Fluorescent Human RAD51 Reveals Multiple Nucleation Sites and Filament Segments Tightly Associated along a Single DNA Molecule

Modesti, Mauro; Ristić, Dejan; Heijden, Thijn van der; Dekker, Cees; Mameren, Joost van; Peterman, Erwin J. G.; Wuite, Gijs J. L.; Kanaar, Roland; Wyman, Claire

doi:10.1016/j.str.2007.04.003

Cited by 73 publications

(104 citation statements)

References 32 publications

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“…They have the potential to unite, if neighboring filaments are in register, but if not, then short gaps of DNA will persist. In this regard, our results agree with those of a recent single-molecule study where a fluorescently modified Rad51 variant was used to visualize nucleoprotein filament formation (27). Fluorescence imaging suggested that nucleation occurred at least once every 2,700 bp, and scanning force microscopy showed that filament segments were Ϸ500 bp.…”

Section: Discussionsupporting

confidence: 90%

“…This result affirms that Rad51 filaments that are prevented from hydrolyzing ATP are stable and do not disassemble. This result differs from a recent report where surface-tethered Rad51 filaments formed in Ca 2ϩ -ATP were dissociated upon buffer exchange to Mg 2ϩ -ATP (27). This difference in behavior is due to differences in buffer conditions (unpublished data).…”

Section: Rad51contrasting

confidence: 99%

“…Specifically, the application of singlemolecule techniques to the study of recombination proteins has revealed aspects of molecular behavior that were not attainable from ensemble methods (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Single-molecule visualization methods have been used to characterize Rad51-nucleoprotein filaments (20,27); in ref. 27, filament assembly could not be imaged in real time although disassembly could and, in ref.…”

mentioning

confidence: 99%

“…Single-molecule visualization methods have been used to characterize Rad51-nucleoprotein filaments (20,27); in ref. 27, filament assembly could not be imaged in real time although disassembly could and, in ref. 20, Rad51 filaments were visualized indirectly through competition with a fluorescent dye.…”

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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: Discussionsupporting

confidence: 90%

Section: Rad51contrasting

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…hRAD51 (isoform K313, variant C319S) fluorescent labelling with Alexa Fluor 555 was performed as was described previously (Modesti et al , 2007). The degree of labelling was estimated to be around 80%.…”

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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Computational Methods and Deep Learning for Elucidating Protein Interaction Networks

Vora¹,

Kalakoti²,

Sundar³

2022

Methods in Molecular Biology

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Fluorescent Human RAD51 Reveals Multiple Nucleation Sites and Filament Segments Tightly Associated along a Single DNA Molecule

Cited by 73 publications

References 32 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

Computational Methods and Deep Learning for Elucidating Protein Interaction Networks

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