Qualitative and Quantitative Studies of Protein-DNA Interactions by Gel Mobility-Shift Assay

Stone, Stuart R.; Hughes, Melya J.; Jost, Jean-Pierre

doi:10.1007/978-3-0348-7561-5_14

Cited by 12 publications

(7 citation statements)

References 21 publications

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“…Binding of RecBCD Enzyme to DNA Ends-We characterized the binding of RecBCD enzyme to ds DNA by gel mobility-shift assay (26), but used two enhancements to the assay to enable us to study the stoichiometry of binding of RecBCD enzyme to ds ends. The migration of enzyme-DNA complexes was compared to that of monomeric and dimeric RecBCD enzyme on the same gel, to determine the multimeric state of the enzyme bound to DNA ends.…”

Section: Discussionmentioning

confidence: 99%

“…The free and retarded radioactive DNA bands were detected and quantitated by PhosphorImager analysis. Results were analyzed by nonlinear regression for varying DNA concentration or by Hill plots for varying enzyme concentration (26).…”

Section: Determination Of Dissociation Constantsmentioning

confidence: 99%

“…4). Titration of the DNA concentration, at a constant protein concentration below the K D , allows measurement both of the K D (the concentration at which half of the maximal amount of DNA is bound) and of the concentration of protein that is active for binding to DNA (26). These measurements were performed with DNA molecules with one ds DNA end bearing a 4-nt 5Ј-overhang and were made in the absence of Mg 2ϩ ions (Table III).…”

Section: Fig 3 Nondenaturing Polyacrylamide Gel Electrophoresis Ofmentioning

confidence: 99%

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Monomeric RecBCD Enzyme Binds and Unwinds DNA

Taylor

Smith

1995

Journal of Biological Chemistry

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RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since M r of 250,000 -655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (M r Ϸ 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, K D estimates ranged from Ϸ 0.1 nM to Ϸ 0.7 nM in the presence of Mg 2؉ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme.The RecBCD enzyme (EC 3.1.11.5) is a large ATP-dependent enzyme that is involved in recombination and repair of DNA in Escherichia coli (reviewed in Ref. 1). It is encoded by the recB, recC, and recD genes, whose gene products have M r of 134,000, 129,000, and 67,000, respectively, as inferred from DNA sequence data and N-terminal peptide analysis (2-5). The enzyme has a potent ATP-dependent exonuclease that is active on either ds 1 or ss DNA and a weak ATP-stimulated endonuclease activity that acts only on ss DNA. It can use the energy of ATP hydrolysis to unwind ds DNA, either transiently or permanently, in a highly processive reaction (6, 7). The enzyme is active on linear, but not circular, ds DNA and thus requires a ds terminus for its unwinding or nuclease activity (8, 9).Published reports suggest that RecBCD enzyme can exist in either a monomeric (B 1 C 1 D 1 ) or a dimeric (B 2 C 2 D 2 ) form.2 The native M r of the enzyme was initially reported to be about 250,000 (9), as estimated by glycerol gradient centrifugation, consistent with the enzyme molecule containing one copy each of the RecB, RecC, and RecD polypeptides (with a predicted M r of 330,000). A higher M r form of the enzyme, apparently dimeric, was observed in sonicates of E. coli (10), together with the previously reported form, but was lost during subsequent purification steps. RecBCD enzyme purified from a strain that overproduced the enzyme had a native M r of 655,000 but which decreased to about 270,000 in the presence of 0.5 M NH 4 Cl (11). The higher M r form was not observed in a subsequent purification (12). More recently, RecBCD enzyme has been produced either by overproduction of the three subunits within E. coli or by mixing of purified subunits (5). M r estimations by gel filtration or native polyacrylamide gel electrophoresis are consistent with the enzyme ...

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Section: Discussionmentioning

confidence: 99%

Section: Determination Of Dissociation Constantsmentioning

confidence: 99%

Section: Fig 3 Nondenaturing Polyacrylamide Gel Electrophoresis Ofmentioning

confidence: 99%

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Monomeric RecBCD Enzyme Binds and Unwinds DNA

Taylor

Smith

1995

Journal of Biological Chemistry

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“…(D) The K d values of the interaction of the B95-8, A205S, and A206S Ztas were determined by EMSA with increasing probe concentration. The data were quantitated by using phosphorimaging, and the concentrations of bound and free probe were determined according to the method described by Stone et al (33). dent manner, whereas another coiled coil peptide of unrelated sequence, SKIP1 (13), was unable to disrupt Zta complex formation with DNA.…”

Section: Fig 2 All Three Natural Variants Within the Dimerization Dmentioning

confidence: 99%

The Zipper Region of Epstein-Barr Virus bZIP Transcription Factor Zta Is Necessary but Not Sufficient To Direct DNA Binding

Hicks

Al-Mehairi

Sinclair

2003

J Virol

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The viral bZIP transcription factor Zta (BZLF1, EB1, ZEBRA) mediates the switch between the latent and lytic cycles of Epstein-Barr virus (EBV). In part, its activity requires the formation of homodimers and interaction with specific DNA sequence elements (ZREs). Zta has an atypical zipper motif that has a lower stability than do typical bZIP proteins. Here we show that a synthetic peptide directed against the zipper can disrupt the DNA-binding function of Zta. This highlights the relevance of this region for the function of Zta and demonstrates that the zipper region is a potential target for therapeutic agents. We also unmask the relevance of a region adjacent to the zipper (CT region), which is required to direct the interaction of Zta with DNA and to transactivate ZRE-dependent promoters in vivo.

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“…Incubation followed at room temperature. The time necessary to reach equilibrium was assessed by EMSA (not shown; Stone et al, 1991). For all DNA fragments tested equilibrium turned out to be fully established at the earliest timepoints that can be determined by this technique (30 s).…”

Section: Separation Of Phosphorylated From Unphosphorylated Stat Protmentioning

confidence: 99%

DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2-terminal domains stabilizes binding of two dimers to tandem DNA sites.

et al. 1996

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Stat1 alpha, Stat1 beta and a proteolytically defined truncated Stat1 (132–713, Stat1tc) have been prepared from recombinant sources. All three proteins were specifically phosphorylated on Tyr701 in vitro and the phosphoprotein purified to homogeneity. This was achieved by employing a new isolation scheme that does not include DNA affinity steps and readily allows for the isolation of tens of milligrams of activated Stat protein. The purified phosphoprotein was free of traces of unphosphorylated polypeptide as detected by mass spectrometry. The phosphorylated Stat1 preparations bound to various DNA recognition sites with the same Keq of approximately 1 × 10(‐9) M; distinction between ‘weak’ and ‘strong’ binding sites is determined by the very rapid dissociation (< 30 s, t1/2) from ‘weak’ sites compared with ‘strong’ sites (approximately 3 min, t1/2). Reports of ‘weak’ tandem binding sites in a natural gene caused us to examine binding to tandem sites leading to the finding that the Stat1 alpha or beta (38 amino acids shorter on the C terminus) bound to two tandem sites (but not two head‐to‐head sites) with a higher stability than to a single recognition site. The N‐terminally truncated protein Stat1tc did not show this cooperative binding, thus implicating the N‐terminal domain in promoting Stat1‐Stat1 dimer interaction.

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Qualitative and Quantitative Studies of Protein-DNA Interactions by Gel Mobility-Shift Assay

Cited by 12 publications

References 21 publications

Monomeric RecBCD Enzyme Binds and Unwinds DNA

Monomeric RecBCD Enzyme Binds and Unwinds DNA

The Zipper Region of Epstein-Barr Virus bZIP Transcription Factor Zta Is Necessary but Not Sufficient To Direct DNA Binding

DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2-terminal domains stabilizes binding of two dimers to tandem DNA sites.

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