Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Mazina, Olga M.; Mazin, Alexander V.

doi:10.1074/jbc.m410244200

Cited by 74 publications

(92 citation statements)

References 53 publications

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“…Human and yeast Rad54 were purified as described previously in ref. 19. Untagged hRad54 was produced by proteolytic cleavage of GST-hRad54 with thrombin, as previously described in ref.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro, Rad54 has several important activities, reflecting its multiple functions at different stages of HR (16). Rad54 stimulates DNA strand exchange (17-19) and DNA heteroduplex extension promoted by Rad51 (20), and it can translocate along double-stranded DNA (dsDNA) by using the energy of ATP hydrolysis (19,(21)(22)(23), displace Rad51 from dsDNA (24), and remodel chromatin (16). We recently showed that Rad54 is a HJ-processing enzyme that promotes branch migration of HJs (25) and D-loop dissociation (26).…”

mentioning

confidence: 99%

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Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Mazina

Mazin

2008

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

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Rad54, a key protein of homologous recombination, physically interacts with a DNA structure-specific endonuclease, Mus81-Eme1. Genetic data indicate that Mus81-Eme1 and Rad54 might function together in the repair of damaged DNA. In vitro, Rad54 promotes branch migration of Holliday junctions, whereas the Mus81-Eme1 complex resolves DNA junctions by endonucleolytic cleavage. Here, we show that human Rad54 stimulates Mus81-Eme1 endonuclease activity on various Holliday junction-like intermediates. This stimulation is the product of specific interactions between the human Rad54 (hRad54) and Mus81 proteins, considering that Saccharomyces cerevisiae Rad54 protein does not stimulate human Mus81-Eme1 endonuclease activity. Stimulation of Mus81-Eme1 cleavage activity depends on formation of specific Rad54 complexes on DNA substrates occurring in the presence of ATP and, to a smaller extent, of other nucleotide cofactors. Thus, our results demonstrate a functional link between the branch migration activity of hRad54 and the structurespecific endonuclease activity of hMus81-Eme1, suggesting that the Rad54 and Mus81-Eme1 proteins may cooperate in the processing of Holliday junction-like intermediates during homologous recombination or DNA repair.branch migration ͉ Holliday junction resolution ͉ homologous recombination H omologous recombination (HR) is responsible for the repair of DNA double-stranded breaks (DSB) and faithful chromosome segregation during meiosis (1). The Holliday junction (HJ) is a central intermediate of HR (2). HJs are thought to form during meiotic recombination, DSB repair, and repair of collapsed replication forks (3, 4). At late stages of HR or DNA repair, HJs, which constitute a physical link between DNA molecules, must be resolved to allow chromosome separation. Enzymes that cleave HJs, called HJ resolvases, are structure-specific endonucleases that introduce coordinated single-strand cuts across the junction (5). Several HJ resolvases from bacteriophages, eubacteria, archaea, eukaryotic viruses, and mitochondria have been identified, isolated, and characterized (5). However, the identity of eukaryotic nuclear HJ resolvases is still elusive.A role for Mus81 in HJ resolution was first proposed in studies on Schizosaccharomyces pombe, where Mus81 was identified as a critical factor for the production of viable spores, survival under conditions that lead to stalling of replication fork progression, and viability in the absence of RecQ helicase, Rqh1 (6). All of the defects in S. pombe mus81 mutants were rescued by expression of RusA, a bacteriophage resolvase that is highly specific for HJs (7). Mus81 protein is widely conserved among eukaryotes, including Saccharomyces cerevisiae (8), S. pombe (6), Arabidopsis thaliana (9), mice (10, 11), and humans (12). Mus81 is related to the XPF family of structure-specific endonucleases, which share a highly conserved motif (V/IERKX 3 D) that constitutes an integral part of the endonuclease catalytic site. Mus81 functions as a heterodimer with a noncatalytic p...

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“…Human and yeast Rad54 were purified as described previously in ref. 19. Untagged hRad54 was produced by proteolytic cleavage of GST-hRad54 with thrombin, as previously described in ref.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Mazina

Mazin

2008

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

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“…Previously, we showed that RAD54 has a similar effect on dsDNA binding by the RAD51 filament (47). Here, we wanted to test whether BLM may stimulate the DNA pairing activity of RAD51 by the same mechanism.…”

Section: Blmmentioning

confidence: 99%

“…Here, we wanted to test whether BLM may stimulate the DNA pairing activity of RAD51 by the same mechanism. As in our previous studies with RAD54 (47), we used a co-aggregation assay that measures formation of low speed sedimenting co-aggregates resulting from the homology-independent uptake of dsDNA by the RAD51-ssDNA filament (48). If BLM were to increase dsDNA binding affinity of the RAD51-ssDNA filament, we would expect to observe an increase in resistance of co-aggregates to solution ion strength.…”

Section: Blmmentioning

confidence: 99%

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Bugreev

Mazina

Mazin

2009

Journal of Biological Chemistry

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Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-singlestranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS), 2 a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7-9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13-15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18 -20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with . BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yea...

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“…Fig. 1B shows a kymograph (a plot of spatial position vs. time) obtained for the real-time assembly of Rad51 (100 nM) on a single DNA molecule in the presence of ATP (1 mM) and calcium acetate (2 mM) to prevent ATP hydrolysis (30,31). At the beginning of the reaction, the DNA molecule is Ϸ14.0 m in length for the flow rate used.…”

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.

133

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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Human Rad54 Protein Stimulates DNA Strand Exchange Activity of hRad51 Protein in the Presence of Ca2+

Cited by 74 publications

References 53 publications

Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Human Rad54 protein stimulates human Mus81–Eme1 endonuclease

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules

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