The SNF2 family of proteins includes representatives from a variety of species: these proteins participate in a variety of roles in cellular processes, such as transcriptional regulation (e.g. MOT1, SNF2 and BRM) and the maintenance of chromosome stability during mitosis (e.g. lodestar) and various aspects of the processing of DNA damage, including nucleotide excision repair (e.g. RAD16 and ERCC6), recombinational pathways (e.g. RAD54) and post-replication daughter strand gap repair (e.g. RAD5). This family also includes many proteins with no known function. To better characterize this family of proteins, molecular phylogenetic techniques have been used to infer evolutionary relationships among the family members. The SNF2 family has been divided into multiple subfamilies, each of which represents what is proposed to be a functionally and evolutionarily distinct group. Subfamily structure can be used to predict the functions of some of the uncharacterized proteins in the SNF2 family (Eisen, 1995).

Yeast Rad54, a homolog of Drosophila Okra

The RAD54 and RAD51 (see Drosophila Rad51-like) genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. The Rad54 protein interacts with the Rad51 protein both in vivo and in vitro, and the NH2-terminal 115 residues of the Rad54 protein are shown to be necessary for this interaction. These data imply that the Rad54 protein is part of a multiprotein yeast recombination complex (Jiang, 1996).

The RAD54 gene of Saccharomyces cerevisiae encodes a putative helicase, which is involved in the recombinational repair of DNA damage. The RAD54 homolog of the fission yeast Schizosaccharomyces pombe (rhp54+) was isolated by using the RAD54 gene as a heterologous probe. The gene is predicted to encode a protein of 852 amino acids. The overall homology between the mutual proteins of the two species is 67%, with 51% identical amino acids and 16% similar amino acids. A rhp54 deletion mutant is very sensitive to both ionizing radiation and UV. Fluorescence microscopy of the rhp54 mutant cells reveals that a large portion of the cells are elongated and occasionally contain aberrant nuclei. FACS analysis shows an increased DNA content in comparison with wild-type cells. Through a minichromosome-loss assay it was shown that the rhp54 deletion mutant has a very high level of chromosome loss. The rhp54 mutation in either a rad17 or a cdc2.3w mutant background (where the S-phase/mitosis checkpoint is absent) shows a significant reduction in viability. It is hypothesized that the rhp54+ gene is involved in the recombinational repair of UV and X-ray damage and plays a role in the processing of replication-specific lesions (Muris, 1996).

Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases. Both proteins are also important members of the RAD52 group, which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae. Rad51 and Rad54 proteins are shown by genetic, molecular and biochemical criteria to interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression is observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. These data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. Since both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms (Clever, 1997).

The Saccharomyces cerevisiae RAD51 and RAD54 genes are both required for the occurrence of homologous recombination and for the repair of double-stranded DNA breaks. Previous studies have indicated that Rad51 protein, together with the single-stranded DNA-binding factor replication protein A (RPA), can promote the formation of heteroduplex DNA, which is a key intermediate in homologous recombination. Rad54 protein has now been purified to near homogeneity and its molecular function has been biochemically tested. Rad54 protein possesses a double-stranded DNA-dependent ATPase activity, and it interacts with the Rad51 protein. Addition of Rad54 protein to reactions containing Rad51 strongly stimulates the rate of pairing between homologous single-stranded and double-stranded DNA molecules. It is concluded that Rad54 acts to overcome kinetic impediments that would limit homologous DNA pairing between recombining chromosomes in vivo (Petukhova, 1998).

To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, a synthetic lethal screen was performed with the yeast pol30-104 mutation. Nine mutations were identified that display synthetic lethality with pol30-104; three mutations affect the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affect three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52 and rad57). pol30-104 is also found to displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. Mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1delta) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments (Merrill, 1998).

The Schizosaccharomyces pombe rhp51+, rad22+ and rhp54+ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Delta and rhp54Delta strains are extremely sensitive to ionizing radiation; the rad22Delta mutant turns out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Delta and rhp54Delta are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Delta mutant is only reduced by a factor of two. Blot-analysis demonstrates that the majority of the leu+ transformants of the wild-type and rad22Delta strains have integrated one or more copies of the vector. Gene conversion events are observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Delta and a rhp54Delta background. Meiotic recombination is hardly affected in the rad22Delta mutant. The rhp51Delta and rhp54Delta strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Delta strain and 27% in the rhp54Delta strain, as compared with wild-type cells. However, in the rhp51Delta mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe (Muris, 1997).

Most mitotic recombination and repair genes of Saccharomyces cerevisiae show no specificity of action for the genome ploidy. A novel repair and recombination gene is described that is specific for recombination and repair between homologous chromosomes. The RDH54 gene is homologous to the RAD54 gene, but rdh54 mutants do not show sensitivity to methyl methanesulfonate at concentrations that sensitize a rad54 mutant. However, the rdh54 null mutation enhances the methyl methanesulfonate sensitivity of a rad54 mutant and single rdh54 mutants are sensitive to prolonged exposure at high concentrations of methyl methanesulfonate. The RDH54 gene is required for recombination, but only in a diploid. Evidence is presented showing that the RDH54 gene is required for interhomolog gene conversion but not intrachromosomal gene conversion. The rdh54 mutation confers diploid-specific lethalities and reduced growth in various mutant backgrounds. These phenotypes are due to attempted recombination. The RDH54 gene is also required for meiosis, since homozygous mutant diploids show very poor sporulation and reduced spore viability (Klein, 1997).

The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project has discovered a homolog of RAD54: RDH54/TID1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. In contrast, the rdh54/tid1 single mutant does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, yet it shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of R4D54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins (Shinohara, 1997).

Repairing a double-strand break by homologous recombination requires binding of the strand exchange protein Rad51p to ssDNA, followed by synapsis with a homologous donor. Chromatin immunoprecipitation has been used to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HMLα donor. Localization of Rad51p to MAT precedes its association with HML, providing evidence of the time needed for the Rad51 filament to search the genome for a homologous sequence. Rad51p binding to ssDNA requires Rad52p. The absence of Rad55p delays Rad51p binding to ssDNA and prevents strand invasion and localization of Rad51p to HMLα. Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HMLα; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand (Sugawara, 2003).

Repair of a double-strand break (DSB) often occurs by gene conversion, a homologous recombination event. Homologous recombination depends on a search for homology by the ends of the broken chromosome to locate an intact donor sequence that could be used as a template for DNA repair. In budding yeast and other eukaryotes, the search for homology is facilitated by the DNA strand exchange protein, Rad51p, the homolog of the bacterial RecA protein. In vitro, Rad51p, like RecA, forms extensive Rad51p-DNA filaments, with 3 bp bound per monomer. The RecA or Rad51p filament can catalyze strand exchange between ssDNA and a homologous dsDNA. This process is greatly facilitated by prior exposure of the ssDNA to a ssDNA binding protein, SSB in bacteria, or RPA in eukaryotes. These ssDNA binding proteins appear to facilitate the polymerization of RecA/Rad51p across ssDNA regions that can form secondary structure (Sugawara, 2003 and references therein).

Incubation of RPA with ssDNA before the addition of Rad51p, however, reduces formation of Rad51 filaments. Either Rad52p or the Rad55p/Rad57p heterodimer can overcome this inhibition and mediate the loading of Rad51p onto ssDNA. Rad54p shares sequence similarity with the Swi2/Snf2 family of chromatin-remodeling proteins and appears to act at a later step in strand exchange, to extend heteroduplex DNA, and to alter DNA conformation during the synaptic and/or postsynaptic phases of strand exchange. In vivo, Rad51p-mediated recombination in S. cerevisiae requires the participation of Rad52p, Rad54p, Rad55p, and Rad57p. Deletions of RAD52 are the most defective in spontaneous and DSB-induced recombination, since this protein is required both for Rad51p-mediated and Rad59p-mediated recombination events. The requirements for Rad55p and Rad57p are the least stringent, as defects are often seen only at low temperature. This defect can be suppressed by the overexpression of Rad51p (Sugawara, 2003 and references therein).

One of the best-studied homologous recombination events is the HO endonuclease-induced switching of the MATa locus, using HMLα as the donor template during gene conversion. A galactose-inducible GAL::HO gene provides the means to induce the DSB synchronously in all cells of the population. Physical monitoring of MAT DNA has shown that the DSB ends are first resected by 5' to 3' exonucleases, presumably so that the 3'-ended ssDNA can recruit the Rad51p strand exchange protein and undergo strand invasion of the donor and initiate new DNA synthesis. Although the formation of a strand invasion intermediate has not been directly demonstrated, the next step in the process, the use of the 3' end of the invading strand as a primer to copy the template DNA, has been detected by PCR. This was observed to occur about 30 min after the appearance of the DSB and 30 min before the recombination event was completed. HO cleavage and 5' to 3' resection are normal in mutant cells lacking the auxiliary recombination proteins Rad52p, Rad54p, Rad55p, and Rad57p, but the primer extension step does not occur; however, in some strain backgrounds there is a small amount of MAT switching in rad54Δ mutants (Sugawara, 2003 and references therein).

To determine more precisely the recombination steps that are prevented in the absence of the auxiliary proteins, chromatin immunoprecipitation (ChIP) was used to follow the ability of Rad51p to associate with ssDNA formed at MAT and with DNA sequences at HML as well as a model for the synapsis of MAT and HML during gene conversion. This approach has enabled distinct functions for Rad52p, Rad54p, and Radd55p/Rad57p to be seen in the Rad51p-mediated process. It is concluded that Rad52p is required for the recruitment of Rad51p to a DSB, whereas the rate of recruitment is reduced or delayed in the absence of Rad55p/Rad57p. At the step of strand exchange, Rad55p/Rad57p is required for recruitment of Rad51p to the donor whereas Rad54p is not, suggesting that synaptic association between MAT and the HML donor does not need Rad54p. Rad54p is required at a postsynaptic step to enable the completion of DNA repair (Sugawara, 2003).

Rad54 protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. This study addresses the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. Saccharomyces cerevisiae Rad54 is shown to exert distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 protein but to a lesser extent. In contrast, the interaction of Rad54 protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 protein hydrolyzes ATP in a reduced mode at 60%-80% of its rate on protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. It is suggested that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination (Kiianitsa, 2002).

Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. It is concluded that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process (Mazin, 2000).

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carries out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibits dsDNA-specific ATPase activity. Rad54 protein stimulates Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation is accompanied by a concurrent increase in the formation of heteroduplex DNA. These results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein does not stimulate DNA strand exchange by increasing presynaptic complex formation. It is concluded that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments (Solinger, 2001a).

Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depends on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins (Solinger, 2001b).

Rad54 protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Rad54 protein dissociates Rad51 from nucleoprotein filaments formed on dsDNA. Addition of Rad54 protein overcomes inhibition of DNA strand exchange by Rad51 protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underline the importance of specific Rad54-Rad51 protein interactions. Rad51 protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. It is suggested that Rad54 protein is involved in the turnover of Rad51-dsDNA filaments (Solinger, 2002).

Saccharomyces cerevisiae Rad51, Rad54, and replication protein A (RPA) proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating single-stranded DNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities. RPA is still indispensable for optimal reaction efficiency, but its role in this instance is to sequester free single-stranded DNA, which otherwise inhibits Rad51 and Rad54 functions. Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction (Van Komen, 2002).

In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, Rad52, Rad55 and Rad57 proteins. In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein. Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dependent ATPase activity and changes DNA topology in an ATP hydrolysis-dependent manner. Rad54 catalyzes bidirectional nucleosome redistribution by sliding nucleosomes along DNA. Nucleosome redistribution is greatly stimulated by the Rad51 nucleoprotein filament but does not require the presence of homologous single-stranded DNA within the filament. On the basis of these data, it is proposed that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination (Alexeev, 2003).

Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. In this study, using physical criteria, the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein has been demonstrated directly. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein (Mazin, 2003).

The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. It is suggested that the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding (Fung, 2006).

The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic interhomologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. This study describes an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, it has been demonstrated that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. Evidence is presented that the Rad51 binding domain resides within the N terminus of Rdh54. Accordingly, N-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54Delta mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54.Rad51 complex formation. The Rdh54 expression and purification procedures described in this study should facilitate the functional dissection of this DNA recombination/repair factor (Chi, 2006).

The meiosis-specific recombinase Dmc1 plays a critical role in DNA strand exchange in budding yeast. Tid1/Rdh54, a member of the Swi2/Snf2 family of DNA translocases, has been shown to stimulate Dmc1-dependent recombination. Tid1 and its budding yeast paralog Rad54 have a variety of biochemical activities that may contribute to their biological function. This study demonstrates that Dmc1 can associate with chromatin in the absence of DNA double-strand breaks (DSBs), and Tid1 suppresses this association. Chromatin immunoprecipitation experiments indicate that an activity shared by Tid1 and Rad54 is required for normal assembly of Dmc1 at DSB sites in preparation for recombination. These results lead to a model in which the ATP hydrolysis-dependent DNA translocase activity of Tid1 acts to promote dissociation of Dmc1 from nonreombinogenic sites on chromatin, with Rad54 being able to substitute for this function in the absence of Tid1. The tendency of Dmc1 to form unproductive interactions with chromatin is proposed to be a consequence of the mechanism of strand exchange. The results raise the possibility that ATP hydrolysis-dependent disruption of nonproductive recombinase-DNA interactions is a feature shared with other homologous recombination systems (Holzen 2006).

Rad52, meiosis and repair in yeast

Homologous recombination in Saccharomyces cerevisiae is critically dependent on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is an ssDNA-binding protein that is also crucial to the recombination process. Rad52 protein is shown to effect the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not (Sugiyama, 1998).

At meiosis, a 5'-end-specific resection (paring back) of DSBs produces 3' single strand DNA tails. Several lines of evidence indicate that this ssDNA is acted upon by RPA, Rad51, Rad52, Rad55, and Rad57 proteins as an early step in homologous recombination. A model is presented for the repair of the resected DSBs. Initially, RPA, a protein that is abundant in the yeast cell, binds the ssDNA produced by resection. Although resection is likely to be concurrent with assembly of the recombination apparatus, RPA plays an essential early step because assembly of the recombination proteins is typically kinetically slower than the binding of an ssDNA binding protein; also, because physical analysis demonstrates that these ssDNA tails are approximately 1 kb long, RPA will be needed to prevent the formation of an intramolecular ssDNA secondary structure, which is inhibitory to both Rad51 and Rad52 protein function. This RPA-ssDNA complex then undergoes one of two alternative fates. One fate is replacement of RPA by Rad51 protein, aided by Rad52 and Rad55-Rad57 proteins, to produce a contiguous Rad51 protein-ssDNA filament. This Rad51 protein-ssDNA complex could then invade homologous dsDNA to produce homologously paired joint molecules with the associated exchange of DNA strands. An alternative fate is the annealing of the RPA-ssDNA complex to complementary ssDNA by Rad52 protein. In this process Rad52 protein interacts with RPA via protein-protein interaction. Interestingly, Rad52 protein, as well as RPA, is required for both these processes. This coincides with the fact that RAD52 function is required for most homologous recombination in S. cerevisiae (Sugiyama, 1998 and references).

Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that similar foci are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required, supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes (Gasior, 1998).

The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. A rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability. The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented (Hays, 1998).

The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination. A RecA homolog, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein. Purified Rad52 protein is shown to bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Electron microscope observations reveal that Rad52 molecules form multimeric rings. An increase in the intensity of fluorescence when Rad52 is bound to epsilonDNA shows an alteration of the structure of ssDNA. RPA binds to Rad52 and enhances the annealing of complementary ssDNA molecules. This enhancement is not observed in Escherichia coli SSB protein or T4 phage gp32 protein. It is concluded that Rad52 forms a ring-like structure and binds to ssDNA. Its structure and DNA binding properties are different from those of Rad51. The interaction of Rad52 with RPA plays an important role in the enhancement of annealing of complementary ssDNAs. It is therefore proposed that Rad52 mediates the RAD51-independent recombination through an ssDNA annealing, assisted by RPA (Shinohara, 1998b).

The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. Rad52 protein is found to stimulate DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required (New, 1998).

In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homolog of the recA gene; the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologs of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Rad52 protein is shown to stimulate Rad51 reactions and binding to Rad51 is necessary for this stimulatory effect. It is concluded that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments (Shinohara, 1998a).

DMC1, the meiosis-specific eukaryotic homolog of bacterial recA, is required for completion of meiotic recombination and cell cycle progression past prophase. In a dmc1 mutant, double strand break recombination intermediates accumulate and cells arrest in prophase. Genes have been isolated which, when present at high copy numbers, suppress the meiotic arrest phenotype conferred by dmc1 mutations. Among the genes isolated were two which suppress arrest by altering the recombination process. REC114 suppresses formation of double strand break (DSB) recombination intermediates. The low viability of spores produced by dmc1 mutants carrying high copy numbers of REC114 is rescued when reductional segregation is bypassed by mutation of spo13. High copy numbers of RAD54 suppress dmc1 arrest, promote DSB repair, and allow formation of viable spores following reductional segregation. Analysis of the combined effects of a null mutation in RED1, a gene required for meiotic chromosome structure, with null mutations in RAD54 and DMC1 shows that RAD54, while not normally important for repair of DSBs during meiosis, is required for efficient repair of breaks by the intersister recombination pathway that operates in red1 dmc1 double mutants. It is concluded that over-expression of REC114 suppresses meiotic arrest by preventing formation of DSBs. High copy numbers of RAD54 activate a DMC1-independent mechanism that promotes repair of DSBs by homology-mediated recombination. The ability of RAD54 to promote DMC1-independent recombination is proposed to involve suppression of a constraint that normally promotes recombination between homologous chromatids rather than sisters (Bishop, 1999).

Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, three distinct foci are observed that colocalize with adjacent GFP chromosome marks. The kinetics of focus formation with recombination intermediates and products were compared when HO-cleaved MATalpha recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. Evidence is presented for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52 (Miyazaki, 2004; full text of article).

Plant Rad54 homologs and RNA-directed DNA methylation

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for Arabidopsis mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily (Drosophila homolog: Okra). In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome (Kanno, 2004).

Whether DRD1 is involved in RNA-directed de novo methylation or acts to maintain RNA-induced non-CpG methylation remains to be determined. However, the heavy loss in drd1 plants of CpNpN methylation, which is not efficiently maintained in the absence of the RNA trigger, suggests a direct relationship between DRD1 activity and RNA signals. Given the relatedness of DRD1 to RAD54, it is intriguing to consider possible mechanistic similarities between RNA-directed DNA methylation and homologous DNA repair. In each case, the respective chromatin-remodeling factor could facilitate a homology search on duplex DNA, nucleosome displacement, and DNA unpairing and thereby allow heteroduplex formation and recruitment of enzyme complexes. In the RNA-directed DNA methylation pathway, this could create an RNA-DNA hybrid that attracts DMTases, thus accounting for the extraordinary specificity of cytosine methylation, which is largely restricted to the region of RNA-DNA sequence similarity. The lack of DRD1 homologs outside of the plant kingdom may mean that RNA-directed DNA methylation occurs only in plants. Alternatively, RAD54 or ATRX-like proteins may serve this function in other organisms (Kanno, 2004).

Mammalian RAD54 homologs

continued: okra Evolutionary homologs part 2/2 |
okra: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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