Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules

Heijden, Thijn van der; Seidel, Ralf; Modesti, Mauro; Kanaar, Roland; Wyman, Claire; Dekker, Cees

doi:10.1093/nar/gkm629

Cited by 101 publications

(171 citation statements)

References 46 publications

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“…This finding could imply that growth occurs by addition of trimers to the filament. Such a conclusion would also seem to be in qualitative accord with a recent study that concluded that Rad51 nucleoprotein filaments grow by addition of Rad51 pentamers (28). However, we note that an implicit assumption in the kinetic models is that growth is indefinite: i.e., that once nucleated, a filament can grow to the DNA ends, or until it encounters another filament.…”

Section: Discussionsupporting

confidence: 88%

“…The explanation for the failure of Rad51 filaments to grow indefinitely is not clear (28). One possibility is that, even under conditions of limited ATP hydrolysis (i.e., in the presence of calcium and ATP), a small amount of ATP is hydrolyzed to produce an ADP-bound Rad51 species.…”

Section: Discussionmentioning

confidence: 99%

“…20, Rad51 filaments were visualized indirectly through competition with a fluorescent dye. Also, magnetic tweezers were used to monitor changes in DNA length due to Rad51 binding in real time, but direct imaging of the assembly process is not possible with this technique (28). Our work complements the previous studies by combining visualization of individual Rad51-DNA complexes and real-time monitoring of their assembly and disassembly.…”

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confidence: 92%

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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: 88%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 92%

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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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“…2A, lanes 3, 5, and 6). It was previously shown that Ca 2þ promotes formation of a stable ATP-bound RAD51-ssDNA filament (high affinity DNA binding state) by inhibiting the RAD51 ATPase; whereas in the presence of Mg 2þ , RAD51 forms a filament that is prone to dissociation because of a buildup of protein-bound ADP (25,27). For RecA, high Mg 2þ (15 mM) and low Mg 2þ (4 mM) concentrations were optimal for the initial DNA strand exchange and for subsequent BM, respectively.…”

Section: Resultsmentioning

confidence: 99%

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

Rossi

Mazina

Bugreev

et al. 2011

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

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The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.H omologous recombination (HR) is the process responsible for maintaining genome stability in all living organisms; it is particularly important for repairing DNA double-strand breaks (1-5). The process of HR involves the enzymatic degradation of broken dsDNA ends into resected DNA duplex with protruding ssDNA tails (6, 7). Following resection, the central protein of HR, the prokaryotic (bacterial recombinase protein) RecA or its eukaryotic homolog Rad51, is loaded onto the ssDNA tails and forms a contiguous nucleoprotein filament (8, 9). Although RecA and Rad51 share only ∼30% sequence homology, the filaments they form and the conformational changes they induce in DNA are nearly identical (10).The RecA/Rad51-ssDNA filament possesses the unique ability to search for homologous dsDNA sequences and promote DNA strand exchange: an invasion of ssDNA into homologous DNA duplex that results in the displacement of the identical ssDNA from the duplex and formation of a joint molecule (JM) ( Fig. S1A) (11). Besides its essential DNA strand-exchange activity RecA/RAD51 recombinases have an ability to extend JMs by a kinetically distinct process known as heteroduplex extension or three-strand branch migration (BM), in which one DNA strand is progressively exchanged for another (11,12). When the extending heteroduplex reaches the ssDNA-dsDNA junction on the invading DNA strand, the 3-stranded JM is converted into a 4-stranded Holliday junction (HJ). Then, specialized DNA translocating proteins, like Escherichia coli RuvAB, bind to HJs and promote their migration by four-strand BM (otherwise referred to as the BM of HJs) (13). These DNA translocases are capable of bypassing sequence heterologies and disrupting nucleoprotein complexes encountered during BM. Finally, DNA repair synthesis by DNA polymerases takes place on JMs followed by their resolution.Surprisingly, the RecA/Rad51 recombinases can also promote four-strand BM (Fig. S1A) (14, 15). This activity may play a significant role in the initial stages of recombination in vivo by helping to form and stabilize HJs, and therefore it is important to u...

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“…74 Similarly, the real time assembly/disassembly kinetics of Rad51 nucleoprotein filaments have been studied by magnetic tweezers. 75 In addition, the conversion of ssDNA to dsDNA by DNA polymerases can be measured using magnetic tweezers and the rates of conversion in turn yield biochemical information about the polymerization reaction. 29,30 A unique aspect of magnetic tweezers is that they can apply torque to an attached molecule.…”

Section: Magnetic Tweezers (Mt)mentioning

confidence: 99%

The importance of surfaces in single-molecule bioscience

2008

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The last ten years have witnessed an explosion of new techniques that can be used to probe the dynamic behavior of individual biological molecules, leading to discoveries that would not have been possible with more traditional biochemical methods. A common feature among these singlemolecule approaches is the need for the biological molecules to be anchored to a solid support surface. This must be done under conditions that minimize nonspecific adsorption without compromising the biological integrity of the sample. In this review we highlight why surface attachments are a critical aspect of many single-molecule studies and we discuss current methods for anchoring biomolecules. Finally, we provide a detailed description of a new method developed by our laboratory for anchoring and organizing hundreds of individual DNA molecules on a surface, allowing "high-throughput" studies of protein-DNA interactions at the single-molecule level.

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Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules

Cited by 101 publications

References 46 publications

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA

The importance of surfaces in single-molecule bioscience

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