Gene Conversion Tracts Stimulated by HOTI-Promoted Transcription Are Long and Continuous

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Copyright 0 1990 by the Genetics Society of Americ Gene Conversion Trcts Stimulted by HOTI-Promoted Trnscription Are Long nd Continuous Kren Voelkel-Meimn nd G.

1 Copyright by the Genetics Society of Americ Gene Conversion Trcts Stimulted by HOTI-Promoted Trnscription Are Long nd Continuous Kren Voelkel-Meimn nd G. Shirleen Roeder Deprtment of Biology, Yle University, New Hven, Connecticut Mnuscript received Februry 12, 1990 Accepted for publiction September 4, 1990 ABSTRACT The recombintion-stimulting sequence, HOTl, corresponds to the promoter of trnscription by yest RNA polymerse I. The effect of HOTl onmitoticinterchromosomlrecombintion ws exmined in diploid strins crrying heterozygous URA3 gene on chromosome ZZZ. The frequency of Ur- recombinnts ws incresed 20-fold when HOTl ws inserted into the chromosome ZIZ copy mrked with URA3, t loction 48 kbp centromere-proximl to URA3. Ur- recombinnts were incresed only 2-fold when HOTl nd URA3 were on opposite homologues. These results suggest tht most HOTl-promoted Ur- recombinnts result from gene conversion nd tht sequences on the HOTl-contining chromosome re preferentilly converted. Chrcteriztion of Ur- recombinnts isolted from strins crrying multiple mrkers on chromosome ZZZ indictes tht HOTl-promoted gene conversion trcts re unusully long (often >75 kbp) nd lmost lwys continuous. Furthermore, conversion trcts frequently extend to both sides of HOTl. We suggest tht HOTl promotes the formtion of double-strnd brek which is often followed by exonucleolytic digestion. Repir of the broken chromosome could then result from gp repir or from replictive repir primed only by the centromere-contining chromosoml frgment. HE recombintion-stimulting sequence, HOTl, T is derived from the ribosoml DNA of Scchromyces cerevisie nd corresponds to sequences required for promotion of trnscription by RNA polymerse I (VOELKEL-MEIMAN, KEIL nd ROEDER 1987; STEWART nd ROEDER 1989). When inserted t novel loctions in the yest genome, HOTl stimultes both intrchromosoml nd interchromosoml recombintion in mitoticlly dividing cells (KEIL nd ROEDER 1984; VOELKEL-MEIMAN, KEIL nd ROEDER 1987; VOELKEL-MEIMAN nd ROEDER 1990). Severl observtions indicte tht trnscription by RNA polymerse I inititing in HOTl nd proceeding through the djcent sequences is responsible for the enhncement of recombintion (VOELKEL-MEIMAN, KEIL nd ROE- DER 1987; STEWART nd ROEDER 1989). We hve previously exmined the effect of HOTl on mitotic interchromosoml recombintion (VOELKEL-MEIMAN nd ROEDER 1990). HOT1 ws inserted just upstrem of the HIS4 gene on chromosome III in diploid strins crrying his4 heterolleles. HOTl stimulted the formtion of His+ prototrophs resulting from gene conversion when present on one or both copies of the chromosome. When HOTl ws present on only one of the two homologs, the his4 gene on the HOT1-contining chromosome ws preferentilly converted. Thus, HOTl defines the chromosome on which it resides s the recipient of genetic informtion during conversion. In this respect, HOTl is similr to sequences tht stimulte meiotic recom- bintion in S. cerevisie (NICOLAS et l. 1989), Schizoscchromyces pombe (GUTZ 197 l), Sordri brevicolis (MACDONALD nd WHITEHOUSE 1979) nd Neurospor crss (ANGEL, AUSTIN nd CATCHESIDE 1970). In this pper, we hve extended our studies of HOTZ-promoted interchromosoml recombintion. Our results indicte tht HOTl stimultes the formtion of recombinnts tht re homozygous for mrkers distl to the site of HOTl insertion. In HOTl heterozygotes, sequences present on the HOT-1-contining chromosome re preferentilly host nd sequences on the homolog re duplicted. Furthermore, these long, continuous conversion trcts frequently include mrker tht lies centromere-proximl to HOTl even when HOTl-promoted trnscription proceeds wy from the centromere. These results suggest tht events inititing in the region of HOTI-promoted trnscription cn extend significnt distnce (25-50 kbp) to both sides of the initition site. We propose tht HOTI-promoted trnscription stimultes the formtion of double-strnd brek which is subsequently repired by brek-nd-replicte pthwy of recombintion. In this pthwy, sequences distl to the brek re lost nd the broken chromosome is repired by repliction using the centromere-contining chromosoml frgment s primer nd the homologous chromosome s templte. MATERIALS AND METHODS Medi: SC, MIN, GNA nd YEPD medi were prepred s described by SHERMAN, FINK nd HICKS (1986). SC + 5- Genetics 126: (December, 1990)

2 852 K. Voelkel-Meimn nd G. S. Roeder TABLE 1 Yest strins Strin Genotype RLKl-3C MAT his4-260 de2-1 ur3-52 K144 MAT leu2-112 his4-712 ur3-52 SR202-3B MAT leu2-i 12 his4-260 de2-1 JEl38-17B MAT leu2 his4-260 ur3 rg4 spol3::ura3 thrl-4 trpl lys2 M520 MAT leu2 his4-712 de2 ur3 spol3::ura3 trpl thrl-4 lys2 M521 MAT leu2 his4-712 de2 spo13::ura3 thrl-4 lys2 MAT leu2-112 his4-712 HML ADE2 ur3-52 " SI243 MAT LEU2 his4-620 HML[URA3] de2-1 ur3-52 MAT leu2-i 12 his4-712 HML[URA3] ADEZ ur3-52 " S1245 MAT LEU2 his4-260 HML de2-1 ur3-52 MAT leu2-112 bik1::hoti-t his4-712 HML[URA3] ADEZ ur3-52 SI250 MAT LEU2 bik1::hoti-t his4-260 HML ADE2::BIKl::deZ ur3-52 MAT leu2-112 bik1::hotl-t his4-712 HML ADEZ ur3-52 S1254 MAT LEU2 bik1::hotl-t his4-260 HML[URA3] ADE2::BIKl::deP ur3-52 MAT bik1::hotl-t leu2-i 12 his4-712 HML ADE2 ur3-52 SI 274 MAT LEU2 BlKl his4-260 HML[URA3] ADE2::BIKI::deP ur3-52 MAT leu2-i bik1::hoti-t 12 his4-712 HML[URA3] ADE2 ur3-52 SI278 MAT@ LEU2 his4-260 BIKl HML ADE2::BIKI::deZ ur3-52 leu2-112 MAT bik1::hotl-t his4-712 ur3-52 TRPl ADE2 " SI 282 MAT LEU2 BIKl his4-260 JIOA::URA3 ADE2::BIKl::deZ trpl-h3 ur3-52 MAT leu2-112 bik1::hotl-t his4-712 JIOA::URA3 ADE2 TRPI ur3-52 " S 1289 BIKl MAT LEU2 his4-260 ADE2::BIKl::deZ trpl-h3 ur3-52 MAT leu2-112 bik1::hotl-c his4-712 JlOA::URA3 ADEZ trpl-h3 ur3-52 SI291 MAT LEU2 bik1::hotl-c his4-260 ADE2::BIKl::de trpl-h3 ur3-52 leu2-112 his4-712 ]IOA::URA3 MAT ur3-52 trpl-h3 ADE2 S1293 MAT LEU2 bik1::hoti-c his4-260 ADE2::BIKI::deP trpl-h3 ur3-52 MAT leu2-112 bik1::hoti-c his4-712 B9G::TRPl lioa::ura3 ADEZ trbl-h3 ur3-52 S 1294 BIKl LEU2 MAT ADE2::BIKI::deZ his4-260 ur3-52 trpl-h3 MAT leu2-112 bik1::hotl-c his4-712 JlOA::URA3 ADE2 trpl -H3 ur3-52 SI 295 his4-260 LEU2 MAT ADE2::BIKI::deP ur3-52 trpl-h3 MAT leu2-112 his4-712 ADE2 trpl-h3 ur3-52 s139i MAT LEU2 his4-260 JIOA::URA3 de2-1 trpl-h3 ur3-52 MAT leu2-112 his4-712 JIOA::URA3 ADE2 trpl-h3 ur3-52 SI392 MAT LEU2 his4-260 de2-1 trpl-h3 ur3-52 MAT leu2-112 bik1::hotl-t his4-712 JIOA::URA3 ADE2 trpl-h3 ur3-52 SI393 MAT LEU2 bik1::hotl-t his4-260 ADE2::BIKI::deZ trpi-h3 ur3-52 leu2-112 bik1::hotl-t MAT ur3-52 trpl-h3 his4-712 ADEZ S1394 MAT LEU2 bik1::hotl-t his4-260 JlOA::URA3 ADE2::BIKl::deP trpl-h3 ur3-52 ur3-52 trpl-h3 ADE2 his4-712 BlKl leu2-112 MAT SI395 MAT LEU2 bik1::hotl-t his4-260 JIOA::URA3 ADE2::BIKl::deZ trpl-h3 ur3-52 MAT leu2-112 his4-712 BIKI JIOA::URA? ADE2 trpl-h3 ur3-52 SI396 MAT LEU2 bik1::hoti-t his4-260 ADE2::BIKl::deZ trpl-h3 ur3-52 leu2-112 bik1::hoti-t MAT trpi-h3 ur3-52 his4-712 ADE2 S1397 MAT LEU2 BIKl his4-260 JlOA::URA3 ADE2::BlKI::deZ trpl-h3 ur3-52 MAT leu2-112 bik1::hotl-t his4-712 JIOA::URA3 ADE2 trpl-h3 ur3-52 SI398 BlKl LEU2 MAT his4-260 ADE2::BIKl::de trpl-h3 ur3-52 MAT leu2-112 his4-712 B9G::TRPl JlOA::URA3 ADEZ trpl-h3 ur3-52 S1399 ur3-52 trpl-h3 de2-1 his4-260 LEU2 MAT MAT leu2-112 bik1::hotl-t his4-712 B9G::TRPl JIOA::URA3 ADE2 trpl-h3 ur3-52 S 1402 BlKl LEU2 MAT ADE2::BIKl::deP his4-260 ur3-52 trpl-h3 MAT leu2-i his4-712 JlOA::URA3 ADE2 trpl-h3 ur3-52 SI41 1 MAT LEU2 his4-260 B9G::TRPI de2-1 trpl-h3 ur3-52 his4-712 JlOA::URA3 leu2-112 ur3-52 BIKI trpl-h3 MAT ADEZ SI413 MAT LEU2 bik1::hotl-t his4-260 B9G::TRPl ADEP::BIKI::de2 trpl-h3 ur3-52 MAT leu2-112 bik1::hotl-t his4-712 OA::URA3 J 1 ADE2 trpl-h3 ur3-52 SI414 MAT hi~4-260 LEU2 BlKl B9G::TRPI ADE2::BIKl::deP trpl-h3 ur3-52 "

-18 853 Long Conversion Trcts URA3 v leu2 c /! kbp ~ 28 hid-260. 1350 kbp.24 49 kbp. " -4 " 695 bp 27 kbp 21 kbp FIGURE 1.-Distribution of mrkers on chromosome III.

3 Long Conversion Trcts URA3 v leu2 c /! kbp ~ 28 hid kbp kbp. " -4 " 695 bp 27 kbp 21 kbp FIGURE 1.-Distribution of mrkers on chromosome III. Shown t the top is the distribution of chromosome III mrkers in prentl diploids used to mesure recombintion. The tringles indicte the sites of insertion of URA3, TRPl nd HOTl. In S1402, these mrkers re present on the his#-712 chromosome s digrmmed. The inserts present in other strins nd the chromosome III homolog on which they reside re indicted in Tble 1. The open rrow indictes the position nd orienttion of the HIS4 gene. The circle indictes the chromosome III centromere. Shown below the chromosomes re the physicl distnces between mrkers. The his#-260 nd his#-712 muttions lie 45 bp nd 1396 bp, respectively, downstrem of the strt of the HIS4 coding region (DONAHUE, FARA- BAUGH nd FINK 1982). HOTl is inserted 650 bp upstrem of HIS#. The sizes given for the URA3-TRPI, TRPI-his#-712, HOTI-LEU2, nd LEU2-CEN3 intervls re pproximte nd re bsed on the dt of NEWLON et l. (1986). The distnce between URA3 nd the left end of the chromosome is 19 kbp (BUTTON nd ASTELL 1986). Digrm is not to scle. fluoro-orotic cid (FOA) ws mde by supplementing SC to 0.5 mm urcil nd 0.88% FOA (BOEKE, LACROUTE nd FINK 1984). Yest strins nd trnsformtions: All of the strins used to mesure recombintion re isogenic diploids derived from two hploid prents, RLK1-3C nd K144 (Tble 1). Derivtives of these strins were constructed by trnsformtion using the method Of IT0 et l. (1983). Trnsformnts were verified by Southern blot nlysis (MANIATIS, FRITSCH nd SAMBROOK 1982). HOTl ws inserted s 570-bp BglII frgment consisting of the 320-bp EcoRI-HpI frgment contining the enhncer of trnscription by RNA polymerse I nd 250-bp SmI- EcoRI frgment contining the 35s rrna initition site (VOELKEL-MEIMAN, KEIL nd ROEDER 1987). A HOTl mutnt (#38) defective in the stimultion of recombintion ws constructed by STEWART nd ROEDER (1989). The plsmids nd procedures used to construct strins crrying wild-type nd mutnt HOTl sequences oriented such tht trnscription by RNA polymerse I should proceed towrd the telomere (HOTl-t nd hotl-t) were described previously (VOELKEL-MEIMAN nd ROEDER 1990). Strins crrying HOTl oriented towrd the centromere were constructed using plsmids L685 nd V106. pl685, described by VOELKEL-MEIMAN, KEIL nd ROEDER (1987), ws used to trnsform K 144 nd its derivtives; pv 106 ws used to trnsform RLK1-3c nd its derivtives. pv106 is the sme s pv127 (VOELKEL-MEIMAN nd ROEDER 1990) except tht the BglII frgment contining HOTl is derived from pl685 nd is inserted in the sme orienttion s tht in pl685. Both plsmids were trgeted for integrtion t HIS4 by clevge with ClI nd Ur+ trnsformnts were selected. Ur- segregnts which hd lost the plsmid due to intrchromosoml recombintion were then selected on SC + FOA. These segregnts were exmined by Southern blot nlysis to identify those tht retined HOTl. The insertion of HOT1 sequences results in disruption of the BIKI gene (TRUEHEART, BOEKE nd FINK 1987). This disruption ws complemented by integrtion of plsmid contining BIKI nd ADEZ t the ADEZ locus on chromosome XV (VOELKEL-MEIMAN nd ROEDER 1990). The URA3 gene ws inserted t two different loctions centromere-distl to HIS4 s described by VOELKEL-MEIMAN nd ROEDER (1 990). In strins designted s HML[URA3], URA3 is inserted t Hind111 site immeditely centromereproximl of the HML locus. In strins designted JIOA::URA3, URA3 is inserted into the BmHI frgment tht lies immeditely djcent to the BmHI frgment contining HML (NEWLON et l. 1986). The distnce between the two sites of URA3 insertion is bout 4.5 kbp. The URA3 gene ws introduced by the one-step gene disruption method of ROTHSTEIN (1983). The TRPl gene ws inserted into chromosome IZI using plsmid V163. pv 163 is derivtive of plsmid B9G which contins 6.6 kbp BmHI frgment of chromosome 111 DNA (NEWLON et l. 1986). TRPl ws inserted t site (either RsI or HZncII) locted bout 1.5 kbp wy from the centromere-proximl BmHI site. The TRPl gene ws inserted s n 820-bp EcoRI-StuI frgment fter filling in the EcoRI end with Klenow frgment. pv163 ws digested with BmHI prior to trnsformtion into yest resulting in sub- stitutive trnsformtion (ROTHSTEIN 1983). For ech of the constructions described, three independent trnsformnts were isolted from ech hploid strting strin. For strins derived by multiple trnsformtion steps, three independent trnsformnts were isolted fter the first trnsformtion. Ech of these ws then trnsformed nd single trnsformnt of ech ws chosen t ech of the subsequent steps. The finl three MAT trnsformnts were mted to the three MAT trnsformnts in ll possible pirwise combintions to generte nine independent diploids. Fluctution tests: Fluctution tests were crried out s described by VOELKEL-MEIMAN nd ROEDER (1 990) except tht cells were plted on SC + FOA insted of SC - His. The method of the medin ws used to clculte recombintion frequencies (LEA nd COULSON 1949). Isoltion nd chrcteriztion of independent His+ nd Ur- recombinnts: A protocol similr to the fluctution test ws used to recover independent Ur- nd His+ recombinnts nd to estimte recombintion frequencies. The nine independent diploids with the sme genotype were grown to sturtion in 2 ml of GNA. Ech of these cultures ws then diluted nd used to set up severl 0.5 ml GNA cultures t 30 cells/ml. To isolte independent recombi- nnts, the cells from sturted cultures were spun down nd resuspended in 1 ml of wter nd then ten 11.1 of ech ws spotted on SC + FOA or SC - His. A single Ur- or His+ colony ws picked nd purified from ech spot. To estimte the frequency of Ur- recombinnts for the experiment shown in Tble 4B, sturted cultures were diluted thousndfold nd 20 ~l of ech ws then ptched on n SC + FOA plte. Ur- derivtives of S 1399, S 1402 nd S 1294 were tested for their genotype t HIS4 s follows. Recombinnts were Footnote to Tble I /IOA::URA3 indictes n insertion of the (IRA3 gene 46 kbp centromere-distl to HIS#; HML[URA3] indictes n insertion of (IRA3 imnleditelv djcent to the HML locus (bout 4.5 kbp further from HIS4 thn the site of the JIOA::URA? insertion). B9G::TRPI indictes n insertion of the TRPI gene 18 kbp distl to HIS#. ADEE:BIKI::de2 indictes dupliction of the ADE2 gene flnking pbr322 nd BIKI. The trpl-h3 muttion results from fill-in of the Hind111 site in the TRPl coding region.

4 854 K. Voelkel-Meimn nd G. S. Roeder ptched on YEPD medium nd then replic plted to SC - tion by RNA polymerse I proceeds towrd the telo- His medium nd UV-irrdited to induce mitotic recombimere (HOTI-t), the frequency of Ur- recombinnts ntion (SHERMAN, FINK nd HICKS 1986). His- recombinnts tht gve rise to His+ prototrophs during mitotic growth is incresed fold (S1394 nd S1393). In strins weressumed to be his4-712/his4-260 in genotype. Ur- heterozygous for HOTI-t, the frequency of Ur- rerecombinnts tht were phenotypiclly His+ were sporulted combinnts depends on whether URA? nd HOTl re nd tetrds were dissected (SHERMAN, FINK nd HICKS on the sme or different copies of chromosome ZZZ. 1986); the his4 lleles present in His- spore clones were When HOTI nd URA3 re in cis (S1395 nd S1398), determined by mitotic recombintion tests s described previously (VOELKEL-MEIMAN nd ROEDER 1990). His- recom- the frequency of Ur- recombinnts is bout 20-fold binnts tht filed to generte His+ recombinnts were force greter thn the bckground level. In contrst, when mted to his4-260 nd hid-712 testers nd the resulting HOTl nd URA3 re in trns (S1397 nd S1396), triploids were tested for their bility to produce His+ recom- Ur- recombinnts re incresed only 2-3-fold. The binnts s follows. First, ptches of His- recombinnts were replic plted to 4 YEPD pltes nd UV-irrdited to induce stimultion of recombintion observed in HOTl hethomozygosityt themtingtypelocus.after 4-6-hr erozygotes is not due to the heterology between the incubtion, ech YEPD plte ws spryed with suspension two copies of chromosome ZZI becuse HOTl frgof cells from one the of four tester strins (SR202-3B, JEl38- ment crrying muttion tht completely destroys 17B, M520 nd M521; Tble 1). After incubtion overnight HOTl ctivity hs no effect on the frequency of Urto llow mting, the pltes were replicted to MIN + Leu + Trp + His medium to select for the triploids resulting from recombinnts (S1282 nd S1289). mting. After growth t 30" for 2-3 dys, the ptches of The frequency of Ur- recombinnts is incresed triploids were replicted to medium lcking histidine nd even when HOTI is oriented such tht trnscription UV-irrdited. His- recombinnts homozygous for the his4- by RNA polymerse I should proceed towrd the 260 llele produced His+ recombinnts only when mted to centromere (HOTI-c in S1291, S1293 nd S1295). his4-712 tester nd vicevers.recombinnts tht produced His+ recombinnts when mted to the hid-712 tester The stimultion is much less thn when HOTl is but not the his4-260 tester could be either his4-260/his4- oppositely oriented but the difference between the cis 260 or his4-260/his4-260, 712 in genotype. Severl of these recombinnts were further chrcterized by tetrd nlysis nd ll (88 out of 88) proved to be his4-260/his The genotypes of ll of the his4-712/his4-712 homozygotes were lso confirmed by tetrd nlysis. Independent His+ recombinnts isolted from S1243, S1245,S1250,S1254,S1274ndS1278werechrcterized by tetrd nlysis (VOELKEL-MEIMAN nd ROEDER 1990). Five to ten tetrds were dissected from ech recombinnt. RESULTS HOTl promotes recombinnts homozygous for distl mrker: The effect of HOTl on interchromosoml recombintion ws exmined in diploid strins crrying the URA? gene ner the left end of one copy of chromosome ZZZ. A 570-bp subclone of HOTl (consisting of the enhncer nd inititor of trnscription by RNA polymerse I; VOELKEL-MEIMAN, KEIL nd ROEDER 1987) ws inserted just upstrem of HIS4 on one or both copies of the chromosome. The distnce between HOTl nd URA3 is bout 48 kbp (Figure 1). In these strins, recombinnts tht hve lost the URA? gene cn result either from interchromosoml crossing over or from gene conversion (Figure 2) nd cn be selected on medium contining FOA (BOEKE, LAC- ROUTE nd FINK 1984). Two sets of diploid strins were constructed, one in which URA3 is inserted on the chromosome crrying MAT nd one in which URA3 is on the MAT chromosome. The effect of HOTl on the frequency of Urrecombinnts is shown in Tble 2. In the bsence of HOTl, the frequency of Ur- recombinnts is bout (S1391 nd S1392). When HOTl is present on both chromosomes nd oriented such tht trnscrip- nd trns configurtions of URA3 nd HOTl is still observed. HOTl-promoted Ur- recombinnts re homozygous for n intervening mrker: To determine whether HOT1-promoted Ur- recombinnts re lso homozygous for sequences in the intervl between HOTl nd URA3, the TRPl gene ws inserted on one copy of chromosome ZII t position bout hlfwy between HOTl nd URA? (Figure 1). Two sets of strins were constructed, one in which TRPl is on the sme chromosome s URA3 nd nother in which URA3 nd TRPl re on opposite homologues. Independent Ur- recombinnts were isolted from con- trol strins nd from HOTl heterozygotes in which HOTl nd URA3 re in cis. These recombinnts were tested for their bility to grow in the bsence of tryptophn; recombinnts tht were phenotypiclly Trp+ were exmined by Southern blot nlysis to determine whether they were homozygous or heterozygous for the TRPl insertion. The results re presented in Tble 3. In strin S1402, URA3, TRPl nd HOTl re ll on the sme chromosome; 91 % of the Ur- recombinnts derived from this strin re Trp- nd the reminder re still heterozygous for the TRPI insertion. In S 14 14, TRPl is on the opposite homologue to URA? nd HOTl; 94% of the Ur- recombinnts isolted from this strin re homozygous for the TRPI insertion nd the reminder re heterozygous for TRPI. Thus, most Ur- recombinnts re homozygous for the proximl TRPl mrker. Sequences present t the site of the TRPl insertion on the URA3-mrked prentl chromosome re lost from Ur- recombinnts.

Conversion Long Trcts 855 c TRPl j! c TRPl TRPl 1 2 FIGURE 2.-Ur- recombinnts due to crossing over or gene conversion, A, Crossing over. B, Gene conversion.

5 Conversion Long Trcts 855 c TRPl j! c TRPl TRPl 1 2 FIGURE 2.-Ur- recombinnts due to crossing over or gene conversion, A, Crossing over. B, Gene conversion. A (i), The prentl diploid fter DNA repliction but before recombintion. The configurtion of URA3 nd TRPl mrkers indicted is tht present in S1414; in the sme strin, HOTl is present on the URA3- mrked chromosome. Ech chromosome consists of two sister chromtids represented by the solid nd dshed lines. A (ii), Diploid fter crossover between TRPl nd the centromere. The dotted lines indicte the position of the crossover. A (iii), Diploids resulting from two possible ptterns of chromtid segregtion. B (i), The prentl diploid in G2. B (ii), Diploid fter gene conversion. The htched box indictes the region of conversion. B (iii), The dughter cells resulting from chromtid segregtion. About 35% of the Ur- recombinnts isolted from the control strins (S1399 nd S1411) re still heterozygous t TRPI, s expected if significnt frction of the events occurring in control strins initite in sequences centromere-distl to TRPl. The distribution of TRPl homozygotes (-/-) nd heterozygotes (+/-) mong Ur- recombinnts derived from S1294, in which HOTl points towrd the centromere, is not very different from tht observed in the corresponding control (S 1399). However, further chrcteriztion of the Ur- Trp- recombinnts derived from S nd S 1294 does revel differences between the two strins (see below). HOTI-promoted Ur- recombinnts result from gene conversion: The observtion tht Ur- recombinnts re incresed more when HOTI nd URA3 re on the sme chromosome suggests tht most HOTIpromoted Ur- recombinnts result from gene conversion rther thn reciprocl crossing over. Reciprocl crossing over hs 50% chnce (Figure 2A) of producing Ur- recombinnt regrdless of which chromosome initites the exchnge event; therefore, the frequency of Ur- recombinnts should be unffected by the linkge between HOTl nd URA3. The pronounced stimultion of Ur- recombinnts in strins in which HOTl nd URA? re in cis cn, however, be explined if most Ur- recombinnts result from gene conversion (Figure 2B) nd if HOTI defines the chromosome on which it resides s recipient. In this cse, most of the convertnts rising from the trns configurtion will be homozygous for the insertion (URA?/URA3) wheres most of the recombinnts resulting from the cis configurtion will be homozygous for sequences derived from the chromosome not mrked with URA3. Although gene conversion is the simplest explntion for the results presented, there re lterntive interprettions. For exmple, HOTl might stimulte reciprocl crossing over but do so only when HOTI nd URA3 re present on the sme chromosome (ie., HOTl nd URA3 might somehow interct with ech other to stimulte exchnge). Experiments to distinguish HOTI-promoted gene conversion from crossing over employed strin S in which HOTl nd URA3 re in cis nd TRPl is on the homolog (Tble 3). If most HOTI-promoted Urrecombinnts result from crossing over, then the production of Ur- (TRPIITRPI) recombinnt should be ssocited with the formtion of Trp- (URA3/ URA3) recombinnt (Figure 2A). Thus, HOTl should promote both clsses of recombinnts eqully. However, if most Ur- recombinnts result from gene conversion (in which the HOTI-contining chromosome is the recipient), then the formtion of Urrecombinnt should not be ssocited with the formtion of Trp- recombinnt (Figure 2B) nd HOTl should hve little or no effect on the frequency of Trp- recombinnts in the S strin bckground. (As shown in Tble 3, sequences t the sites of the URA3 nd TRPl insertions remin linked in t lest 90% of HOTI-promoted recombinnts.) Severl independent cultures of S were plted for single colonies on complete medium nd these colonies were then replic plted to medium lcking urcil nd medium lcking tryptophn to detect Urnd Trp- recombinnts. The dt, presented in Tble 4A, support the notion tht HOTI-promoted recombinnts result from gene conversion for two resons. First, the totl number of Ur- recombinnts re-

6 856 K. Voelkel-Meimn nd TABLE 2 Effect of HOT1 on the frequency of Ur- recombinnts Sruin s1391 S1394 S1395 S 1397 URA3 Genotype HOTI-t URA3 HOTl-t cy URA3 HOTI-t HOTI-t URA3 cy SI282 ura3 hotl-t SI392 SI 393 SI396 S1398 SI289 SI291 s1293 S1295 URA? (Y cy URA3 HOTI-t HOTI-t cy UR,4 3 HOTI-t URA3 HOTI-t URA3 hotl-t cy cy URA3 HOTl-c HOTI-c URA3 HOTI-c (IRA3 HOTI-c cy Ut-frequency Fold increse 0.8 x IO-^ 1.0 x 25.3 X 10" 31.6 X 16.6 X lo-' 20.8 X 2.1 X 10-~ 2.6 X 0.7 X 0.9 X 1.1 x 10-~ 1.0 x 49.0 x IO-^ 44.5 X 2.4 X X 19.2 X 10" 17.5 X 0.9 X 10-~ 0.8 X 9.0 X X 1.2 X 10-~ 1.1 x 2.7 X 10-~ 2.5 X Ech of the frequencies shown is the medin frequency from severl independent cultures. For stins S1291, S1293 nd S1295, 16 cultures were tested; 9 cultures were tested for ech of the other strins. The fold increses in strins S1394, S1395, SI 397 nd SI 282 re reltive to SI 391; other strins re compred to S1392. The strins listed in this Tble (nd their isogenic derivtives) were nlyzed in t lest two fluctution tests, with similr results. For exmple, nlysis of SI 396 (in which HOTl nd URA3 re in trns) yielded medin frequency of 2.1 X in one experiment nd 2.4 X in nother; nlysis of n isogenic Trp+ derivtive of S 1396 (S1413, see Figure 1 nd Tble 1) yielded medin frequency of 2.4 X An indiction of the reproducibility of the results obtined with HOTl heterozygotes in which HOT1 nd URA3 re in cis cn be obtined by compring S1395 nd S1398 in this tble with ech other nd with S1402 nd SI414 in Tble 3. covered in this experiment is much greter (bout 10 times) thn the number of Trp- recombinnts. The second line of evidence comes from those cultures displying frequency of Ur- recombinnts significntly higher thn the medin frequency (cultures e nd g in Tble 4A). These represent cultures in which recombintion event occurred erly in the growth of the culture nd the products of this event were propgted through subsequent mitotic divisions. If this erly event ws reciprocl crossover, then both Ur- nd Trp- recombinnts should be present t the G. S. Roeder sme frequency (since the event would produce one of ech). However, if the erly event ws gene conversion, then culture which is jckpot for Urrecombinnts should not be jckpot Trp- for recombinnts. In this experiment, the two cultures with high frequency of Ur- recombinnts did not disply correspondingly high frequency of Trp- recombinnts. To detect dditionl exmples of these erly events, severl independent cultures of S14 14 were grown to sturtion nd then spot-tested to identify those with high frequency of Ur- recombinnts. The cells in these cultures were plted for single colonies nd then tested for their Ur nd Trp phenotypes by replic plting. As shown in Tble 4B, the production of Ur- recombinnt ws unccompnied by the formtion of Trp- recombinnt in ll seven of the events exmined. Thus, most HOTIpromoted Ur- recombinnts result from gene conversion. HOTZ-promoted conversion trcts re continuous but do not lwys initite t HOTl: In strins S1402 nd S1294, URA3, TRPl nd HOTl re ll present on the sme chromosome. Ur- Trp- recombinnts derived from these strins represent conversion trcts tht cover t lest the URA3 nd TRPl mrkers. To more precisely loclize the right-hnd endpoints of these conversion trcts, the genotypes t HIS4 of Ur- Trp- recombinnts were determined. The strting strin is heterollelic t HIS4 with the centromeredistl his4-712 llele on the HOTI-contining chromosome nd the his4-260 muttion on the homolog (Figure 1). Genotypes t HIS4 were determined s described in the Mterils nd Methods. The results re presented in Tble 5A. Three clsses of Ur- Trp- recombinnts re expected if conversion trcts re continuous. Convertnts tht hve their right-hnd endpoint between TRPI nd the site of the hid-712 muttion will still be heterollelic t HIS4 (his4-712/his4-260). Convertnts with one endpoint in the intervl between his4-712 nd hid-260 will be phenotypiclly His+ nd genotypiclly heterozygous for the hid-260 muttion (HIS4/his4-260). Convertnts with the right-hnd endpoint on the centromere-proximl side of the site of the his4260 muttion will be homozygous for his The results presented in Tble 5A indicte tht 92% of the Ur- Trp- recombinnts from the control strin (S1399) nd 98% of the Ur- Trp- recombinnts from the HOTI-contining strins (S1402 nd S1294) cn be ccounted for by continuous conversion trcts. The reminder result from discontinuous conversion trcts, from chromosome loss or from conversions t HIS4 ssocited with crossing over between HIS4 nd TRPl. Ur- derivtives of S1399, S1402 nd S1294 tht re still heterozygous for the TRPI insertion my

Long Conversion Trcts 857 TABLE 3 TRPZ genotypes of Ur- recombinnts Frequency (%) Strin S1399 S 1402 S1294 TRPl URA3 Ur- Fold TRPl TRPl TRPl Prentl genotype frequency increse +I- +I+ -1- URA3 TRPl

7 Long Conversion Trcts 857 TABLE 3 TRPZ genotypes of Ur- recombinnts Frequency (%) Strin S1399 S 1402 S1294 TRPl URA3 Ur- Fold TRPl TRPl TRPl Prentl genotype frequency increse +I- +I+ -1- URA3 TRPl HOTl-t URA3 TRPl HOTl-c 1.0 X 10-~ 1.0 x X 14.9 X X x S1411 SI414 URA3 TRPl ura3 HOT14 TRPl 1.3 X 1.0 x X 11.5 X The recombintion frequencies shown re the medin frequencies derived from nine independent cultures with one exception; for S1294, 16 cultures were exmined. The fold increses in S1402 nd SI294 re reltive to S1399; SI414 is compred to S1411. The numbers of Ur- recombinnts tested for their Trp phenotypes were 225 for S1399, 272 for S1402, 216 for S1294, 52 for S1411 nd 50 for S1414. The numbers of Trp+ recombinnts tested by Southern blot nlysis were 90 for S1399, 10 for S1402, 28 for S1294, 52 for S1411 nd 50 for S1414. TABLE 4 Frequencies of Ur- nd Trp- derivtives of S1414 s determined by replic plting Culture Urn- Trp- A b C d e f g h I No. colonies tested B In the experiment shown in A, the nine independent diploids corresponding to SI 414 (see MATERIALS AND METHODS) were grown to sturtion, plted onto synthetic complete medium nd then replic plted to medium lcking urcil nd medium lcking tryptophn. In the experiment shown in B, nine cultures were grown up for ech of the nine S1414 diploids; ech of these ws spottested for the frequency of Ur- recombinnts s described in MATERIALS AND METHODS. The seven cultures with the highest frequencies were then plted for single colonies nd the resulting colonies were replic plted to detect Ur- nd Trp- recombinnts. In cultures e nd g (Tble 4A) nd 1-7 (Tble 4B), the frequency of Ur- recombinnts observed is significntly greter thn the frequency of Trp- derivtives (P < 0.05). See text for detils. result from events (conversions or crossovers) tht re confined to TRPl-distl sequences. Alterntively, they my be the products of discontinuous conversion trcts in which URA3 nd one or more sites proximl to TRPl (but not TRPl ) re converted. To distinguish these possibilities, Ur- Trp+ recombinnts were tested for their genotypes t HZS4. The vst mjority re still heterollelic (Tble 5B) indicting tht neither the his4-712 nor the his4-260 muttion hs been converted. Thus, the recombintion events tht generte Ur- Trp+ recombinnts re lrgely confined to sequences distl to TRPl. HOTI-promoted conversion trcts frequently include sequences to both sides of HOTl : S 1402 nd S1294 derivtives tht re Ur- Trp- nd homozygous for his4260 could result from conversion trcts whose right-hnd endpoints lie in the intervl between his4260 nd HOTl or from conversion trcts tht extend further to the right. These recombinnts were exmined by Southern blot nlysis to determine whether or not they were still heterozygous for the HOTl insertion. In ddition, the diploids were sporulted nd tetrds were nlyzed in order to determine the genotype t the LEU2 gene, locted bout hlfwy between HIS4 nd the centromere (Figure 1). The results re presented in Tble 6. About 6% of the hid-260 homozygotes derived from S 1402 nd 3% from S 1294 re still heterozygous for HOT1 indicting tht the conversion trct hs one endpoint in the intervl between HOTl nd the his4-260 muttion. More thn 90% do not crry HOTl on either chromosome, indicting continuous conversion trct tht includes HOTl. About hlf of these re still heterozygous t LEU2 nd the reminder re homozygous for the wild-type LEU2 gene. The ltter clss represent continuous conversion trcts extending

8 ~~ 858 K. Voelkel-Meimn nd G. S. Roeder TABLE 5 HIS4 genotypes of Ur- recombinnts Alleles recovered (%) No i Strin Prentl genotype i i tested A Trp- SI 399 S 1402 S1294 LIRA3 TRPl his4-712 his URA3 TRPI his4-712 HOTI-t his4-260 URA3 TRPl his4-712 HOTI-c his B Trp+ SI399 S1402 SI294 URA3 TRPl his4-712 his4-260 (IRA3 TRPl his4-712 HOTI-t his4-260 URA3 TRPl his4-712 HOTI-c his The percentges of Ur- Trp- nd Ur- Trp' recombinnts with different genotypes t HIS4 re given. Derivtives described s his4-260 were identified s 2n - 1 strins bsed on severl criteri: (1) they mte efficiently with MAT testers, (2) they fil to sporulte nd (3) when trnsformed with MAT plsmid, sporulted nd dissected, they produce 2 vible:2 ded spores per tetrd. TABLE 6 HOTl nd LEU2 genotypes of his4-260/his4-260 recombinnts genotype Recombinnt his4-260 HOTl leu2 his4-260 LEU2 his4-260 his4-260 his4-260 his4-260 leu2 LEU2 LEU2 LEU2 Frequency (%) S1402 SI 294 LIRA3 TRPl his4-712 HOTl-t leu2 URA3 TRPl his4-712 HOTl-c leu2 LEU2 his4-260 his4-260 LEU his4-260 HOT1 leu his4-260 HOTl LEU2 The percentges of Ur- Trp- his4-260/his4-260 recombinnts with different genotypes t HOTl nd LEU2 re given. The numbers of recombinnts nlyzed by tetrd dissection nd Southern blot hybridiztion were 34 for SI402 nd 38 for S ll the wy from the centromere-proximl side of LEU2 through URA3. A smll number (3%) of the S 1402 recombinnts represent discontinuous conversion trcts in which HOTl ws converted in the opposite direction to URA3, TRPl nd HIS4 or conversion t HOTl ws followed by crossing over. Ur- Trp- recombinnts tht re his4-712/his4-260 or HIS4/his4-260 in genotype cn be explined by long conversion trcts tht hve their right-hnd endpoints in the TRPl-his4-712 or the his4-712-his4-260 intervl, respectively. If this explntion is correct, then these recombinnts should show no signs of conversion t sites proximl to hzs To explore this possibility, number of his4-712/his4-260 nd HZS4/his4-260 recombinnts derived from S 1402 nd S1294 were nlyzed by Southern blot nlysis to determine their genotypes t HOTl nd by tetrd dissection to determine their genotypes t LEU2. All but 2% (1 out of 52) were still heterozygous t HOTl nd ll but 4% (2 out of 45) were still heterozygous for the leu2 muttion. Summry of Ur- dt: A summry of the nlysis of Ur- recombinnts derived from the control strin, S1399, nd the HOTl heterozygotes, S1402 nd S1294, is shown in Figure 3. As indicted in Figure 2, Ur- recombinnts cn result from either gene conversion or crossing over. The fct tht HOTl stimultes recombintion 10 times more when HOTl nd URA3 re in cis ZIS. in trns suggests tht bout 90% of the Ur- recombinnts derived from S1402

Long Conversion Trcts 859 A. SI399 8. SI402 e. Si294 D. SI402 E. S1294 FIGURE 3.-Distributions of recombintion events.

9 Long Conversion Trcts 859 A. SI SI402 e. Si294 D. SI402 E. S1294 FIGURE 3.-Distributions of recombintion events. The histogrms indicte the distributions of right-hnd conversion endpoints or crossover sites for Ur- recombinnts derived from S1399, S1402 nd S1294. A, B nd C show the frequencies of events in the URA3-TRP1 (LIRA-TRP), TRP1-his4-712 (TRP-712), hid-712-his4-260 ( ) nd hid-260-centromere (260-CEN) intervls. In D nd E, the his CEN3 intervl is further subdivided into the his4-260-hotz (260-HOT), HOTI-LEU2 (HOT-LEU) nd LEU2-CEN3 (LEU-CEN) intervls. Vlues were clculted from the informtion in Tbles 3, 5 nd 6 nd the text. For exmple, the frequency of events occurring in the HOTl- LEU2 intervl in S1294 is the percent of Ur- recombinnts tht re Trp- (61%) multiplied by the percent of Ur- Trp- recombinnts tht re homozygous for his4-260 (76.3%) multiplied by the percent of his-260 homozygotes tht re homozygous t HOT1 (-/-) but still heterozygous t LEU2 (42.1%) multiplied by the frequency of Ur- recombinnts (3.8 X 1 O-4). result from gene conversion. By the sme resoning, hlf to three-qurters of the Ur- recombinnts derived from S1294 re due to conversion. It is unknown wht frction of the Ur- recombinnts derived from the control strin re due to conversion versus reciprocl exchnge. For those Ur- recombinnts tht result from gene conversion, the left end of the conversion trct must lie to the left of URA3. Figure 3 shows the distribution of right-hnd conversion endpoints (for Ur- recombinnts due to gene conversion) or of crossovers (for Ur- recombinnts due to crossing over). In the control strin, events re more or less eqully distributed mong the URA3-TRP1, TRPI-his4-712 nd hid-260-cen3 intervls. In S 1402, contining HOTI-t, the frequencies of events in ll intervls re incresed nd there is shift in the distribution of events towrd more centromere-proximl intervls (Figure 3). In S1294, contining HOTI-c, the frequencies of events in ll intervls re incresed compred to the control but the shift in distribution is not s pronounced s tht observed for S1402. Long conversion trcts mong His+ recombinnts: We hve previously exmined the effect of HOTl on the production of His+ prototrophs in strins crrying his4 heterolleles (his4-712/his4-260) nd distl, heterozygous URA3 mrker (VOELKEL-MEIMAN nd ROEDER 1990). The frequency of His+ recombinnts ws incresed bout 9-fold when HOTl ws inserted just upstrem of HIS4 on both chromosomes nd bout 7-fold when HOTl ws present only on the hid-712 chromosome. About 70% of the His+ recombinnts were still heterozygous for URA3 indicting tht the distl mrker ws not converted. It should be noted tht the frequency of His+ recombinnts (10-4) in HOTl-contining strins is fold lower thn the frequency of Ur- recombinnts (2-5 X lo-'). Thus, His+ Ur- recombinnts represent only smll frction of totl Ur- recombinnts. Approximtely 30% of His+ recombinnts were homozygous for the distl mrker (either URA3IURA3 or Ur-). These recombinnts could result either from gene conversion t HIS4 ccompnied by crossing over or from long conversion trcts extending from HIS4 through URA3 (Figure 4). To distinguish these possibilities, independent His' recombinnts, unselected for their genotype t URA3, were nlyzed by tetrd dissection. When gene conversion t HIS4 is ccompnied by crossover, the His+ gene will segregte into cell tht is homozygous for the distl

860 K. Voelkel-Meimn nd G. S. Roeder A B 1 1 J 1 """"""""""" """""""-""". """"""""""" Reversed Linkge Homozygotes FIGURE 4.-His+ Ur- recombinnts resulting from crossing over or gene conversion.

10 860 K. Voelkel-Meimn nd G. S. Roeder A B 1 1 J 1 """"""""""" """""""-""". """"""""""" Reversed Linkge Homozygotes FIGURE 4.-His+ Ur- recombinnts resulting from crossing over or gene conversion. A, Conversion t HIS4 ccompnied by crossing over. B, Coconversion of his4712 nd URA3. A (i), The prentl diploid in GP. A (ii), Diploid fter conversion of his4-712 ccompnied by crossover between his4-712 nd URA3. A (iii), Diploids resulting from two possible ptterns of chromtid segregtion. B (i), Prentl diploid. B (ii), Diploid fter gene conversion spnning the hid-712 nd LIRA3 mrkers. B (iii), Dughter cells resulting from chromtid segregtion. The recombintion event digrmmed in A represents only one of severl possible types of gene conversion ssocited with crossing over. Both conversion of his4-712 ccompnied by crossover between HIS4 nd LIRA3 (digrmmed) s well s conversion of his4-260 ccompnied by crossover between HIS4 nd LEU2 will give rise to His+ recombinnts tht re homozygous for the distl mrker originlly present on the his4-260 chromosome (U2/U2 in Tble 7). These re the two mjor clsses of recombinnts expected if conversion trcts re continuous nd if crossing over occurs t one end of the conversion trct. His+ recombinnts tht re homozygous for the distl mrker on the his4-712 chromosome (Ul/UI in Tble 7) cn result from conversion of his4-260 ccompnied by crossover in the HIS4 to URA3 intervl or from conversion of his4-712 ccompnied by crossover in the HIS4 to LEU2 intervl. These recombinnts presumbly result from discontinuous conversion trcts in which the his4 llele tht is converted is seprted from the crossover by n unconverted his4 muttion. The open rrow indictes the direction of HIS4 trnscription; the sterisks indicte his4 muttions. Other symbols re s in Figure 2. Drwing is not to scle. mrker 50% of the time (Figure 4A). The other hlf of the time, the His+ gene will segregte into cell which is still heterozygous for URA3 but displys reversed (nonprentl) linkge between the unconverted his4 llele nd either URA3 or LEU2 (depending on whether the crossover occurred in the URA3- HIS4 or HIM-LEU2 intervl). Thus, if most His+ URA3IURA3 nd His+ Ur- recombinnts result from conversion ssocited with crossing over, the number of His+ recombinnts displying reversed linkge should be equl in number to the sum of the two clsses of homozygotes. To determine whether this is the cse, His+ recombinnts were sporulted nd tetrds were dissected nd nlyzed. The his4 lleles present in hploid His- spore clones were determined by llele tests (VOELKEL-MEIMAN nd ROEDER 1990). The linkge between URA3 nd HIS4 nd between HIS4 nd LEU2 ws determined; from this inform- tion, the linkge between URA3 nd LEU2 ws deduced. Three sets of diploid strins were exmined: (1) control strins lcking HOTl, (2) HOTl homozygotes nd (3) strins crrying HOTl only on the chromosome mrked with his4712 nd leu2. The results re summrized in Tble 7. In none of the strins ws 1: 1 rtio of homozygotes to reversed linkges observed. The rtio ws 2.1: 1 (1 5:7) in the control strin, 1.9:l (37:19) in the HOTl homozygote nd 2.4:l (29: 12) in the HOTl heterozygote. In both of the HOTl-contining strins, the results re significntly different from those expected for 1:l rtio (P < 0.01). Although the control strin displys 2.1:l rtio of homozygotes to reversed linkges, this rtio is not significntly different from 1 : 1 rtio (P < 0.1) due to the low frequency of recombinnts of both types. The excess of homozygotes to reversed linkges

Long Conversion Trcts 86 1 TABLE 7 Genotypes of His+ recombinnts S1274ndS1278 S1250ndS1254 S1243ndS1245 U1 712 leu2 (12 260 LEU2 U1 712 HOT1 leu2 Ul 712 HOTl leu2 U2 260 HOTl LEU2 U2 260 LEU2 UI leu2

11 Long Conversion Trcts 86 1 TABLE 7 Genotypes of His+ recombinnts S1274ndS1278 S1250ndS1254 S1243ndS1245 U1 712 leu2 ( LEU2 U1 712 HOT1 leu2 Ul 712 HOTl leu2 U2 260 HOTl LEU2 U2 260 LEU2 UI leu2 Prentl linkge U2 LEU2 (12 leu2 Reversed linkge Ul LEU2 U1 leu2 U 1 homozygotes LEU2 U2 leu2 U2 homozygotes U2 LEU2 Other Totl homozygotes (U 1 nd U2) Totl recombinnts tested Ul nd U2 refer to the distl mrker (URA? or no (IRA?) present on the hid-712 leu2 chromosome nd the his4-260 LEU2 chromosome, respectively. For ll three kinds of strins (controls, HOTl homozygotes nd HOT1 heterozygotes), two sets of diploids were constructed nd nlyzed, one with URA3 on the his4-712 chromosome nd one with URA? on the hid-260 chromosome (see Tble 1). For exmple, URA? is linked to hid-260 in S1243 nd to his4-712 in S1245. Thus, U//U1 homozygote derived from S1243 is Ur- wheres U//U/ homozygote derived from S1245 is URA3IURA3. The vst mjority (>go%) of His+ recombinnts crried HIS4' gene on one chromosome nd either his4-712 or hid-260 on the homoloc: v smll number were heterozygous for the double muttion (his4-260,712) or homozygous HIS4+ (VOELKEL-MEIMAN nd ROEDER 1990). suggests tht, t lest in HOTl-contining strins, some recombinnts my result from gene conversion both t HIS4 nd URA3. A long, continuous conversion trct will generte His+ recombinnt tht is homozygous for the distl mrker if the his4712 chromosome is the recipient nd if the conversion event initites in the intervl between his4-712 nd hid-260 nd extends to the left of the URA3 gene. The resulting His+ recombinnt will be heterozygous for the his4-260 muttion nd homozygous for the distl mrker originlly present on the chromosome mrked with his4260 (U2). As shown in Tble 7, lmost three-qurters of the homozygous recombinnts re homozygous for U2; the mjority of these (bout 90%) re heterozygous for his4260 (dt not shown). Thus, the mjority of the recombinnts hve the phenotype expected to result from coconversion. The dt in Tble 7 demonstrte tht, in the presence of HOTl, 52% (HOTl homozygotes) to 61% (HOTl heterozygotes) of the His+ recombinnts result from conversions t HIS4 (but not URA3 or LEU2) unssocited with crossing over. Another 32% to 21 % result from gene conversion ssocited with reciprocl crossing over [2 (reversed linkges)/totl x 1001 nd 15% result from conversion of both HIS4 nd URA3 [(homozygotes - reversed linkges)/totl x In the control strin, 75% of the His' recombinnts re due to conversion unccompnied by crossing over; 14% re due to conversion ssocited with crossing over nd 7% re due to coconversion. HOTl increses the frequencies of ll three clssesof events but it stimultes conversions ssocited with crossing over nd coconversions even more thn it stimultes short conversion trcts tht re unccompnied by crossovers. DISCUSSION HOT2 promotes long, continuous conversion trcts: We hve exmined the effect of HOTl in diploid strins crrying heterozygous URA3 mrker on the left rm of chromosome ZII. We found tht the frequency of Ur- recombinnts in HOTl heterozygotes is severely ffected by the linkge between HOTl nd URA3. Ur- recombinnts re incresed drmticlly only in strins in which HOTl nd URA3 re in cis. Furthermore, the stimultion of Ur- recombinnts observed when HOTl nd URA3 re present on the sme chromosome is unccompnied by corresponding increse in the frequency of recombinnts homozygous for sequences on the URA3-mrked homolog. These results indicte tht most HOTl-promoted Ur- recombinnts result from gene conversion nd tht sequences on the HOTl-contining chromosome re preferentilly converted, consistent with our previous studies of gene conversion t HIS4 (VOELKEL-MEIMAN nd ROEDER 1990). HOTl-promoted conversion trcts were exmined in strins crrying multiple mrkers on the left rm of chromosome IZI. Most of the conversion trcts giving rise to Ur- recombinnts were t lest 30 kbp in length nd mny were longer thn 77 kbp; the

862 K. Voelkel-Meimn nd verge conversion trct ws 43 kbp long in SI402 (HOTl-t) nd 36 kbp in S1294 (HOTl-c). [Note tht the verge length clculted is minimum vlue.

12 862 K. Voelkel-Meimn nd verge conversion trct ws 43 kbp long in SI402 (HOTl-t) nd 36 kbp in S1294 (HOTl-c). [Note tht the verge length clculted is minimum vlue. For exmple, conversion trcts tht include URA3 nd TRPl but not ny of the other scorble mrkers re ssumed to be 30 kbp in length-the size of the URA3 insertion (1.2 kbp) plus the distnce between URA3 nd TRPl (28 kbp) plus the size of the TRPl insertion (0.8 kbp). In relity, the region of conversion might hve extended lmost to the site of the his4-712 muttion in which cse the trct would be 48 kbp long.] The frction of events tht cn be ccounted for by continuous conversion trcts bsed on the dt vilble is 98% for S1294 (HOTl-c) nd 93% for S1402 (HOTl-t). The frequency of discontinuous conversion trcts my be slightly underestimted since recombinnts were not tested for conversion of ll mrkers proximl to locus which did not undergo conversion. It could be rgued tht most HOT1-promoted conversion trcts re much shorter thn 40 kbp but tht the selection imposed (which demnds loss of mrker fr from the presumed initition site) leds to n rtifctul infltion of conversion trct length. If most HOTl-promoted conversion trcts initite ner HOT1 but extend for distnce of only one or few kbp, then mrker inserted ner HOTl would be expected to undergo higher frequency of conversion thn distnt mrker. In fct, strins inwhich URA3ws inserted less thn 3 kbp downstrem of HOTl-t produced 2-3-fold fewer Ur- recombinnts thn strins crrying URA3 t the more distl loction (dt not shown). This is the result expected if most HOTlpromoted conversion trcts extend ll the wy to the end of the chromosome nd if hlf of the conversion events initite in or proximl to the HIS4 gene nd the other hlf re confined to HZS4-distl sequences (s indicted by the nlysis of S1402 recombinnts). Other investigtors hve mesured conversion trct lengths during mitotic interchromosoml recombintion. JUDD nd PETES (1988) selected Ur- convertnts from diploid strin heterozygous for ur3 muttion nd then exmined these recombinnts for conversion of flnking restriction sites tht were heterozygous in the prent. In bout hlf of the Urrecombinnts exmined, conversion trcts were shorter thn 4 kbp; the other hlf were longer but their lengths could not be ccurtely determined. Diploid strins heterozygous for n uxotrophic muttion (+/-) hve lso been used to screen for sectored colonies tht re prototrophic on one side nd uxotrophic on the other. These sectored colonies cn result from reciprocl recombintion, inwhich cse the prototrophic hlf of the colony is homozygous (+/+), or from nonreciprocl recombintion, in which cse the prototrophic sector is heterozygous (+/-). When spontneous sectored colonies were exmined, G. S. Roeder the mjority resulted from nonreciprocl recombintion; furthermore, gene conversion t loci linked to the mrker used to detect recombintion ws fre- quently observed (JOHNSTON 1971 ; BRUSCHI nd ES- POSITO 1983). For exmple, JOHNSTON (197 1) found tht 10 of 18 colonies sectored for de8 were due to nonreciprocl recombintion; hlf of these lso showed conversion of the TRP4 gene locted bout 40 cm centromere-proximl to ADE8. Mechnism of HOTI-promoted gene conversion: We previously considered two models to ccount for the fct tht HOTl-contining chromosome preferentilly received informtion during gene conversion. In one of these models, HOTl-promoted gene conversion is ssumed to result from symmetric strnd trnsfer (MESELSON nd RADDINC 1975) with the HOTl-contining chromosome cting s recipient (Figure 5, A nd B). According to this model, HOTl stimultes recombintion by rendering the djcent sequences more susceptible to strnd invsion. In order for the symmetric strnd exchnge model to explin the long conversion trcts described here, the region of heteroduplex DNA must frequently extend ll the wy from LEU2 through URA3. Furthermore, ll of the mrkers included in the heteroduplex must be repired in the sme direction. This could result if the mismtches were left unrepired nd then resolved by DNA repliction (Figure 5A) or if the mismtch repir system corrected ll mismtches in the sme direction (Figure 5B). Both of these modes of repir seem unlikely for the following resons. First, mismtch repir in mitosis is very efficient. ESPOSITO nd WACSTAFF (1981) estimted minimum efficiency of 60-70% for mismtched bse pirs t the TRPl, LEU1 nd HIS1 loci. In studies of repir of heteroduplex plsmid DNA trnsformed into yest (BISHOP nd KOLODNER 1986), single bp mismtches, s well s 8-bp nd 12-bp insertion mismtches, were repired in 73-98% of the trnsformnts nlyzed. Unfortuntely, there is no informtion bering on the efficiency of repir of lrge insertion mismtches such s those tht would be creted t URA3, TRPl nd HOTl; nevertheless, filure of repir t sixmismtched sites (URA?, TRPl, his4-712, his4-260, HOTl nd LEU2), three of which represent single bp mismtches, seems unlikely. In order for ll mismtches to be corrected in the sme direction by the mismtch repir system, they would presumbly hve to be included in the sme repir trct. Studies of heteroduplex repir during trnsformtion indicte tht re- pir trcts re frequently shorter thn 1 kb (BISHOP nd KOLODNER 1986). During mismtch correction in vitro in extrcts of mitotic cells, excision-resynthesis trcts re only nucleotides long (MUSTER-NAS- SAL nd KOLODNER 1986). A model tht better explins HOTl-promoted gene

13 Long Conversion Trcts 863 A (i) U T - #.,.,.,,.,,,,,..,.,.,. r,.,., #,. I; 3.<,,,.*,,,.- ~~,~ 1 Asymmetric strnd trnsfer,.,;,. b...*. Double-strnd brek nd gp expnsion I Mismtch repir I Two-sided gp repir FIGURE 5.-Models for HOTZ-promoted gene conversion. A, Asymmetric strnd exchnge followed by repir by repliction. B, Asymmetric strnd exchnge followed by mismtch repir. C, Double-strnd brekge followed by two-sided repir. D, Double-strnd brekge followed by one-sided repir. A (i), Prentl diploid in G,. A (ii), Diploid fter symmetric strnd exchnge. A (iii), Diploid fter DNA repliction. B (i), Prentl diploid. B (ii), Diploid fter symmetric strnd exchnge. B (iii), Diploid fter mismtch repir. C (i), Prentl diploid. C (ii), Diploid fter double-strnd brekge nd gp expnsion. C (iii), Diploid fter two-sided gp repir. D (i), Prentl diploid. D (ii), Diploid fter double-strnd brekge nd gp expnsion. D (111). Diploid fter one-sided gp repir. The model in A nd B ws first proposed by MESELSON nd RADDING (1975); the model in C is the double-strnd-brek repir model (SZOSTAK et l. 1983) nd D is modifiction of this model. See text for dditionl informtion. Ech line represents single strnd of duplex DNA molecule; the strnds from one duplex re indicted by solid lines nd the strnds from the other re htched. U, T nd L indicte the URA3, TRP 1 nd LEU2 genes, respectively; the corresponding - signs indicte the bsence of the URA3 or TRP I gene or the recessive leu2 muttion. For homoduplexes, the genotype is indicted between the two strnds; for heteroduplexes, the genotype of ech strnd is given. In the exmples shown, the region of symmetric strnd exchnge (A nd B) nd the double-strnd gp (C nd D) include the URA3, TRPI nd LEU2 mrkers. For simplicity, only two recombining DNA molecules re shown even though recombintion my occur in G2, when four chromtids re present, s digrmmed in Figures 2 nd 3. conversion events proposes tht HOTl stimultes the formtion of double-strnd brek in the region of HOTI-promoted trnscription (Figure 5, C nd D). This model cn ccount for the long, continuous conversion trcts described in this pper if it isssumed tht brekge is frequently followed by extensive exonucleolytic digestion. The gp generted could then be repired (SZOSTAK et l. 1983) if the free ends creted on both sides of the brek invde the homolog nd initite repir synthesis (Figure 5C). An lterntive to gp repir is brek-nd-replicte pthwy of recombintion nlogous to tht used by bcteriophge T4 (MOSIG 1987). According to this version of the model, only the centromere-contining chromosoml frgment invdes the homolog nd primes repliction (Figure 5D). Sequences distl to the brek might be enzymticlly degrded; if not degrded, then the frgment would be lost t mitosis due to the bsence of centromere. Ur- derivtives tht re mting-competent might be expected to result either from chromosome loss or from conversion trcts tht include the MAT locus. However, only one exmple of mting-competent segregnt ws detected mong over 500 independent HOTI-promoted Ur- recombinnts nlyzed (dt not shown) nd this segregnt resulted from chromosome loss. If most Ur- recombinnts result from double-strnd-brek-induced recombintion, then the low frequency of chromosome loss indictes tht double-strnd brek repir is very efficient. Furthermore, the dt suggest tht the region of exonucleolytic digestion (nd subsequent repir) rrely includes the MAT locus. This could be due to the distnce involved (bout 150 kbp from HOTl to MAT) or to the presence of specific sequence (such s the centromere) tht might inhibit gp expnsion. Do HOTI-promoted recombintion events initite in HOTI-trnscribed sequences? The distributions of recombintion events in HOTI-contining strins suggest tht not ll HOTI-promoted recombintion events initite in sequences undergoing trnscription by RNA polymerse I. Previous studies hve suggested tht trnscription inititing in HOTl cn extend for distnce of bout 12 kbp (STEWART nd ROEDER 1989). Thus, most events re expected to initite in the TRPI-HOT1 intervl in S1402 (HOTIt) nd between HOTl nd LEU2 in S1294 (HOTI-c). Conversion trcts tht hve their right-hnd endpoints proximl to the presumed region of initition cn be ccounted for by double-strnd brekge followed by

864 K. Voelkel-Meirnn nd G. S. Roeder exonucleolytic digestion. However, conversions whose right-hnd endpoints re distl to the region of trnscription re more difficult to explin.

14 864 K. Voelkel-Meirnn nd G. S. Roeder exonucleolytic digestion. However, conversions whose right-hnd endpoints re distl to the region of trnscription re more difficult to explin. Nevertheless, these events re stimulted by HOTI. For exmple, events in the URA3-TRPI intervl re incresed 3-4-fold for both S 1402 nd S In S 1402, these distl events cn be explined by proposing tht some HOTI-promoted trnscripts re significntly longer thn 12 kbp nd therefore extend pst TRPI. This explntion does not, however, pply to SI 294. There re severl possible explntions for the bility of HOTl to stimulte events confined to sequences distl to the region of HOTI-promoted trnscription. First, HOTI-promoted trnscription might somehow interfere with the spontneous (i.e., not HOTI-promoted) recombintion events tht would normlly occur in the region of HOTI-promoted trnscription. This inhibition might result in redistribution of the spontneous events such tht these re incresed in frequency in sequences distnt from the site of HOTl insertion. Alterntively, HOTl my be ble to ct t distnce to trigger the initition of events outside the region of trnscription. For exmple, n enzyme my enter the DNA in the region of HOTI-promoted trnscription nd then trvel some distnce before introducing brek. The RecBC enzyme of Escherichi colis n exmple of n enzyme tht trvels some distnce before cutting (PONTICELLI et l. 1985); this enzyme requires free duplex end in order to enter DNA molecule (STAHL et l. 1983). Another possibility is tht HOTl promotes the formtion of nick (in the region of trnscription) which is expnded to single-strnd gp; the exposed single strnd could subsequently be cleved by n endonuclese to generte double-strnd brek. If the second cut occurs t some distnce from the first, then the double-strnd brek could be distnt from the region of HOTIpromoted trnscription. Models similr to these hve been proposed to explin the bility of HO-induced breks to stimulted recombintion t distnce (Ru- DIN nd HABER 1988; NICKOLOFF et l. 1989; RAY, MACHLIN nd STAHL 1989). Another puzzling observtion is tht HOTI-t stimultes the formtion of Ur- recombinnts significntly more thn HOTI-c. The simplest explntion for this result is tht HOTl-c is less ctive thn HOTlt due to context effect. For exmple, the DNA tht lies proximl to HOTl might fortuitously contin sequences tht terminte trnscription by RNA polymerse I. In this cse, the region of HOT1-promoted trnscription might be shorter in strins contining HOTI-c thn HOTI-t. Alterntively, when HOTl is oriented towrd the centromere, the region of trnscription my be so fr from URA3 tht conversion events inititing in the trnscribed region rrely include URA3. If HOTl promotes trnscription over distnce of 12 kbp, then HOTI-t trnscribes sequences kbp from URA3 nd HOTI-c trnscribes sequences kbp from URA3. To ccount for the 4-fold difference in recombintion frequency between S1402 nd S1294, it is necessry to rgue tht HOTl promotes the conversion of mrker kbp wy &fold less often thn it promotes conversion of mrker kbp wy. Given the ex- treme lengths of the conversion trcts observed, it seems unlikely tht the frequency of events decreses so bruptly. A third possibility is tht HOTI-c promotes events with the sme efficiency s HOTI-t but these conversion trcts hve directionlity such tht they often include sequences downstrem (to the right of HOTl-c) nd less infrequently sequences upstrem. Since the strins used in this study crry no selectble mrkers to the right of HOTI, we hve been unble to explore this possibility. Coincident conversion of HIS4 nd URA3: When gene conversion t HIS4 is ccompnied by reciprocl crossing over in the G:! stge of the cell cycle (Figure 4A), hlf of the resulting His+ recombinnts re ex- pected to disply nonprentl configurtion of mrkers flnking HIS4 (reversed linkge) nd hlf should be homozygous for distl mrker tht ws originlly heterozygous. HABER nd HEARN (1985) observed 1 : 1 rtio of reversed linkges to homozygotes in their studies of gene conversion t HIS4 using strins nd selections very similr to those described here. Three fctors might led to n ltertion in the expected 1:l rtio of reversed linkges to homozygotes. First, if ny crossovers occur in GI, then reversed linkges will exceed homozygotes becuse crossovers in GI generte only recombinnts disply- ing reversed linkge. In fct, ROMAN (1980) observed 2-fold excess of reversed linkges in his studies of X-ry induced recombintion t ADE6. A second fctor which cn ffect the rtio isif the recombinnt chromtids segregte nonrndomly mitosis. t In studies of mitotic recombintion in Drosophil, PIMPI- NELLI nd RIPOLL (1986) observed 2-fold bisin fvor of the recombinnt chromosomes segregting to opposite poles. In the experiments reported here, such bis would result in 2-fold excess of homozygotes to reverse linkges, which is precisely the result observed. An lterntive explntion for the excess of reverse linkges to homozygotes is tht recombinnts homozygous for the distl mrker result not from crossing over but from nonreciprocl recombintion. If rndom chromtid segregtion is ssumed (s it will be throughout the reminder of this discussion), then the observed 2:l rtio of homozygotes to reversed linkges suggests tht bout hlf of the homozygotes result from crossing over nd hlf from gene conversion. Note tht this is minimum estimte of the frequency of coincident conversions since it

15 Conversion Long Trcts 865 ssumes tht ll crossovers occur in Gz. If some crossovers occur in GI (leding to n increse in the number of reversed linkges), then the number of homozygotes resulting from conversion must be greter. Recombinnts tht hve undergone conversion both t HIS4 nd URA3 could result either from coconversion (both mrkers included in the sme conversion trct) or from coincident (but seprte) conversion events. The observtion tht the mjority of His+ recombinnts tht re homozygous for the distl mrker hve the genotype expected for long continuous conversion trcts (U2 HIS4/U2 his4-260; see Tble 7 nd RESULTS) is consistent with the hypothesis tht HIS4 nd URA3 re coconverted. However, this result is inconclusive becuse the sme clss of recombinnts is expected to be the predominnt product of conversions ssocited with crossing over (see Figure 4 nd legend). In the bsence of suitble mrkers in the intervl between HIS4 nd URA3, it is impossible to determine whether simultneous conversions of HIS4 nd URA3 represent continuous conversion trcts or coincident conversions. It should be noted tht very different kinds of HOTI-promoted recombinnts re recovered depending on whether His+ or Ur- recombinnts re selected. The mjority (80-85%) of His+ recombinnts result from reltively short conversion trcts (tht include one of the his4 lleles but not the other his4 muttion nd not URA3 or LEU2) with or without n ssocited crossover. Only 15% re due to coincident conversions (possibly coconversions). In contrst, lmost ll Ur- recombinnts result from long, continuous conversion trcts. These results cn be explined by the models shown in Figure 5, C nd D, if it ssumed tht most His+ recombinnts result from limited gp expnsion wheres Ur- recombinnts result from more extensive exonucleolytic digestion. Most His+ nd perhps some Ur- recombinnts result when sequences on both sides of the brek invde the homolog nd promote repir synthesis (Figure 5C). Mny Ur- recombinnts nd perhps smll frction of His+ recombinnts result from one-ended repir synthesis (Figure 5D). While this is n economicl interprettion of our dt, the possibility tht His+ nd Ur- recombinnts result from fundmentlly different mechnisms cnnot be ruled out. For exmple, Ur- recombinnts might result from breknd-replicte pthwy of recombintion wheres most His+ recombinnts might result from symmetric strnd exchnge. As noted bove, His+ recombinnts re fold less frequent thn Ur- recombinnts. Other studies of intrgenic recombintion in heterollelic diploids hve provided evidence for coinci- dent conversions of distnt mrkers. ESPOSITO nd coworkers used diploid strins crrying heterolleles t the TRPS locus on chromosome VI1 nd distl heterozygous de5 muttion (ESPOSITO 1978; GOLIN nd ESPOSITO 1983; BRUSCHI nd ESPOSITO 1983). They selected Trp+ colonies tht were sectored for the de5 muttion. The mjority of these recombinnts resulted from gene conversion t TRPS ccompnied by crossing over between TRPS nd ADES but bout 15% resulted from conversion both t TRPS nd ADE5. In slightly less thn hlf of these coconvertnts, mrkers in the intervl between TRPS nd ADES (cyh2 nd metl3) were converted in the sme direction s de5. In the reminder, one or both of the intervening mrkers went unconverted, ws converted in the opposite direction to de5 or displyed 4:O (insted of 3: 1) segregtion. In studies of diploids heterollelic t the LEUl nd TRP5 loci (TRP5 is distl to LEUl on chromosome VII), GOLIN nd ESPOSITO (1984) observed tht coincident (LeuC Trp+) convertnts rose 1200 times more frequently thn expected for independent events. Furthermore, n insertion muttion in the intervl between LEUl nd TRPS ws coconverted in bout 40% of the Leu+ Trp+ recombinnts (GOLIN nd FALCO 1988). The uthors suggest tht coincident conversion my involve extensive heteroduplex DNA; long region of gp repir is lso consistent with their results. The mjority (bout 80%) of Leu+ Trp+ recombinnts displying coconversion of the intervening mrker were not homozygous for mrkers distl to TRPS nd thus could not hve resulted from breknd-replicte pthwy of recombintion. It should be noted, however, tht it would be impossible to generte recombinnts of the type selected by breknd-replicte pthwy since the production of Leu+ Trp+ recombinnt requires conversion of one (but not both) lleles t both LEUl nd TRP5. GOLIN nd TAMPE (1 988) hve reported tht coincident conversion lso occurs t unlinked loci; for exmple, coincident conversions t LEU1 (on chromosome VU) nd TYRl (on chromosome II) were 4-8 times more frequent thn expected for independent events. Coinci- dent conversions of unlinked mrkers indicte the existence of subpopultion ofcells tht undergo recombintion t high frequency. Are HOTl-promoted events different from spontneous events? In strins heterozygous for HOTI, HOTl defines the chromosome on which it resides s recipient during meiotic gene conversion. The inequlity of the two prticipting chromosomes (one with nd one without HOTI) mkes it possible to demonstrte tht most Ur- recombinnts result from gene conversion nd to demonstrte tht the HOTIcontining chromosome is recipient. In strins lcking HOTl, recombintion events presumbly initite with equl frequency on both chromosomes; consequently, recombinnts homozygous for sequences on ech homolog re produced with equl frequency. The

16 866 K. Voelkel-Meimn nd G. S. Roeder equlity of the two prticipting chromosomes mkes it impossible to determine whether the Ur- recombinnts rising in control strins result from crossing over or gene conversion. It is possible (perhps even likely) tht most of the spontneous recombinnts result from the sme mechnism s the Ur- recombinnts rising in HOTI-contining strins. HOTl my ct only to increse the frequency of the events nd to effect some ltertion in their distribution. His+ recombinnts my lso result from similr mechnism in both control nd HOTl-contining strins. In both cses, the vst mjority of His+ recombinnts result from gene conversion (s opposed to crossing over) (VOELKEL-MEIMAN nd ROEDER 1990); in control strins, it is impossible to determine whether the inititing chromosome is donor or recipient. There re, however, some differences between the His+ recombinnts rising in control strins nd those derived from HOTI-contining strins. In control strins, the frequency of His prototrophs resulting from simple conversion is slightly incresed nd the frequencies of conversions ssocited with crossing over nd of coincident conversions t HIS4 nd URA3 re decresed (bout 2-fold). These minor differences do not preclude the possibility tht spontneous nd HOTI-promoted events shre common mode of initition (e.g., double-strnd brek). Summry: The results presented here demonstrte tht HOTl promotes conversion events in which ll mrkers distl to the site of HOTl insertion re converted. These results cn most simply be explined by brek-nd-replicte pthwy of recombintion. This work ws supported by Ntionl Institutes of Helth grnt GM28904 nd Ntionl Science Foundtion grnt PCM We thnk TOM MENEES, BRAD OZENBERGER, JOANNE ENGEBRECHT nd BETH ROCKMILL for helpful comments on the mnuscript. LITERATURE CITED ANGEL, T., B. AUSTIN nd D. G. CATCHESIDE, 1970 Regultion of recombintion t the HIS3 locus of Neurospor crss. Aust. J. Biol. 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