Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function

Ristić, Dejan; Modesti, Mauro; Heijden, Thijn van der; Noort, John van; Dekker, Cees; Kanaar, Roland; Wyman, Claire

doi:10.1093/nar/gki640

Cited by 117 publications

(172 citation statements)

References 42 publications

(48 reference statements)

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“…After Ϸ300 s of continuous incubation with Rad51, the DNA is extended by 9.0 m, which is an apparent 65% increase in length (see also Movie S1). This extension is similar to results obtained for RecA (24) and is slightly greater than seen in structural studies, because of the differences in persistence lengths for duplex DNA and the Rad51 nucleoprotein filament (32).…”

Section: Real-time Visualization Of Rad51 Nucleoprotein Filament Assesupporting

confidence: 87%

See 1 more Smart Citation

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

Hilario

Amitani

Baskin

et al. 2009

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

132

161

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Rad51 protein (Rad51) is central to recombinational repair of double-strand DNA breaks. It polymerizes onto DNA and promotes strand exchange between homologous chromosomes. We visualized the real-time assembly and disassembly of human Rad51 nucleoprotein filaments on double-stranded DNA by single-molecule fluorescence microscopy. Rad51 assembly extends the DNA by Ϸ65%. Nucleoprotein filament formation occurs via rapid nucleation followed by growth from these nuclei. Growth does not continue indefinitely, however, and nucleoprotein filaments terminate when Ϸ2 m in length. The dependence of nascent filament formation on Rad51 concentration suggests that 2-3 Rad51 monomers are involved in nucleation. Rad51 nucleoprotein filaments are stable and remain extended when ATP hydrolysis is prevented; however, when permitted, filaments decrease in length as a result of conversion to ADP-bound nucleoprotein complexes and partial protein dissociation. Dissociation of Rad51 from dsDNA is slow and incomplete, thereby rationalizing the need for other proteins that facilitate disassembly.nucleation ͉ RecA protein ͉ recombination ͉ self-assembly ͉ single-molecule G enomes are continually attacked by both endogenous and exogenous agents that damage DNA. DNA damage in the form of DNA breaks can lead to chromosome translocations, cell cycle arrest, and apoptosis. Homologous recombination is an essential biological process that ensures the accurate repair of DNA breaks and thereby contributes to genomic integrity. The recombinational repair of DNA with a break occurs by a multistep process (1-3). The first step requires resection of the broken duplex DNA by a helicase and/or nuclease to produce a region of 3Ј-terminated single-strand DNA (ssDNA) at the ends of the break (1, 4). These ssDNA tails serve as substrates for the assembly of a DNA strand exchange protein, such as RecA in bacteria or Rad51 in eukaryotes (2,3,5). This nucleoprotein filament finds homology in an intact DNA molecule and promotes DNA strand invasion to form an intermediate, termed a joint molecule. Pairing by both processed ends of the broken DNA, and their subsequent replication, results in formation of Holliday junctions. These Holliday junctions undergo branch migration and are resolved enzymatically to produce the repaired DNA.Rad51 protein (Rad51) assembles on either single-or doublestranded DNA (dsDNA) to produce a nucleoprotein filament that, at saturation, comprises 1 Rad51 monomer for every 3 nucleotides or base-pairs of DNA. Electron microscopy and X-ray crystallography show the Rad51 nucleoprotein filament to be a right-handed helical structure in which the DNA is stretched by Ϸ50% over its normal B-form length (6, 7). This filament displays ATP hydrolysis activity when assembled on either ssDNA or dsDNA. Rad51 promotes the homologous pairing of ssDNA with dsDNA; however, migration of the nascent DNA heteroduplex is relatively slow. This DNA strand exchange activity is substantially enhanced by RPA, and is regulated by several mediator proteins (Brca2...

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Section: Real-time Visualization Of Rad51 Nucleoprotein Filament Assesupporting

confidence: 87%

“…Human Rad51 was shown to form stable nucleoprotein filaments under conditions of reduced ATP hydrolysis (32); therefore, we examined the Rad51 nucleoprotein assembly in the presence of different divalent metal and nucleotide cofactor partners to assess the effects of ATP hydrolysis. Fig.…”

Section: Real-time Visualization Of Rad51 Nucleoprotein Filament Assementioning

confidence: 99%

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

Hilario

Amitani

Baskin

et al. 2009

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

132

161

View full text Add to dashboard Cite

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“…Our data clearly indicate that, next to the ADP‐bound state, the NPF can exist in two ATP‐bound states that differ in contour length. Remarkably, the reported hRAD51‐dsDNA filament length of 1.5 nm per hRAD51 monomer (Ristic et al , 2005) matches that of the ATP‐compact state for hRAD51‐ssDNA (1.4 ± 0.3 nm); this correspondence might be significant for homology search and strand exchange processes during homologous recombination. Interestingly, the work on RecA‐ssDNA (Kim et al , 2014) reported significant reloading of ATP within the filament.…”

Section: Discussionmentioning

confidence: 65%

“…The hysteresis area A is determined from Fig 3C as 205 ± 2 pN μM.The binding site size m is three bases (Ristic et al , 2005). …”

Section: Methodsmentioning

confidence: 99%

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

et al. 2018

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An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 kBT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.

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“…Like their prokaryotic counterparts E. coli RecA and T4 UvsX, Rad51 and Dmc1 form a filamentous structure, referred to as the presynaptic filament, on ssDNA [18,19]. Assembly of the presynaptic filament requires ATP binding by the recombinase protein [19,20], and ATP hydrolysis by the DNA-bound recombinase promotes the turnover of the presynaptic filament [21][22][23][24]. All the subsequent biochemical steps, including the capture of the duplex DNA partner and the search for homology in the engaged ssDNA and dsDNA molecules, that lead to D-loop formation occur within the confines of the presynaptic filament [17,19,20].…”

Section: Introductionmentioning

confidence: 99%

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

et al. 2007

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Rad54, a member of the Swi2/Snf2 protein family, works in concert with the RecA-like recombinase Rad51 during the early and late stages of homologous recombination. Rad51 markedly enhances the activities of Rad54, including the induction of topological changes in DNA and the remodeling of chromatin structure. Reciprocally, Rad54 promotes Rad51-mediated DNA strand invasion with either naked or chromatinized DNA. Here, using various Saccharomyces cerevisiae rad51 and rad54 mutant proteins, mechanistic aspects of Rad54/Rad51-mediated chromatin remodeling are defined. Disruption of the Rad51-Rad54 complex leads to a marked attenuation of chromatin remodeling activity. Moreover, we present evidence that assembly of the Rad51 presynaptic filament represents an obligatory step in the enhancement of the chromatin remodeling reaction. Interestingly, we find a specific interaction of the N-terminal tail of histone H3 with Rad54 and show that the H3 tail interaction domain resides within the amino terminus of Rad54. These results suggest that Rad54-mediated chromatin remodeling coincides with DNA homology search by the Rad51 presynaptic filament and that this process is facilitated by an interaction of Rad54 with histone H3.

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Human Rad51 filaments on double- and single-stranded DNA: correlating regular and irregular forms with recombination function

Cited by 117 publications

References 42 publications

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

Two distinct conformational states define the interaction of human RAD 51‐ ATP with single‐stranded DNA

Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

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