Homologous Recombination in Real Time: DNA Strand Exchange by RecA

Heijden, Thijn van der; Modesti, Mauro; Hage, Susanne; Kanaar, Roland; Wyman, Claire; Dekker, Cees

doi:10.1016/j.molcel.2008.03.010

Cited by 85 publications

(98 citation statements)

References 44 publications

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“…Single-molecule detection enables the observation of the dynamic behaviour of individual molecules. Hence, single-molecule techniques are an essential tool to obtain fundamental information on biological processes and to a better understanding of the cellular function [5,6].…”

Section: Introductionmentioning

confidence: 99%

Tethered particle motion mediated by scattering from gold nanoparticles and darkfield microscopy

Dietrich

2009

J. Nanophoton

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Abstract. We measured light scattered from gold nanoparticles with darkfield microscopy in order to perform single molecule detection based on tethered particle motion (TPM). This combination results in a signal to noise ratio of about 40 dB, which allowed us to use 80 nm diameter gold particles as reporters instead of the typically used polystyrene particles whose sizes are up to 1 µm. The particle size is crucial in TPM experiments as it can induce a volume-exclusion effect, which results in a stretching force acting on the DNA tether. This affects both the biophysical and statistical properties of the anchored DNA and hence the interpretation of the experimental data. We demonstrated that the gold nanoparticles and darkfield microscopy can be used to characterize the confined Brownian motion of dsDNA-tethered gold particles with a spatial precision of 3 nm. Physical parameters such as the spring constant of the tethered DNA fragment and the persistence length can be derived from the two dimensional (2D) (x, y) projected image data. We have applied this method to various MgCl 2 and glycerol concentrations as a proof of principle.

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Section: Introductionmentioning

confidence: 99%

Tethered particle motion mediated by scattering from gold nanoparticles and darkfield microscopy

Dietrich

2009

J. Nanophoton

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“…Last, when studying reactions accompanied by a change in DNA topology, use can be made of the coupling between topology and length in plectonemic DNA (78,79,93). The removal or addition of supercoils to DNA leads to a strong, force-dependent change in length, on the order of 50 nm per coil, or 5 nm per base.…”

Section: Future Prospectsmentioning

confidence: 99%

“…DNA-enzyme interactions are often sensitive to torque applied to the DNA, particularly when the reaction is accompanied by a change in DNA topology, and structural and kinetic insight into the reaction can be obtained in these cases from Optical tweezers: method to trap and manipulate an object with a tightly focused laser beam torque spectroscopy studies (27,47). Moreover, torque plays a regulatory role in many aspects of cellular biology, for example, in reactions at the heart of transcription, replication, and recombination (27,48,52,93), and the cell has intricate machinery in place to regulate supercoiling of cellular DNA (48). Magnetic tweezers are thus a powerful tool to address questions related to a wide range of biological systems.…”

Section: Introductionmentioning

confidence: 99%

Recent Advances in Magnetic Tweezers

Vlaminck

Dekker

2012

Annu. Rev. Biophys.

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328

255

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Since their first demonstration of manipulation and mechanical coiling of a single DNA molecule, magnetic tweezers have become a popular and widely used single-molecule manipulation technique. Magnetic tweezers have yielded a wealth of information on the elastic properties of (coiled) DNA and have provided critical insights into the dynamic activity of DNA-processing enzymes. Recent years have seen a wave of technological creativity, and a flurry of new methods and technological advances have been introduced to magnetic tweezers, which will enable more and previously inaccessible information to be extracted from biophysical systems. In this review, we survey the most important recent advances and reflect on more interesting twists to come.

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“…In bacteria, a central role in the homologous recombination is played by RecA proteins (11,14,15). They belong to a class of recombination proteins that are conserved across different organisms, from bacteria to mammals (RadA in archea, and Rad51 and Dmc1 in eukaryotes) (3).…”

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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Homologous Recombination in Real Time: DNA Strand Exchange by RecA

Cited by 85 publications

References 44 publications

Tethered particle motion mediated by scattering from gold nanoparticles and darkfield microscopy

Tethered particle motion mediated by scattering from gold nanoparticles and darkfield microscopy

Recent Advances in Magnetic Tweezers

On the Mechanism of Homology Search by RecA Protein Filaments

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