Proc. Nati. Acad. Sci. USA Vol. 83, pp. 7386-7390, October 1986 Genetics Two mechanisms for directional gene conversion (heteroduplex DNA/mismatch repair/meiotic recombination/Ascobolus immersus) HANAFY HAMZA*, ANGELOS KALOGEROPOULOS, ALAIN NICOLASt, AND JEAN-LUC ROSSIGNOL Interactions Moldculaires Gdnomiques, Universitd Paris-Sud, 91405F Orsay-Cedex, France Communicated by Franklin W. Stahl, June 12, 1986 ABSTRACT G234 is a large silent deletion located in the (the longer strand) whether it was that of the donor or middle of gene b2, which controls spore pigmentation in recipient strand. We conclude that the mechanism that Ascobolus immersus. Its gene conversion directionality was produces gene conversion events from hDNA containing studied in asci, which show evidence of heteroduplex DNA at single-base mismatches differs from that which operates on flanking markers, and was compared to the behavior of closely hDNA containing large nonhomologies. We propose that linked single-base-pair insertions or deletions. We found that mismatch correction arises via single-strand excision and via with the G234 deletion, the genotype of the donor strand in the double-strand-break repair, respectively. heteroduplex is preferentially recovered, irrespective of its G234 or wild-type nature, whereas with single-base-pair inser- MATERIALS AND METHODS tions or deletions, the direction of conversion favors one genotype, whether it was the donor or the recipient strand. We Principle for Gene Conversion Studies in Ascoboius. Each conclude that there exists two mechanisms for directional gene meiosis gives rise to an ascus containing four pairs of sister conversion, the "donor-directed" conversion mechanism be- spores-the products of two meiotic divisions followed by a ing epistatic to the "genotype-directed" one. We discuss these single postmeiotic mitosis. This means that for any gene the data with regard to models for mismatch repair. eight strands of DNA that comprised the four DNA duplexes at meiosis I are recovered in separate spores. Genetic studies in fungi have provided a description of the Gene conversion events lead to non-Mendelian segrega- mechanism of genetic recombination, in particular the proc- tions (NMS). NMS is defined as departures from the 4:4 (four ess of gene conversion (1-5). In this paper, we examine the wild type:four mutant) Mendelian segregation of the meiotic number of mechanisms that produce gene-conversion events products in mutant x wild type crosses. NMS corresponds to and affect its directionality. postmeiotic segregation (PMS)-i.e., 5:3 and aberrant 4:4- Gene conversion of some Ascobolus mutants is directional and to conversion asci (6:2). In aberrant 4:4 asci, two meiotic (6, 7). In the progeny ofwild-type x mutant crosses, putative products segregate at the first postmeiotic division. In 5:3 single-base-pair (1 bp) insertion mutations give an excess of segregations (5+:3m or 3+:5m), only one meiotic product conversion towards the mutant [(two wild type:six mutant) > segregates at the first postmeiotic division; 6:2 segregations (six wild type:two mutant), or 2+:6m > 6+:2m], whereas are of two kinds: 6+:2m or 2+:6m, depending on the 1-bp deletion mutations give an excess towards wild type direction of the conversion event towards wild type or (6+:2m > 2+:6m). Studies in the b2 ascospore color locus mutant, respectively. Spore color pigmentation and morphol- suggested that this disparity (i.e., excess ofconversion in one ogy markers can be scored by visual inspection of asci. This direction) results from heteroduplex formation, followed by allows us to analyze large numbers of meiotic events. an obligatory mismatch correction involving preferential Crosses. Media and culture methods have been described excision of the shortest strand (8, 9). By comparison, the (15, 16). All pairs of crosses but one (experiment II) were conversion behavior of the large deletion mutation b2,G234 performed in isogenic conditions. We used the protocol of was found unusual on two respects: (i) the G234 deletion in Hastings et al. (17) in experiments I, III, IV, and V and that gene b2 exhibits a strict parity in the direction of conversion of Rossignol and Paquette (18) in experiments VI-VIII. (6+:2m = 2+:6m), whereas closely linked 1-bp insertion or Mutations. The strains used belong to the stock 28 of deletion mutations exhibit a 10-fold disparity, and (ii) G234 Ascobolus immersus (19). The white-spore mutations 17 and imposes its own behavior on all tightly linked point mutations A4 map in the left and right part of the gene b2, respectively including 1-bp insertions or deletions (10). This suggested (20). The G234 deletion mutation has a wild-type phenotype that the conversion of point and nonpoint mutations may (brown spores). It lies in the middle part of b2, close to the arise via distinct pathways. E group of intragenic suppression (10). The double mutations In order to discriminate between various recombination EOEI and E2EJ show a pink-spore phenotype distinguishable models (11-14) presented in the Discussion section, we have either from wild type and from the white-spored single-site studied the formation ofheteroduplex DNA (hDNA) flanking mutations EO, El, and E2. These mutations are assumed to be these various mutations and found that asymmetric hDNA 1-bp additions (El) and 1-bp deletions (EO and E2). Recom- can form with similar frequencies on either side of them. bination between EO, El, E2, and G234 is extremely rare (10). Then we investigated the directionality ofgene conversion by Rndl.2 and vag4 mutations were used as additional mark- examining the segregation ofG234 or point mutations in these ers for the detection of aberrant 4:4 asci (21) and the individual asci that show evidence of flanking hDNA. We found that with G234, the genotype ofthe donor strand (in the Abbreviations: hDNA, heteroduplex DNA; NMS, non-Mendelian heteroduplex) was preferentially recovered, irrespective of segregation; PMS, postmeiotic segregation; 1-bp, single base pair; its G234 or wild-type nature, whereas with 1-bp insertion or x+:ym, x wild type:y mutant progeny; xC:yW, x colored-spore:y deletion, the direction of conversion favored one genotype white-spore segregation; xB:yP:zW, x brown-spore:y pink-spore:z white-spore segregation. *Present address: Faculty of Agriculture, Department of Genetics, The publication costs of this article were defrayed in part by page charge Monofiya University, Shebin el Kom, Egypt. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department of Molecular Biology, Massachusetts in accordance with 18 U.S.C. §1734 solely to indicate this fact. General Hospital, Boston, MA 02114. 7386 Downloaded by guest on September 25, 2021 Genetics: Harnza et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7387 distinction of sister spore pairs. RndJ, vag4, and b2 genes are a) genetically unlinked (22). A C. Accuracy of the Spore Color System. Colored spores (195; a) .1-i 17 m A4 0 brown or pink) randomly isolated from mixed pairs in 0 o IV were and strains were experiment germinated, analyzed 0 O0 m by backcrosses for their genotype. Among 121 brown spores, 17 * A4 118 were confirmed as carrying the mutation G234, and 69 a)0) among 74 pink spores were confirmed as carrying the double SE'a) mutation E2EJ. Therefore, the fraction ofphenocopies is low L LI + + + 0 S enough (4%) to infer an EE genotype safely from a pink spore c .a, and a G genotype from a brown spore. 0 + + + Genotype of Mixed Spore Pairs. The genotype of the white S 4.'- spores at 17 and A4 was determined by backcrossing the S + + + white-spore strains with 17 and A4. The white spore should .IJ r- have the same genotype as its colored partner because the 0 middle-marker configurations studied never give PMS in their conversion pattern (10, 17). This assumption was o o , 7 + AA verified by backcrossing with wild-type and El strains. 0 17 + A4 o Altogether, 1080 -mixed pairs isolated in 5 colored-spore:3 C; white-spore segregation (5C:3W) and aberrant 4C:4W asci from experiments I, IV, and VII were analyzed. All but 2 5c showed the same middle-marker genotype for the two sister 0 + spores. Thus, the genotype ofthe middle marker in the mixed a) *0+:* + S r pair can be inferred from the phenotype ofthe colored spore. 0 0 In experiment III, the G234 or wild-type middle-marker genotype of the colored spore was determined by crossing to o + * + a strain carrying GJ: the absence of PMS in the progeny indicates a G234 genotype, whereas its presence indicates the wild-type genotype (23). B RESULTS FIG. 1. Protocol for studying gene conversion at a middle marker by selecting for flanking hDNA formed on one duplex. Asymmetrical Data reported in this paper are based on eight experiments hDNA formed at the two flanking mutations 17and A4 during meiosis (I-VIII). Each experiment comprised a pair ofcrosses (a and was detected in crosses 17A4 x + + among 5C:3W spore asci (24). b) listed in Table 1. All crosses involved two flanking markers In these asci, the mixed pair of spores reflects hDNA, with the and a middle marker. The flanking markers 17 and A4, colored spore reflecting the invading strand and the white spore in were used to reflecting the recipient one. The drawings correspond to cases where coupled cis configuration, detect meiosis in the white spore in the mixed pairs is 17 A4. (A) Middle-marker which hDNA was formed on either one (asymmetric hDNA) mutation (m) on the recipient molecule. (B) Middle marker mutation or both (symmetric hDNA) ofthe interacting DNA duplexes. (m) on the donor molecule. 5C:3W asci indicated asymmetric heteroduplex formation (Fig.