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.

Saccharomyces cerevisiae mutants defective in plasmid-chromosome recombination.

We have studied the recombinational repair of a double-strand break (DSB) in a plasmid-borne ade2::HO-site by an intact ade2 allele following the induction of a galactose-inducible GAL-HO gene. If GAL-HO expression is not attenuated by the presence of a low level of glucose in the galactose medium, deleterious effects are observed. Our comparison of the effects of several rad mutations on the relative efficiencies of DSB repair at both the ade2::HO-site and at the chromosomal MAT locus indicate that the two processes share common functions. Not surprisingly, most of the recombination-defective mutants found using our assay are alleles of genes in the RAD52 epistasis group. The recombination and repair deficiencies vary among the different mutant groups and also among mutants within a group. In general, there is a correlation between the extents of the recombination and repair defects. Our screen also turned up a novel rfa1 allele with a pronounced deficiency in DSB repair and recombination and a srs2 mutation which causes only a mild defect.

Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks.

A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endonuclease. Besides isolating a number of new alleles in known rad genes, we identified a novel allele of the RFA1 gene, rfa1-44, which encodes the large subunit of the heterotrimeric yeast single-stranded DNA-binding protein RPA. Characterization of rfa1-44 revealed that it is, like members of the RAD52 epistasis group, sensitive to X rays, high doses of UV, and HO-induced DSBs. In addition, rfa1-44 shows a reduced ability to undergo sporulation and HO-induced gene conversion. The mutation was mapped to a single-base substitution resulting in an aspartate at amino acid residue 77 instead of glycine. Moreover, all radiation sensitivities and repair defects of rfa1-44 are suppressed by RAD52 in a dose-dependent manner, and one RAD52 mutant allele, rad52-34, displays nonallelic noncomplementation when crossed with rfa1-44. Presented is a model accounting for this genetic interaction in which Rfa1, in a complex with Rad52, serves to assemble other proteins of the recombination-repair machinery at the site of DSBs and other kinds of DNA damage. We believe that our findings and those of J. Smith and R. Rothstein (Mol. Cell. Biol. 15:1632-1641, 1995) are the first in vivo demonstrations of the involvement of a eukaryotic single-stranded binding protein in recombination and repair processes.

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines.

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.

Resistance of cytotoxic T lymphocytes to the lytic effects of their toxic granules.

A cytotoxic T lymphocyte (CTL) characteristically kills target cells one after the other by releasing toxic granules that contain one or more cytolytic components. To determine how CTLs avoid destroying themselves when they release granules and lyse target cells, 7 murine CD8+ CTL cell lines were compared with 19 other cell lines for susceptibility to lysis by the isolated toxic granules. Murine CD8+ CTLs were clearly the most resistant cells: granules did not lyse them even after they were exposed to azide, cyanide, and 2-deoxyglucose, conditions that were found to enhance the susceptibility of all the other cells tested, including other T cells. Thus, resistance of CD8+ CTLs to cytotoxic granules appears to be independent of cellular ATP. To reconcile these findings with other observations that, under some circumstances, CTLs can be lysed by other CTLs, we suggest a model in which a CTL releases only a limited proportion of its toxic granules at each antigen-specific encounter with a target cell; the amount released is sufficient to kill most target cells but to leave the CTL undamaged and with enough granules to attack other target cells.