RecA binding to a single double-stranded DNA molecule: A possible role of DNA conformational fluctuations

Léger, Jean-François; Robert, Jérôme; Bourdieu, Laurent; Chatenay, D.; Marko, John F.

doi:10.1073/pnas.95.21.12295

Cited by 156 publications

(153 citation statements)

References 22 publications

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“…For the Bto-S model, these segregated transitions are different in nature (unpeeling versus B-to-S conversion) for temperatures T ≤ 25 • C at 150 mM NaCl (and for higher temperatures at higher salt concentrations). These results suggest that DNA-elongating proteins, such as RecA, could induce within DNA stretching transitions whose character differs with sequence composition, conferring upon the elongated RecA-DNA complex [3,4] a sequencedependent elasticity. Our results also suggest that precise melting of localized regions of DNA can be effected by stretching molecules whose sequences are appropriately designed.…”

Section: Resultsmentioning

confidence: 94%

“…The resulting 'overstretched' form of the molecule is approximately 1.7 times longer than helical B-DNA. Overstretching is of crucial importance for the biological function of DNA: the bacterial protein RecA elongates DNA by a factor of 1.5 upon binding [3,4,5], a mechanism central to homologous recombination and to chromosomal segregation during cell division [6]. However, the nature of the overstretched state remains a source of considerable controversy: some think overstretched DNA in vitro to be a hybridized form called S-DNA (the 'Bto-S' picture) [7,8,9,10,11,12], while a competing picture considers overstretching to signal a conversion to unhybrizided single strands (the 'force-melting' picture) [13,14,15,16,17,18].…”

Section: Introductionmentioning

confidence: 99%

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Stretching chimeric DNA: A test for the putative S-form

Whitelam

Pronk

Geissler

2008

The Journal of Chemical Physics

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Double-stranded DNA 'overstretches' at a pulling force of about 65 pN, increasing in length by a factor of 1.7. The nature of the overstretched state is unknown, despite its considerable importance for DNA's biological function and technological application. Overstretching is thought by some to be a force-induced denaturation, and by others to consist of a transition to an elongated, hybridized state called S-DNA. Within a statistical mechanical model we consider the effect upon overstretching of extreme sequence heterogeneity. 'Chimeric' sequences possessing halves of markedly different AT composition elongate under fixed external conditions via distinct, spatially segregated transitions. The corresponding force-extension data display two plateaux at forces whose difference varies with pulling rate in a manner that depends qualitatively upon whether the hybridized S-form is accessible. This observation implies a test for S-DNA that could be performed in experiment. Our results suggest that qualitatively different, spatially segregated conformational transitions can occur at a single thermodynamic state within single molecules of DNA.

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Section: Resultsmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Stretching chimeric DNA: A test for the putative S-form

Whitelam

Pronk

Geissler

2008

The Journal of Chemical Physics

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“…The correlation between sequence specific DNA ''bendability'' and binding affinity has been demonstrated very nicely for the binding of a repressor protein to sequences with differing elastic properties (34). Micromanipulation experiments on RecA binding to DNA demonstrate that the binding affinity of RecA increases for mechanically prestretched DNA, suggesting that spontaneous thermal stretching fluctuations may be playing a role in the binding of RecA to DNA, and that RecA binds preferentially to thermally stretched DNA (9).…”

Section: Discussionmentioning

confidence: 99%

“…One fundamental question underlying the binding mechanism is whether the protein binds to DNA and then distorts it or whether the DNA is capable of undergoing spontaneous fluctuations in which the distorted conformations are energetically accessible, and the protein binds with high affinity to these distorted conformations (9). Sharply bent or kinked DNA conformations that are thermally accessible could be playing an important role in the binding of proteins to DNA (10,11).…”

mentioning

confidence: 99%

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

Kuznetsov¹,

Sugimura

Vivas³

et al. 2006

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

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Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNAbinding sites. Previous kinetics measurements on several DNAbending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending͞unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from Ϸ2 ms ؊1 at Ϸ37°C to Ϸ40 ms ؊1 at Ϸ10°C and correspond to an activation energy of Ϸ14 ؎ 3 kcal͞mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend͞kink DNA before forming a tight complex with IHF.DNA bending kinetics ͉ laser temperature jump ͉ protein-DNA interactions ͉ time-resolved FRET measurements

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“…As a result, the homologous search is more efficient for longer filaments and for less extended (more coiled) DNA chains. A general question on the role of DNA coiling in the protein search has been addressed theoretically (23,(32)(33)(34). However, a quantitative model of the intersegment transfer for the homology search has not been developed yet.…”

Section: Introductionmentioning

confidence: 99%

On the Mechanism of Homology Search by RecA Protein Filaments

Kochugaeva

Shvets

Kolomeisky

2017

Biophysical Journal

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Genetic stability is a key factor in maintaining, survival and reproduction of biological cells. It relies on many processes, but one of the most important is a homologous recombination, in which the repair of breaks in double-stranded DNA molecules is taking place with a help of several specific proteins. In bacteria this task is accomplished by RecA proteins that are active as nucleoprotein filaments formed on single-stranded segments of DNA. A critical step in the homologous recombination is a search for a corresponding homologous region on DNA, which is called a homology search. Recent single-molecule experiments clarified some aspects of this process, but its molecular mechanisms remain not well understood. We developed a quantitative theoretical approach to analyze the homology search. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the homology search is presented. It is found that the search dynamics depends on the degree of extension of DNA molecules and on the size of RecA nucleoprotein filaments, in agreement with experimental single-molecule measurements of DNA pairing by RecA proteins. Our theoretical calculations, supported by extensive Monte Carlo computer simulations, provide a molecular description of the mechanisms of the homology search.

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RecA binding to a single double-stranded DNA molecule: A possible role of DNA conformational fluctuations

Cited by 156 publications

References 22 publications

Stretching chimeric DNA: A test for the putative S-form

Stretching chimeric DNA: A test for the putative S-form

Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

On the Mechanism of Homology Search by RecA Protein Filaments

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