In Saccharomyces cerevisiae, HO endonuclease-induced mating-type (MAT) switching is a specialized mitotic recombination event in which MAT sequences are replaced by those copied from a distant, unexpressed donor (HML or HMR). The donors have a chromatin structure inaccessible for both transcription and HO cleavage. Here we use physical monitoring of DNA to show that MAT switching is completely blocked at an early step in recombination in strains deleted for the DNA repair genes RAD51, RAD52, RAD54, RAD55 or RAD57. We find, however, that only RAD52 is required when the donor sequence is simultaneously not silenced and located on a plasmid. RAD51, RAD54, RAD55 and RAD57 are still required when the same transcribed donor is on the chromosome. We conclude that recombination in vivo occurs between DNA molecules in chromatin, whose structure significantly influences the outcome. RAD51, RAD54, RAD55 and RAD57 are all required to facilitate strand invasion into otherwise inaccessible donor sequences.

We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains. Both the MATa-stk (J. E. Haber, W. T. Savage, S. M. Raposa, B. Weiffenbach, and L. B. Rowe, Proc. Natl. Acad. Sci. USA 77:2824-2828 and MATa-survivor (R. E. Malone and D. Hyman, Curr. Genet. 7:439-447, 1983) alleles result from a T-*A base change at position Zll of the MAT locus. These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MATa switches to MATa. Mating-type switching in a MATa-stk strain relative to a MATa Z1lT strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter. We confirmed by Southern analysis that the Zl1A mutation reduced the efficiency of double-strand break formation compared with the Z1lT variant; the reduction was more severe in MATa than in MATa. In MATa, the Zl1A mutation also creates a matel (sterile) mutation that distinguishes switches of MATa-stk to either MATa or matkl-stk. Pedigree analysis of cells induced to switch in G, showed that MATa-stk switched frequently (23% of the time) to produce one matod-stk and one MATa progeny. This postswitching segregation suggests that Zil was often present in heteroduplex DNA that was not mismatch repaired. When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of matal -stkIMATa sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%). We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.Mating-type switching in the yeast Saccharomyces cerevisiae involves replacement of a or a DNA sequences at the MAT locus by opposite mating-type sequences derived from one of two silent donor loci, HMRa and HMLax (Fig. 1).cis-acting mutations that reduce or abolish MAT switching have been obtained (9,33,38,41). These mutations prevent efficient cleavage of the MAT locus by HO endonuclease, which initiates replacement of Ya or Ya by a gene conversion mechanism. Two types of mutations have been described. MATa-inc and MATa-inc (inconvertible) mutations are lesions within the HO recognition-cleavage site that are lost (healed) during rare switches to the opposite mating type because the double-strand break repair mechanism invariably replaces the region including these base pair changes. A second class of "stuck" mutations was described by Haber et al. (9). These mutants exhibited reduced switching from MATa to MATa but differed from inc mutants in that they were not healed in switching from MATa-stk to MATa (which switched normally) and back to MATa-stk (9). The MATa-stk mutations appeared to reduce the efficiency of HO endonuclease cleavage because HO MATa-stk strains carrying the rad52-1 mutation did not die, while wild-type MATa strains died when HO endonuclease-induced double-strand breaks were unable to be repaired.Another M...

Homothallic switching of yeast mating type (MAT) Recombination intermediates can be studied in detail during synchronously induced switching of MAT (Fig. 1A) and related substrates (10-13). After HO endonuclease cleavage of MAT, the DNA to the right of the double-strand break is converted to a long single-stranded tail by a 5' -* 3' exonuclease that advances by about 1-2 nt/sec (10,12,13). This 3' single-stranded tail apparently invades the intact donor locus, HMLa, and forms heteroduplex DNA. The subsequent elongation of this strand to copy the opposite mating-type (Ya) sequences can be detected by the use of PCR (Fig. 1B) (10).We found that this step (designated Ya-MATdlsg joining) occurs -30 min before the completion of recombination as measured both by a second PCR reaction (MATproximai-Ya joining; Fig. 1C) and by the appearance of the completed product (MATa).Heteroduplex DNA formed during MAT switching has not been detected physically, but its existence can be shown genetically by the appearance of sectored colonies after switching, an event we refer to as PSS (4, 5). For example, switches of the MATa-stk mutation (a T --A mutation at position MAT-Zll) lead either to MATa (normal a mating; Tzii) or matal-stk (sterile; Az,,), depending on the fate ofthe stk mutation (Fig. 1A). By comparing the outcomes of switching in a wild-type strain with a pmsl mutant derivative that is defective in mismatch repair, we showed that nearly all switching events between the mutant Az,, of MATa-stk and the normal Tzll allele at HML involve the formation of heteroduplex DNA formation followed by mismatch repair (5). In a mismatch repair-proficient strain, the Az,, mutation is replaced by Tzll in 85-90%o of all DNA strands, so that 75% of the switches yielded colonies in which all progeny were MATa. Most of the remaining switches exhibit PSS, in which one MATa cell and one matal-stk cell are formed. In contrast, with a pmsl mutant strain, the fraction of switches in which the Az,, mutant is converted to Tzll on both DNA strands decreases from 75% to 15%, with a concomitant increase in MATa/matal-stk sectored colonies (5). These results showed that a mutation only 8 bp from the 3' end of the HO endonuclease cleavage site is usually not removed by exonucleolytic digestion but is included in heteroduplex DNA. Moreover, the repair of the mismatch was shown to be highly preferential; although the information at HMLa is used to convert MAT, reciprocal events, in which the mutation at MAT was introduced into HMLa, are not observed (4, 5).A critical question in understanding exactly how recombination occurs during MAT switching (or in any other homologous recombination event) is, when does mismatch correction occur? As shown in Fig. 2, there are two alternative pathways whereby MATa-stk can switch to MATa. In the first possible pathway (Fig. 2 A, B, and E), heteroduplex DNA is formed between the invading 3'-ended single strand of MAT (stk) DNA ( Fig. 2A) and the resident HMLa locus; then the invading strand is corrected to ...

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We analyzed humoral immune responses to nonhuman leukocyte antigen (HLA) after cardiac transplantation to identify antibodies associated with allograft rejection. Protein microarray identified 366 non-HLA antibodies (>1.5 fold, P < .5) from a discovery cohort of HLA antibody-negative, endothelial cell crossmatch-positive sera obtained from 12 cardiac allograft recipients at the time of biopsy-proven rejection. From these, 19 plasma membrane proteins and 10 autoantigens identified from gene ontology analysis were combined with 48 proteins identified through literature search to generate a multiplex bead array. Longitudinal sera from a multicenter cohort of adult cardiac allograft recipients (samples: n = 477 no rejection; n = 69 rejection) identified 18 non-HLA antibodies associated with rejection (P < .1) including 4 newly identified non-HLA antigenic targets (DEXI, EMCN, LPHN1, and SSB). CART analysis showed 5/18 non-HLA antibodies distinguished rejection vs nonrejection. Antibodies to 4/18 non-HLA antigens synergize with HLA donor-specific antibodies and significantly increase the odds of rejection (P < .1). The non-HLA panel was validated using an independent adult cardiac transplant cohort (n = 21 no rejection; n = 42 rejection, >1R) with an area under the curve of 0.87 (P < .05) with 92.86% sensitivity and 66.67% specificity. We conclude that multiplex bead array assessment of non-HLA antibodies identifies cardiac transplant recipients at risk of rejection. | 2769 BUTLER ET aL.