this complex will stimulate the latent endonuclease activity of MutH to cleave the transiently unmethylated error-containing daughter strand, thereby marking this strand for removal. We have previously shown that the proper discrimination of the unmethylated strand from the methylated strand can be mapped to a single amino acid residue in MutH, i.e. Tyr 212 . Biochemical analyses indicated that only MutL, not MutS, interacts direc...

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An Efficient Method for the Preparation of Long Heteroduplex DNA as Substrate for Mismatch Repair by the Escherichia coli MutHLS System

Cited by 18 publications

References 13 publications

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

Chemical Trapping of the Dynamic MutS-MutL Complex Formed in DNA Mismatch Repair in Escherichia coli

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

Mapping Protein-Protein Interactions between MutL and MutH by Cross-linking

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