Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A

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. We find that Rad52 protein stimulates 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.     

Stimulation by Rad52 of yeast Rad51-mediated recombination

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 homologue of the recA gene and 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. Homologues 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. Here we show that Rad52 protein stimulates Rad51 reactions and that binding to Rad51 is necessary for this stimulatory effect. We conclude that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments.     

Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA

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. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, 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 (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729-10734, 1996). 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.     

The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences

The Rad51 protein of Saccharomyces cerevisiae is a eukaryotic homolog of the RecA protein, the prototypic DNA strand-exchange protein of Escherichia coli. RAD51 gene function is required for efficient genetic recombination and for DNA double-strand break repair. Recently, we demonstrated that RecA protein has a preferential affinity for GT-rich DNA sequences-several of which exhibit enhanced RecA protein-promoted homologous pairing activity. The fundamental similarity between the RecA and Rad51 proteins suggests that Rad51 might display an analogous bias. Using in vitro selection, here we show that the yeast Rad51 protein shares the same preference for GT-rich sequences as its prokaryotic counterpart. This bias is also manifest as an increased ability of Rad51 protein to promote the invasion of supercoiled DNA by homologous GT-rich single-stranded DNA, an activity not previously described for the eukaryotic pairing protein. We propose that the preferred utilization of GT-rich sequences is a conserved feature among all homologs of RecA protein, and that GT-rich regions are loci for increased genetic exchange in both prokaryotes and eukaryotes.     

A novel nucleic acid-binding protein that interacts with human rad51 recombinase

Using the yeast two-hybrid system, we isolated a cDNA encoding a novel human protein, named Pir51, that strongly interacts with human Rad51 recombinase. Analysis in vitro confirmed the interaction between Rad51 and Pir51. Pir51 mRNA is expressed in a number of human organs, most notably in testis, thymus, colon and small intestine. The Pir51 gene locus was mapped to chromosome 12p13.1-13. 2 by fluorescence in situ hybridization. The Pir51 protein was expressed in Escherichia coli and purified to near homogeneity. Biochemical analysis shows that the Pir51 protein binds both single- and double-stranded DNA, and is capable of aggregating DNA. The protein also binds RNA. The Pir51 protein may represent a new member of the multiprotein complexes postulated to carry out homologous recombination and DNA repair in mammalian cells.     

Interaction of Rad51 with ATP and Mg2+ induces a conformational change in Rad51

The presumptive first step in the Rad51-promoted formation of joint molecules is binding of the protein to ssDNA in the presence of ATP and Mg2+. In this paper, we report that Rad51's ability to bind DNA is rapidly inactivated when incubated at 30-37 degrees C but is stabilized by the presence of ATP and Mg2+. Although unable to promote binding to DNA, ATP-gamma-S also prevents inactivation of Rad51 at 37 degrees C. AMP-P-N-P lacks this property, while ADP protects partially but only at 5-10 times higher concentrations than ATP. These observations correlate with the dissociation constant of those nucleotides for Rad51 determined by equilibrium dialysis. Rad51 binds ATP and ATP-gamma-S with a 1:1 stoichiometry and Kds of 21 and 19 microM, respectively. The presence of DNA significantly increases the affinity of Rad51 for ATP, while DNA has a smaller effect on the affinity of ATP-gamma-S. Competition binding studies show that ADP and AMP-P-N-P bind with a 5- and 55-fold lower affinity, respectively, than ATP. The CD spectrum of Rad51 with negative double minima at around 210 and 222 nm is characteristic of an alpha-helical protein. Upon binding ATP and Mg2+, the CD spectrum is altered in the regions 194-208 and 208-235 nm, changes that are indicative of a more structured state; this change does not occur with Rad51 that has been inactivated at 37 degrees C. We surmise that the active conformation is more resistant to inactivation at elevated temperature. Our data suggest that one of the roles of ATP and Mg2+ in Rad51-mediated strand exchange is to induce the proper protein structure for binding the two DNA substrates.     

The DNA binding properties of Saccharomyces cerevisiae Rad51 protein

Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homologue the Escherichia coli RecA protein, we analyzed the DNA binding properties of the Rad51 protein. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of protein. Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6-9 nucleotides or base pairs. Therefore, Rad51 protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it.     

Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA. In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme. Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1. Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins. We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6. This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins. Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18. Each of these regions of Rad6 is indicated to form an amphipathic helix.     

The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A

The human Rad51 protein is homologous to the RecA protein and catalyses homologous pairing and strand transfer reactions in vitro. Using single-stranded circular and homologous linear duplex DNA, we show that hRad51 forms stable joint molecules by transfer of the 5' end of the complementary strand of the linear duplex to the ssDNA. The polarity of strand transfer is therefore 3' to 5', defined relative to the ssDNA on which hRad51 initiates filament formation. This polarity is opposite to that observed with RecA. Homologous pairing and strand transfer require stoichiometric amounts of hRad51, corresponding to one hRad51 monomer per three nucleotides of ssDNA. Joint molecules are not observed when the protein is present in limiting or excessive amounts. The human ssDNA binding-protein, hRP-A, stimulates hRad51-mediated reactions. Its effect is consistent with a role in the removal of secondary structures from ssDNA, thereby facilitating the formation of continuous Rad51 filaments.     

Human Rad51 protein can form homologous joints in the absence of net strand exchange

The eukaryotic homologs of RecA protein are central enzymes of recombination and repair, and notwithstanding a high degree of conservation they differ sufficiently from RecA to offer insights into mechanisms and biological roles. The yield of DNA strand exchange reactions driven by both Escherichia coli RecA protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC composition, HsRad51 promoted less exchange than RecA. When 40% of bases were GC pairs, the rate constant for strand exchange by HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA. The ability of HsRad51 to form joints in the absence of net strand exchange was confirmed by experiments in which heterologous blocks at both ends of linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization. These findings suggest that HsRad51 acting alone on human DNA in vivo is a pairing protein that cannot form extensive heteroduplex DNA.     

The self as a mediator between personality and adjustment.

The self can be conceptualized as a mediating agent that translates personality into situated goal-directed activities and adaptation. This research used a level-of-analysis approach to link personality dimensions (Level 1) to self-systems (Level II) and to teacher ratings of adjustment in African American, Mexican American, and European American students (N?=?317). The authors hypothesized that links among aspects of self-esteem and teacher ratings of adjustment would be domain specific, and those links to dimensions of the 5-factor model would reflect the domain specificity. Structural equation modeling corroborated hypotheses about domain specificity in links between adjustment and 5-factor dimensions. Results were discussed in terms of levels of analysis for personality structure, personality development, and age-related adaptations to social contexts. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Psychache as a mediator in the relationship between perfectionism and suicidality.

E. S. Shneidman (1993) has proposed that psychache (i.e., unbearable psychological pain) is directly associated with suicide and mediates the effects of all other relevant psychological factors. The present research tested this proposition by examining whether psychache mediates the relationship between perfectionism and suicidality. Furthermore, the link between perfectionism and psychache was examined for mediation by unfulfilled psychological needs. Participants were 264 undergraduate students. Structural equation modeling with bootstrapped estimates determined that psychache fully mediated the relationship between socially prescribed perfectionism and suicidality. Additionally, the relationship between socially prescribed perfectionism and psychache was partially mediated by unfulfilled psychological needs. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Self-esteem as a mediator between perfectionism and depression: A structural equations analysis.

This study of college students (N?=?464) examined the association between adaptive and maladaptive dimensions of perfectionism and 2 mental health outcomes (self-esteem and depression). Confirmatory factor analysis was used to develop and assess the measurement model used in this study. Structural equations modeling was used to test a mediational model derived from prior theory and research. Analyses supported the existence of 2 perfectionism factors. Path models revealed that adaptive perfectionism was not directly or indirectly (through self-esteem) associated with depression. Maladaptive perfectionism was negatively associated with self-esteem and positively associated with depression. Self-esteem also buffered the effects of maladaptive perfectionism on depression. Distinguishing adaptive from maladaptive perfectionism is discussed in the context of recommendations for practice and future research. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically 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 a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects 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. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.     

Fit as a mediator of the relationship between work hours and burnout.

The authors studied number of hours worked and estimated its relationship to burnout in a nonrandom sample of 141 married physicians. It was hypothesized that this relationship is mediated by a process called fit, conceptualized as the extent to which workers realize the various components of their work–family strategies. Results of structural equation modeling supported the mediation hypothesis. Employees whose work hours are more or fewer than they and their partner prefer and whose work hours are distributed differently than they and their partner prefer will be more disengaged, distracted, and alienated at work than will their counterparts who are working their preferred schedules. Thus, the relationship between number of hours worked and burnout depends on the extent to which work schedules meet the needs of the worker, her or his partner, and their children, if any. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Functional and physical interaction between Rad24 and Rfc5 in the yeast checkpoint pathways

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1. RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although the rad24Delta mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect in rfc5-1 mutants. Furthermore, overexpression of RAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.     

Visualisation of human rad52 protein and its complexes with hRad51 and DNA

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.     

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.     

Bodily shame as a mediator between abusive experiences and depression.

The role of bodily shame as a mediator between sexual or physical abuse and depression was investigated in a community sample of 101 women who had been followed for 8 years. In general, childhood and adult abuse were related to the occurrence of depression in the study period but when both were considered together, only adult abuse showed an independent association. However, childhood and adult abuse were both independently related to chronic or recurrent depression. Bodily shame was related to childhood abuse, and this association could not be accounted for by bodily dissatisfaction or low self-esteem. Bodily shame, but not childhood abuse, was related to chronic or recurrent depression when both factors were considered together and current depressive symptoms were controlled. (PsycINFO Database Record (c) 2010 APA, all rights reserved)