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Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic

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Effects of Mutagen-Sensitive Mus Mutations on Spontaneous Mitotic Copyright 0 1992 by the Genetics Society of America Effects of Mutagen-Sensitivemus Mutations on Spontaneous Mitotic Recombination in Aspergillus Ping Zhao and Etta Kafer Department of Biology, McGill University, Montreal, Canada H3A 1Bl Manuscript received September 26, 1990 Accepted for publication December3, 199 1 ABSTRACT Methyl methane-sulfonate (MMS)-sensitive, radiation-induced mutants of Aspergillus were shown to define nine new DNA repair genes, musK to muss. To test mus mutations for effects on mitotic recombination, intergenic crossing over was assayed between color markers and their centromeres, and intragenic recombination between two distinguishable adE alleles. Of eight mutants analyzed, four showed significant deviations frommus+ controls in both tests. Two mutations,musK and musL, reduced recombination, while musN and musQ caused increases. In contrast, musO diploids produced significantly higher levels onlyfor intragenic recombination. Effects were relatively small, but averages between hypo- and hyperrec mus differed 15-20-fold. In musL diploids, mostof the rare color segregants resulted from mitotic malsegregationrather than intergenic crossing over.This indicates that the musL gene productis required for recombination and thatDNA lesions lead to chromosome loss when it is deficient. In addition, analysis theof genotypes of intragenic (ad’) recombinants showed that themusL mutation specifically reduced single allele conversion but increased complex conversion types (especially recombinants homozygousfor ad+).Similar analysis revealed differences between the effects of two hyperrec mutations;musN apparently caused high levels solely of mitotic crossingover, while musQ increased various conversion types but not reciprocal crossovers. These results suggest that mitotic gene conversion and crossing over,while generally associated, are affected differentially in some of the mus strains of Aspergillus nidulans. ENETIC analysis of recombination has revealed transactions (e.g., ligase, analyzed in several species; G a large number of different genes which affect or topoisomerase, recently identified as the product recombination and many of these are also important of the HPRl gene in yeast; ACUILERAand KLEIN for DNA replication and/or repair. However, even 1990). In contrast, mutations which reduce recombi- the number of genes which specifically affect recom- nation are more likely to cause defects in proteins bination was foundto be very high, since several specifically requiredfor recombination. The latter different pathways of homologous recombination can was demonstrated, e.g., for the RAD52 gene in yeast occur in a single species (MAHAJAN1988; reviewed by in which disruption abolishes certain types of recom- SMITH1989). So far, few enzymes involved primarily bination (MALONEet al. 1988). By “interspecies com- inrecombination have been characterized. Only in plementation” the RAD52 protein was shown to sub- Escherichia coli and its phages has a variety of products stitute for thefunctions of two phage T4 recombina- from such genes been identified (Cox and LEHMAN tion mutants (CHENand BERNSTEIN1988). Using a 1987). different approach, recombination function was de- In eukaryotes, mutants which increase or reduce duced for “endo-exonuclease” of Neurospora which recombination have also revealed an increasing num- showed antigenic relatedness to the recC component ber of different types of recombination in assays that of the recBCD enzyme in E. coli (FRASER,KOA and use a variety of substrates [as reviewed, e.g., for yeast CHOW 1990). Antibody against this Neurospora en- by ORR-WEAVER andSZOSTAK (1985); or for mam- zyme crossreacts with nucleases from many eukaryotes malian cell systems by BOLLAG,WALDMAN and LISKAY and was used to clone the corresponding genein yeast (1989)~ In some cases, mutants which cause increased (CHOWand RESNICK1988) (reviewed by FRIEDBERC recombinationcould be used to identify enzymes, 1988). It precipitates almost 100% of the correspond- when the corresponding genes were cloned and se- ing nuclease of Aspergillus which shows all the inter- quences comparedto those of prokaryotic gene prod- esting properties of the Neurospora enzyme (KOA, ucts. Such mutants may accumulate recombinogenic FRASERand KAFER 1990). lesions which result in channeling of DNA repair into For the analysis of genetic recombination in Asper- recombination pathways. Theirgene products fre- gillus, we have analyzed alluvs mutants which are quentlyfunction more generally in variousDNA cross-sensitive to methyl methane-sulfonate(MMS) for Genetics 130: 717-728 (April, 1992) 718 P. Zhao and E. Kafer effects on recombination (KAFER and MAYOR1986). (mus, uvs, or ribo, ad, etc.). Such crosses frequently were We found that a few of them closely resembled the heterozygous for translocations and produced increased fre- quencies of specific disomic types. These aneuploids were well analyzedUV-sensitive mutantsof E. coli and used to map the break points of translocations which caused yeast.Some uvs mutantswere hyperrec types and no mutant effects (KAFER 1977). showedproperties of excision-defectivemutants, Parasexual cycle, mitotic analysis and construction of diploid while others resembled rec- types. However, we also strains: Diploids heterozygous for nutritional and conidial identified “novel” types; e.g., hyperrec mutants which color markers were selected from forced heterokaryons in which rare fusion of vegetative nuclei occurs. Diploids of appear to cause chromosomal aberrations resembling Aspergillus are quite stable, but mitotic recombinants are mus mutations in Drosophila(GATTI 1979). To obtain formed at low frequency and occasionallyhaploids are null mutations in additional recombination genes for found, completing the “parasexual” cycle (PONTECORVOet unambiguous tests of epistatic relationships, we in- al. 1953). Such haploid products of nondisjunction show ducedfurther MMS-sensitive mutants by y-rays. practically no crossing over. They were increased by treat- ment with the spindle poisons benomyl or chloral hydrate Among such mutants, various types with alteredlevels and used to map mus mutations and translocations to chro- of recombination have been obtained in several spe- mosomes and to analyze diploid genotypes (KAFER 1977). cies. Also many rec- mutations are known to be hy- Diploid tester strains, mostly homozygous for mus muta- persensitive to this alkylatingagent (among themrecC tions or mus+, were constructed from equivalent pairs of of E. coli and rad52 of yeast). It was expected, there- suitably marked haploid strains (Table 1; for gene symbols and genetic map, see CLUTTERBUCK1987). All strains were fore, that atleast some of our mutations would affect heterozygous for the main markers required for tests of recombination andpossibly include a case with altered inter- and intragenic recombination (especially pabaA yA levels of endo-exonuclease. Of the recovered muta- adE8 / adE20; see last part of “Materials and Methods”). tions in Aspergillus, several were defective in DNA Notall test diploids were translocation free. Three mus repair, cross-sensitive to various mutagens (mus-221 mutations could not be separated from translocations which unavoidably were homozygous in such diploids [muss224 to mus-234). Among these, two new alleles of uvsB T3(III;VII), mus0226 T2 (III;VII) and musP234 TI(II;VII)]. have been identified (uvsB22I and B2??). The others In addition, a widespread spontaneous translocation, complemented and/or recombined freely with all uvs Tl(I;III) with break points on theleft arms of chromosomes mutations that caused MMS sensitivity (KAFER and I and III, was inadvertently retained in strains of musR223 MAYOR1986). and muss224 (diploids 2743 and 2744; Table 1). No effects on recombination were found for any of these translocations We now report results from testsof mitotic recom- in tests which used the yA and adE markers on the right arm bination for eight mus mutations which represent new of chromosome I. DNA repairgenes of Aspergillus nidulans. Five of As “positive” controls, similar diploids were constructed them showed significant differences from wild-type for two UV-sensitive mutations, namely the hyperrec uvs- controls, generallyfor intergenic crossingover as well F201 (SHANFIELDand KAFER 1969) and the rec- mutant uusCII4 UANSEN 1970). as intragenic recombination. Measurements of intergenicrecombination frequen- cies: For each experiment, cultures were started on solid MATERIALS AND METHODS complete medium (CM),using conidia from one of 20 original parallel cultures for inoculation. After incubation Media and special procedures:Standard Aspergillus me- (3 days at 37” and 2-4 days at room temperature) conidia dia and genetic methods were used as developed by PON- were harvested. Suspensions of1 07/ml were prepared which TECORVO et al. (1953). For minor modificationsof the media, served for all recombination assays. To measure survival and for methods specific to the genetic analysis of mitotic and intergenic recombination, samples were plated on CM recombination and DNA repair, detailed procedures were (aiming at 30-40 colonies per plate, based on hemacyto- as described previously (SCOTTand KAFER 1982; KAFERand meter counts). Plates were incubated at 37” for 3 days (4- MAYOR1986). 5 days at room temperature formusO and Q which conidiate Genetic analysis; constructionof strains: In A. nidulans, poorly at 37 ”). Colonies were counted and survival relative methods of genetic mapping include
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