Dataset Information

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament.

ABSTRACT: Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament's torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca(2+) ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament.

SUBMITTER: Lee M

PROVIDER: S-EPMC3737536 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament.

Lee Mina M Lipfert Jan J Sanchez Humberto H Wyman Claire C Dekker Nynke H NH

Nucleic acids research 20130522 14

Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of ...[more]

PMID: 23703213

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Project description:Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion. Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined. Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55-Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55-Rad57 heterodimer associates with the Rad51-single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51-Rad55-Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.

Dataset Information

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament.

Publications

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament.

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