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Stimulation of Meiotic Recombinationin Yeast by an ARS Element Alison J. Rattray and Lorraine S. Symington Institute for Cancer Research and Department of Microbiology, Columbia UniversityCollege of Physicians and Surgeons, New York, New York 10032

Manuscript received August 5, 1992 Accepted for publication January22, 1993 ABSTRACT In a previous study, meiotic recombination events were monitored in the 22-kb LEU2 to C E N 3 region of chromosome III of Saccharomyces cerevisiae. One region (the hotspot) was shown to have an enhanced level of both gene conversion events and reciprocal crossovers, whereas a second region (the coldspot)was shown to havea depressed levelof both types of recombination events.In this study we have analyzedthe effectsof a replication origin,ARS307, located about2 kb centromere proximal to the hotspot region,on the distributionof meiotic recombination events.We find that a deletion of this origin results in a reduction of both gene conversions and reciprocal crossovers in the hotspot region, and that a 200-bp fragment of this ARS element can stimulate both types of recombination events when relocated to the coldspot region. Although the magnitude of stimulation of these events is similar in both orientations, whether the ARS is functional or not, the distribution of events is dependent upon the orientationof the element.

N yeast and other fungiwhere

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all four meiotic products can be recovered, two types of recombination events are observed when scoring two heterozygous linked markers.A reciprocal exchange between two markers yields a tetrad with two spores in the parental configuration, and two spores in which the markershave exchanged partners. In these tetrads the markerssegregate 2:2. Gene conversion events are a nonreciprocal transfer of information and are scored as departures from 2:2 segregation, in which 3:l or 1:3. If the heterozygousmarkerssegregate gene conversion events are initially scored, these are found to be associated with the exchange of flanking markers about 50% of the time (FOGEL,MORTIMER and LUSNAK1981;FOGELet al. 1979;HURST,FOGEL and MORTIMER 1972). Conversely, when crossovers are scored, theseare found to beassociated with gene conversion events at least 50% of the time (BORTSand HABER1987; SYMINGTON and PETES 1988; WILLIS and KLEIN 1987).Therefore, these two processes are consideredto be mechanistically related. All three currently favored models envision gene conversion as an intermediate during the formation of reciprocal crossovers (HOLLIDAY 1964;MESELSONand RADDING 1975;RADDING1982;SZOSTAKet al. 1983). There is considerable variation in the frequency at which a given marker undergoes meiotic gene conversion (FOGELet al. 1979). These variations are attributed to the proximity of recombination initiation sites, or hotspots. Very few recombination hotspots have been studied at the molecular level. T h e a d e 6 M26 hotspot in Schizosacchamyces pombe is associated with a single nucleotidechange (PONTICELLI, SENA Genetics 134: 175-188 (May, 1993)

and SMITH1988;SZANKASI et al. 1988)and correlates with a DNA binding activity that specifically recognizes the M26 allele (W. WAHLSand G. SMITH,personal communication). However, thehotspot does not appear to be active when a 3-kb fragment of a d e 6 , containing the M26 allele, is moved to adifferent chromosomal location (PONTICELLI and SMITH1992). T w o hotspots have been found to be associated with double-strand breaks (DSBs): the ARG4 locus (SUN et al. 1989),and a hotspot created by insertion of the LEU2 gene at theHIS4 locus (CAO,ALANIand KLECKNER 1990).At least one factor important for the ARG4 hotspot is a poly(dA-dT) tract (SCHULTES and SzosTAK 199 1). ZENVIRTH et al. (1992)used pulse field gel electrophoresis to map DSBs in meiotic chromosomes and found break sites associated with other known hotspotsincluding the HIS4 hotspot (WHITEet al. 199 l), and with the hotspot for events initiated in the B-M region of chromosome ZZZ (SYMINGTON et al. 1991),which is the subject of this study. T h e hotspot at HIS4 is also associated with the binding site for the Rap1 protein (WHITEet al. 1991). Little is known about the dependence of recombination on DNA replication. In eukaryotic viral systems the two processes are intimately related. For example, recombination in adenovirus absolutely requires replication of the input DNAs (YOUNGet al. 1984) as does heteroduplex formation in pox virus (FISHERet al. 199 1). Inbacteriophage T4 recombinationhotspots are coincident with, and dependent upon, the replication origins (YAP and KREUZER1991). Other eukaryotic hotspots have been defined as preferred sites for the integration of viral and/or transforming

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A. J. Rattray and L. S. Symington

CHROMOSOME 111 HML I

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FIGURE1.-Map of Chromosome III. A map of chromosome III and a physical map of the region between LEU2 and CEN3 are shown. G4B, D8B and C2G refer to recombinant plasmids containing EarnHl fragments derived from this chromosome (NEWLONet al. 1986). Within the D8B fragment are a recombination hotspot and coldspot. The C2G fragment contains the initiation region (I.R.) for most of the recombination events in the hotspot (SYMINGTON et al. 1991),an origin of replication (ARS307, solid box; NEWLONet al. 1986; GREENFEDER and NEWLON1992),and the centromere (CEN3; indicated by the open circle). The physical map does not show all sites for each restriction enzyme listed. Key: B, EamHI; Bc, EclI; Bs, EstEII; C, ClaI; G , EglII; M, MluI; R, EcoRI; Sp, SpeI; X, Xhol; Xb, XbaI.

DNAs. In general these appear to be nuclease sensitive regions of high AT content (KING et al. 1985; ROHDEWOHLD et al. 1987; SHIH, STOVEand COFFIN1988; VIJAYA,STEFFENand ROBINSON1986).Sequences required for the amplification of DNAs in eukaryotic systems are also associated with regions of high A T content (BEITEL,MCARTHUR and STANNERS 1991; HYRIEN et al. 1987; LEGUOY et al. 1989). In the current study we have analyzed the dependence of recombination on a replication origin located near the previously identified hotspot between LEU2 and CEN3 of chromosome III. ARS307 has been shown to functionin vivo in both mitosis (GREENFEDER and NEWLON1992) and meiosis (I. COLLINSand C. NEWLON, personal communication). Weshow that one-half to two-thirds of the events in the hotspot are dependent upon the presence of ARS307. This element is movable, and insertion of as little as 200 bp into the coldspot stimulates recombination in the vicinity of the coldspot. Furthermore, this stimulation appears to function independently of DNA replication. MATERIALSANDMETHODS Geneticmethods, DNAmanipulations andSouthern analysis were as described previously (SYMINGTON and PETES 1988). Plasmids: A complete list of the plasmids used, and their relevant source, is shown in Table 1. Most of these plasmids are derived from plasmids D8B and C2G [constructed by NEWLONet al. (1986)], which contain contiguous BamHI fragments from chromosome ZZI in the plasmid YIp5 (see Figure 1). All of the sites used in the plasmid constructions are identified in Figure 1. Plasmid C2GAARSwas agift from C. NEWLON.It is identical to C2G except fora 500-bp deletion of ARS307 (formerly CPGIARS) from theEcoRI to ClaI sites. Plasmid pLS83 contains the EcoRI fragment of C2G containing ARS307 cloned into theLYSP gene of plas-

mid p3L6, which complements the slow growth phenotype of strains with the CPGAARS deletion. Plasmid pLS123 is the BamHI to XhoI fragment of D8B containing thecoldspot region, on a nonepisomal URA3 plasmid. Plasmids pLS125, pAL20-7, pAL21-1, pAL21-4 and pAL21-6 are all derivatives of plasmid pLS123 where the indicated ARS element has been blunt-end ligated into the XbaI site of pLS123. T h e presence of the wild-type or mutant ARS element in these plasmids was confirmed by DNA sequencing. To test for ARS activity by a high frequency transformation assay, DNA fragments from the coldspot region with or without CEN vector the insertedARS elements were cloned into the pRS326. T h e DNA fragments used for these constructions are illustrated in Figure 8, and theplasmids generated listed in Table 1. Yeast strains: A complete list of the yeast strains used for this study is given in Table 2. Construction of the restriction enzyme sitemutations of thehaploid parental strains(AS 13, AS14 and AS20), has been described in detail previously (SYMINGTON and PETES1988; STAPLETON and PETES199 1). Cells with the CPGAARS chromosomal deletion grow very slowly (C. NEWLON, personal communication), and therefore derivative strains of AS 13 andAS 14 were constructed (LS72 and LS74, respectively) with the 3.4-kb EcoRI fragment from the ARS307 region placed at the LYSP locus, by two step transplacement with pLS83. Initially, Ura+ transformants were selected, then Lys- derivatives were counterselected by their resistance to the lysine analog ru-aminoadipate (SHERMAN,FINK and HICKS 1986). This selects for cells which have lost the plasmid sequences by intramolecularrecombination (WINSTON,CHUMLEYandFINK 1983), replacing the wild-type LYS2 gene with the disrupted version. Strains LSl1 1and LS79 were constructed by two step transplacement of LS72 and LS74 (respectively), with plasmid CPGAARS, effectively deleting the 500-bp fragment containing ARS307. Transformants in which the plasmid hadintegratedintothegenome were selected as Ura+ colonies. Ura+ transformants were then plated onto medium containing 5-fluoroorotic acid (5-FOA) (BOEKE, LACROUTE and FINK 1984)to isolate Ura- cells which had excised the URA3-containing plasmid sequences by intramolecular recombination. Derivatives in which the plasmid-borne insertion and/or deletion remainedin the genomewere detected

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ARS-Stimulated Recombination TABLE 1 Plasmids Source

Name

Description of plasmid

C2G D8B CPGA'ARS p3L6 pLSl1 pLS83

8.4-kb BamHI fragment from chromosome ZZZ cloned into YIp5 10.6-kb BamHI fragment from chromosomeZZZ cloned into YIp5 500-bp EcoRI to ClaI deletion of ARS307 from C2G (see Figure 1) LYS2 gene in YEp24 2.1-kb EcoRI 2-p fragment deleted from YEp24 Blunt-ended 3.4-kb EcoRI fragment from C2G, including ARS307, cloned into bluntended BamHI cleaved p3L6 4.3-kb BamHI to XhoI fragment of the coldspot from D8B cloned into BnmHI and SalI cleaved pLSl1 Blunt-ended 0.5-kb EcoRI to ClaI fragment of ARS307 cloned into blunt-ended XbaI cleaved pLS123 Wild-type ARS307 sequence cloned into pVHA (200-bp fragment of ARS307 cloned between BamHI and SalI linkers) Identical to pVH402 except for the single base-pair T to G transversion in the ARS consensus sequence at position 9, creating an Ars- phenotype A derivative of pRS306 (SIKORSKI and HIETER1989) containing the SUP1 I gene and CEN4 Blunt-ended 0.2-kb BamHI to SalI fragment of ARS307 from pVH402 cloned into blunt-ended XbaI cleaved pLSl23, with the T-rich strand of the ARS consensus sequence oriented 5'-3' relative to the X2 site ( 2 0 0 4 Blunt-ended 0.2-kb BamHI to SalI fragment of ars307 from pVH428 cloned into bluntended XbaI cleaved pLS123, ( 2 0 0 9 Two copies of blunt-ended 0.2-kb BamHI to SalI fragment of ars307 from pVH428 cloned into blunt-ended XbaI cleaved pLS123 (200 > 200 >) Blunt-ended 0.2-kb BamHI to SalI fragment of ars307 from pVH428 cloned into blunt-ended XbaI cleaved pLSl23, (200>) 1.75-kb Xbal to EcoRI fragment from D8B cloned into pKSII- (Stratgene) 3. I-kb BglII to XhoI fragment from pLS123cloned into pRS326 3.3-kb BglII to XhoI fragment from pAL21-6 cloned into pRS326 3.3-kb BglII to XhoI fragment from pAL20-7 cloned into pRS326 3.3-kb BglII to XhoI fragment from pAL21-I cloned into pRS326 3.5-kb BglII to XhoI fragment from pAL21-4 cloned into pRS326 0.62-kb BglII to SphI fragment from pAL20-7 cloned into pRS326 0.62-kb BglII to SphI fragment from pAL2I-I cloned into pRS326 0.64-kb BglII to Sal1 fragment from pAL20-7 cloned into pRS326 0.64-kb BglII to Sal1 fragment from pAL2I-I cloned into pRS326 0.33-kb ScaI to SalI fragment from pAL20-7 cloned into pRS326 0.33-kb ScaI to Sal1 fragment from pAL21-1 cloned into pRS326 0.2-kb BamHI to SalI fragment from pAL20-7 cloned into pRS326 0.2-kb BamHI to SalI fragment from pAL21-I cloned into pRS326 -

pLS 123 pLS125 pVH402 pVH428 pRS326 pAL20-7

PALL]-I pAL2 1-4 pAL21-6 pAL3O pAL44 pAL45 pAL46 pAL47 pAL48 pAL53' pAL54 pAL57 pAL58 pAL63 pAL64 pAL67 pAL68

et al. (1986) NEWLON NEWLON et al. (1986) C. NEWLON C. FALCO L. SYMINCTON This work

This work This work (1 990) VANHOUTENand NEWLON

VANHOUTENand NEWLON(1 990) J. THEIS and C. NEWLON

This work

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' A refers to a b

deletion. 200< and 200> refer to the orientation of the ARS fragment, with the direction of the arrow indicating the 5' to 3' orientation of the T-rich strand of the ARS consensus sequence. ' The sites used for the construction of plasmids pAL53 to pAL68 are diagrammed in Figure 8.

by Southern analysis. All other haploid strains were constructed by two step transplacement of either AS14 and AS20, or LS79 and LSl 1 1with plasmids containing different segments or sequences of ARS307 cloned into the XbaI site of plasmid pLS123 selecting Ura+ transformants, and counterselecting on 5-FOA. All diploid strains were made by mating the appropriatehaploids to producehomozygous deletions and/or insertions of the ARS307 fragments. Diploids were selected by plating on minimal media containing only adenine, uracil, and if necessary, lysine, and were confirmed by Southern analysis. T h e presence of the homozygous point mutation in strain LS326 was confirmed by amplifying a chromosomal fragment containing the insert with oligonucleotides flanking the insertionsite, and sequencing the amplified fragment. Tetrad analysis: Tetrad dissection was performed as described by SHERMAN,FINK and HICKS(1986). Tetrads

with a crossover between LEU2 and CEN3 were identified by second division segregation of LEU2 withrespect to both of the centromere linked markers TRPl and ARC4 (SYMINGTON and PETES1988) which were also heterozygous in all ofour diploid strains. DNA was isolated by the glass bead method (HOFFMANand WINSTON1987) from all four spore cultures of the crossover tetrads, and examined by Southern analysis for each of the heterozygous restriction enzyme site markers. The results of the Southern analysis were then summarized as shown in Figures 3 and 6. The percentage of the population undergoing a gene conversion event at any one of the heterozygous sites (Figures 4A and 7A) was determined by summing the number of conversion events at a given site and dividing this number by the total number of tetrads. For example, of the 62 tetrads analyzed from LS47 10 showed conversion of the SpeI site. Since the crossover frequency of this strain is 12.5%, the 62 crossover

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A. J. Rattray and L. S. Symington TABLE 2

Description of yeast strains _ _ _ _ _ _ ~ ~

~~

Relevant

AS13 AS14 AS20 LS47 LS72 LS74 LS79 LS86 LSlll LSll7 LS118 LS119 LS120 LS121 LS122 LS3 13 LS3 14 LS3 15 LS3 16 LS3 17 LS3 18 LS3 19 LS320 LS321 LS322 LS323 LS326 W303

~

genotype or description

Strain

Source

a leu2 ade6 ura3; has three restriction site mutations in the 23-kb LEU2 to CEN3 region a trfl adc6 uro3 arg4 tyr7; has 10 restriction site mutations centromere proximal to LEU2 Insertion of an additional restriction site mutation centromere proximal to LEU2 in AS 13 Diploid formed by crossing AS 1 3and AS 14 Integration of C2G EcoRI (ARS307)fragment at LYS2 by two-step transplacement of AS20 with pLS83 Integration of C2G EcoRI (ARS307)fragment at LYSZ by two-step transplacement of AS14 with pLS83 Diletion of ARS307 (EcoRI-ClaI)in LS74 by two-step transplacement with pC2GMRS Diploid homozygous for AARS307 formed by crossing LS79 and LSl 1 1 Deletion of ARS307 (EcoRI-ClaI)in LS72 by two-step transplacement with pC2GMRS Insertion of ARS307 (EcoRI-ClaI) in CS-XbaI" of AS14 by two-step transplacement with pLSl25 Insertion of ARS307 (EcoRI-ClaI)in CS-XbaI of AS20 by two-step transplacement with pLS125 Insertion of ARS307 (EcoRI-ClaI)in CS-XbaI of LS79 by two-step transplacement with pLS125 Insertion of ARS307 (EcoRI-ClaI)in CS-XbaI of LS111 by two-step transplacement with pLS125 Diploid homozygous for CS::ARS307 formed by crossing LS 117 and LS118 Diploid homozygous for MRS307 andCS::ARS307 formed by crossing LS119 and LS120 Insertion of ars307 (200>) in CS-XbaI of LS79 by two-step transplacement with pAL21-6 Insertion of ARS307 (200 200>) in CS-XbaI of LS79 by two-step transplacement with pAL21-4 Insertion of ars307 ( 2 0 0 3 in CS-XbaI of LSl 1 1by two-step transplacement with pAL21-6 Insertion of ARS307 (200 200>) in CS-XbaI of LSl 1 1by two-step transplacement with pAL21-4 Diploid homozygous for MRS307 andCS::ars (200>) produced by crossing LS3 13 andLS317 Diploid homozygous for MRS307 and CS::ARS (200 200>) produced by crossing LS3 16 and LS320 Diploid homozygous for AARS307 and CS::ars (200