A Novel Function of Rad54 Protein

Mazin, Alexander V.; Alexeev, Andrei; Kowalczykowski, Stephen C.

doi:10.1074/jbc.m212779200

Cited by 158 publications

(62 citation statements)

References 57 publications

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“…11 and 12). The interaction with the presynaptic Rad51-ssDNA filament targets Rad54 to the pairing site (20,39,40), where Rad51 will be dsDNA-bound after DNA-strand exchange (Fig. 3c).…”

Section: Discussionmentioning

confidence: 99%

Terminal association of Rad54 protein with the Rad51–dsDNA filament

Kiianitsa

Solinger

Heyer

2006

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

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Rad54 protein is a Snf2-related dsDNA-specific ATPase essential for homologous recombination mediated by Rad51 protein, the eukaryotic RecA ortholog. Snf2-related enzymes couple ATP hydrolysis with translocation on dsDNA to remodel or dissociate a wide variety of protein-dsDNA complexes. Rad54 and Rad51 interact through species-specific contacts and mutually stimulate their biochemical activities. Specifically, Rad51 bound to dsDNA, the product of homologous recombination after DNA-strand exchange, stimulates the Rad54 ATPase up to 6-fold, leading to the turnover of Rad51 in the product complex. Electron microscopy visualized the Rad51-Rad54 interaction on dsDNA, showing that an oligomeric form of Rad54 associates preferentially with termini of the Rad51-dsDNA filament. Our data support a mechanism of processive dsDNA-Rad51 filament dissociation by the translocating Rad54 protein.recombination ͉ ATPase ͉ Snf2-like proteins H omologous recombination (HR) is a high-fidelity, templatedependent pathway involved in the nonmutagenic tolerance of DNA damage, the repair of complex DNA damage, and the recovery of stalled and collapsed replication forks (1, 2). The Rad52 group proteins define the HR pathway. Initially, RPA, the eukaryotic ssDNA-binding protein, binds to ssDNA exposed by doublestrand break processing or discoordination of leading and lagging strands at stalled replication forks. Mediator proteins (Rad52, Brca2, and Rad51 paralogs) orchestrate the assembly of Rad51 protein on ssDNA to form the nucleoprotein filament, which performs homology search and DNA-strand invasion to prime repair synthesis by using the undamaged sister chromatid or homolog as a template. After resolution of the pairing intermediates, HR has restored the contiguity of the chromosomes with a crossover or noncrossover outcome.Eukaryotes contain a large number of Snf2-related proteins (3, 4). This group of proteins shares structural and sequence similarity with SF2 DNA helicases, but they are unable to catalyze strand separation typical for DNA helicases (5). Instead, these proteins likely translocate on dsDNA inducing topological changes. Using the energy of ATP hydrolysis, their action leads to the remodeling or dissociation of protein-dsDNA complexes (4, 6). Prominent members of this group are the chromatin-remodeling factors Snf2, Isw1, Isw2, or Chd1. Each eukaryotic genome codes for a variety of Snf2-related proteins. Saccharomyces cerevisiae alone has 17 paralogs, and a number of these proteins appear to function outside a nucleosome remodeling context; for example, the Mot1 protein was found to dissociate the TATA box-binding protein from dsDNA (7). DNA repair pathways are richly endowed with specific, largely nonredundant Snf2-related proteins that function in transcriptioncoupled repair (Cockayne's Syndrome B͞Rad26 in S. cerevisiae; ref. Rad54 is a critical member of the Rad52 group, and its absence leads to catastrophic sensitivity to double-strand breaks in yeast (2,11,12). This protein displays robust dsDNA-specific ATPase ac...

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“…11 and 12). The interaction with the presynaptic Rad51-ssDNA filament targets Rad54 to the pairing site (20,39,40), where Rad51 will be dsDNA-bound after DNA-strand exchange (Fig. 3c).…”

Section: Discussionmentioning

confidence: 99%

Terminal association of Rad54 protein with the Rad51–dsDNA filament

Kiianitsa

Solinger

Heyer

2006

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

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“…The Rad55-Rad57 heterodimer also possesses a recombination mediator activity (18,41,45), and Rad54 is capable of stabilizing the Rad51 presynaptic filament (21,46). In addition, the Rad59 protein binds DNA (47) and associates with Rad51, Rad52, and RPA in a complex (48).…”

Section: New Insights Concerning the Role Of Rad52 In Hr-rad52mentioning

confidence: 99%

Molecular Anatomy of the Recombination Mediator Function of Saccharomyces cerevisiae Rad52

Seong

Sehorn

Plate

et al. 2008

Journal of Biological Chemistry

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A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52 with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function. Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNAbound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad52 in the rad52 ⌬327 background enhances the association of Rad51 protein with a HO-made DNA double-strand break and partially complements the methylmethane sulfonate sensitivity of the mutant cells. Our results provide a mechanistic framework for rationalizing the multi-faceted role of Rad52 in recombination and DNA repair.

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“…In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of HR (6 -8). Rad54 protein is shown to interact with and stabilize the Rad51 nucleoprotein filament, stimulating its DNA pairing activity (9,10). Interestingly, although Rad52 protein plays a pivotal role in DSB repair in Saccharomyces cerevisiae, the role of vertebrate and Schizosaccharomyces pombe Rad52 is much less significant (11)(12)(13).…”

mentioning

confidence: 99%

Hypersensitivity of Nonhomologous DNA End-joining Mutants to VP-16 and ICRF-193

Adachi

Suzuki

Iiizumi

et al. 2003

Journal of Biological Chemistry

139

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A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA doublestrand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4 ؊/؊ and KU70 ؊/؊ cells, which are defective in nonhomologous DNA endjoining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54 ؊/؊ cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.DNA double-strand breaks (DSBs) 1 can be caused by a variety of exogenous and endogenous agents, posing a major threat to genome integrity. If left unrepaired, DSBs may cause cell death (1, 2). Vertebrate cells have evolved two major pathways for repairing DSBs, homologous recombination (HR) and nonhomologous DNA end-joining (NHEJ) (2)(3)(4)(5).With the use of homologous DNA sequences, HR allows for accurate repair of DSBs. In eukaryotic cells, the HR reaction is performed by a wide variety of proteins including Rad51, Rad52, and Rad54 (2). In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of HR (6 -8). Rad54 protein is shown to interact with and stabilize the Rad51 nucleoprotein filament, stimulating its DNA pairing activity (9, 10). Interestingly, although Rad52 protein plays a pivotal role in DSB repair in Saccharomyces cerevisiae, the role of vertebrate and Schizosaccharomyces pombe Rad52 is much less significant (11)(12)(13).In contrast to accurate repair by HR, NHEJ can lead to imprecise joining of DSB ends. It has been well established that NHEJ is responsible for V(D)J recombination in lymphocytes (3, 5). The NHEJ reaction relies on Ku (a heterodimer of Ku70 and Ku86), DNA-PKcs, Artemis, Xrcc4, and DNA ligase IV (the LIG4 gene product) (3,5,14). Extensive biochemical studies propose a model for the mechanism of NHEJ (3,5,14,15). First, Ku binds to the ends of a DSB and recruits the DNAPKcs⅐Artemis complex. This complex would then trim the ends to make the ends ligatable. A...

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A Novel Function of Rad54 Protein

Cited by 158 publications

References 57 publications

Terminal association of Rad54 protein with the Rad51–dsDNA filament

Terminal association of Rad54 protein with the Rad51–dsDNA filament

Molecular Anatomy of the Recombination Mediator Function of Saccharomyces cerevisiae Rad52

Hypersensitivity of Nonhomologous DNA End-joining Mutants to VP-16 and ICRF-193

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