Copyright 0 1993 by the Genetics Society of America

Ends-In vs. Ends-Out Recombination in Yeast

P. J. Hastings*, Carolyn McGill? Brenda Shafert and Jeffrey N. Strathem+

*Department of Genetics, University of Alberta, Edmonton, Alberta T6G 2E9Canada, andtNCZ-Frederick Cancer Research and Dewelopment Center, ABL-Basic Research Program, Laboratory of Eukaryotic Gene Expression, Frederick, Maryland 21 702-1201 Manuscript received September 1, 1992 Accepted for publication August 30, 1993

ABSTRACT Integration of linearized plasmids into yeast chromosomes has been used as a model system for the study of recombination initiated by double-strand breaks. The linearized plasmid DNA recombines efficiently into sequences homologous to the ends of the DNA. This efficient recombination occurs both for the configuration in which the break is in a contiguous region of homology (herein called the ends-in configuration) and for “omega” insertions in which plasmid sequences interrupt a linear region of homology (herein called the ends-out configuration). The requirements for integration of these two configurations are expected to be different. We compared these two processes in a yeast strain containing an ends-in target and an ends-out target for thesame cut plasmid. Recoveryof ends- in events exceeds ends-out events by two- to threefold. Possible causes for the origin of this small bias are discussed. The lack of an extreme difference in frequency implies that cooperativity between the two ends does not contribute to the efficiency with which cut circular plasmids are integrated. This may also be true for the repairof chromosomal double-strand breaks.

HE repair of double-strandbreaks (DSBs) by when paired with the homologous target (“ends-out”). T homologous recombination requires both sides Becausedirect cooperation of endsseems difficult of the break tobe involved in therecombination with theends-out configuration, ends-out events process. Whether the two sides have similar roles in might be expected to occur less efficiently than ends- the recombination process has not been determined. in events.We report here that the two topologiesyield The DSB-repair model as describedby SZOSTAKet al. similar frequenciesof integration with a two- to three- (1983) incorporates a symmetricintermediate in fold bias favoring the ends-in configuration. which ends from bothsides of the DSB have invaded the unbroken chromatid. It is possible that the rules MATERIALS AND METHODS for invasion of the twosides are not the same(CAMP- BELL 1984; ROTHSTEIN 1984; THALERand STAHL Plasmids: We filled inthe EcoRI site of pBR322 (BOLIVAR et al. 1977) and inserted a XhoI linker to create pBR322X. 1988). For example, it is possible that the invasion by The URA? gene was inserted as a XhoI fragment into the the first side alters the uncut chromosomeso that it is XhoI-Sal1 sites of pBR322X to create pBRpU3A. A 2-kb a better target for the strand(s) from the other sideof fragment of chromosome ZZZ extending from the Hind111 the break. It has been suggested that the invasion of site centromere proximal of MAT to within 50-bp of the a region of homology by DNA from one side of the MAT Y region was inserted into pBRpU3A as a XhoI frag- ment. The trpl gene (a 857-bp EcoRI to BglII fragment) break sets up a replication fork that may open the and the his3 gene (a 1075-bp fragment extending from a DNA thatis homologous to the other sideof the break BamHI site inserted 210 bases upstream of the ATG to an (SZOSTAKet al. 1983). In other words, invasion of one EcoRI site inserted 201 bases downstream of the HIS3 end facilitates invasion of the other end. This coop- termination codon) were inserted at an EcoRI site in the erativity would minimize the formation of chromo- MAT fragment. The resultant plasmid, pCM54, carries the trpl-488 allele (an in-frame UAG codon) and the his3-192 somal rearrangements that would result if the two allele (a frame-shift caused by filling in an NdeI site) (MCGILL DNAs from opposite sides of a DSB recombine with et al. 1990). A Sal1 plus XhoI partial digest of pGM54 was different targets. self-ligated to create pCMTRPl and pGMHIS3. SmaI XhoI To determinewhether there is cooperativity be- fragments from these two plasmids were combined to create the inside out version of pCM54 designated pCM54EVT tweenthe two sides of a DSB,we comparedtwo (for everted). These plasmids are diagramed in Figure 1. different topologiesof the DNA ends.We determined Yeast strains: The yeast strains (Table 1) used as recipi- the efficiencyof integration of a cut plasmid at a ents for transformation with pGM54 and pGM54EVT have target where the ends point toward each other when the genotype MATa::[trpl-U89 his3-6211 lys2::[trpl-089his% paired with a region of homology (“ends-in”) as com- 6211 leu2-A1 tyr7-1 trpl-A1 his3-A200 ura3-52 and are re- lated to the previously described strain GRY558 (MCGILL pared with the integration of that same cutplasmid at et al. 1990) by the insertion of the trpl-his3 module at lys2. a target where the ends point away from each other They have the trpl-089 and his5621 alleles inserted next

Genetics 135: 973-980 (December, 1993) 974 P. J. Hastings et al.

TABLE 1 x XIS Yeast strains pGM54 MATU + - - + MATVWX +-+ + Strain Genotype GRY558 MATa::[frpl-O89 his3-6211 LYS2 leu2-AI 477-1 trpl- A1 his3-A200 ura3-52 GRYl148 MATa::[frpl-089 his3-6211 lys2::[his3-621 trpl-O89/ pGMTRPl leu2-AI qr7-1 trpl-AI his3-A200 ura3-52 GRYI 150 MATa::[trp1-089 his3-6211 lys2::[trpl-089 his3-6211 leu2-AI fyr7-1 trpl-A1 his3-A200 ura3-52 GRYll74 MATa canl LYS2 leu2-A1 ade2-101 trpl-A1 XISB R x XIS NK his3-A200 ura3-52 GRY 1 152 MATa lys2-801 ura3-52 pGMHIS3 A~DId his3 GRY 1 153 MATa lys2-801 ura3-52 - +MATVWX + + GRY 1163 MATa::[trpl-488 his3-1921 LYS2 leu2-AI ade2-101 trpl-A1 his3-A200 ura3-52 canl

the resulting diploid clearly identifies a given transformant as the result of insertion of the plasmid at MATa or atlys2. +- +MATVWXMATU+- ++ The MAT and lys2 targets were chosen because they allow direct analysis of spore patches without tetrad dissection. FIGURE1 .-Plasmids utilized in this study. pGM54 includes the This replica-plating protocol allowed the analysis of many trpl and his3 genes inserted into an EcoRI site in a DNA fragment more transformants than could have been handled by tetrad from the centromere proximal side of MAT. Note the unique genetics. The protocol involved making patches of the Ura+ BamHI site used to linearize pGM54. MATU is the 527 base chro- transformants onto SC-ura [synthetic complete medium mosome 111 region centromere proximal to that EcoRl site. MATV (SHERMAN,FINK and HICKS 1986) minus uracil]. The is the 428 base region between the EcoRI site and the start of patches were mated to a lawn of CRY 1174 (or CRY 1 163) MAW.pGMHIS3 and pGMTRPl are SalI-XhoI deletion deriva- selecting on SD + his + trp + leu (minimal medium plus tives of pGM54 used in the construction of the everted version of histidine, tryptophane and leucine). This medium selected this interval designated pGM54EVT.Note the unique Xhol site diploids carrying the URA3 and ADEP alleles from the used to linearize pGM54EVT. B = EarnHI, Bg = EgllI, K = KpnI, transformed strain and the 7YR7 and LYS2 alleles from R = EcoRI, Sa = SalI, Sp = SpeI, Xb = XbaI, XN = filled in NdeI CRY 1 174. After days,2 the mating plate was replica-plated X = Xhol, XIS = XhoI/SalI ligation. to SD + his + trp + leu to enrich for thediploids. After one additional day of growth, the patches were replica-plated to to the MAT locus in the same orientation as the trpl and two SPOR plates (1.5% potassium acetate, 0.25% yeast his3 genes in the plasmid pCM54 (Figure 2A). GRYl 150 extract, 0.1 % glucose, 2% agar plus nutritional supplements has the trpl-089 and his3-621 alleles plus MAT DNA, de- at one-fourth the concentration used in synthetic complete rived from a plasmid (pCM6 13) similar to pCM54, inserted medium) and incubated for threedays to induce meiosis and into the lys2 gene on chromosome II. The 4-kb MAT::trpl- sporulation. The sporulated patches were mated to MATa 089 his3-621 insertion was made as a substitution for 2.4-kb ura3 lys2 and MATa ura3 lys2 strains (GRYl 152and of lys2 DNA (FLEIG,PRIDMORE and PHILIPPSEN1986) from CRY 1 153, respectively) by replica-plating the patches onto a BglII site (at which a Xhol linker was inserted) to a XhoI lawns of the testers spread on YEPD. After 1 day,the spore- site (Figure 2B). CRY 1 148 differs from GRYl 150 in that mating plates were replica-plated to selective plates. TO the MAT::trpl-089 his3-621 insertion at lys2 in GRYl 148 determine whether the URA3 plasmid was inserted atMATa, has the inverted structure similar to pCM54EVT (Figure we compared the ability of the spore patches to mate and 2C). The 4-kb BamHI insertion into lys2 in CRY 1 148 was complement the Ura requirement of the a and a tester made as a substitution for 2.8-kb of lys2 from a BgllI site to strains (CRY 1 152 andCRY 11 53).For insertions at MATa, a BamHI site. The insertions into the lys2 DNA were made there were far more a Ura+ spores than a Ura+ spores. In in a plasmid derivative of pDP6 (FLEIG, PRIDMOREand contrast, URA3 insertions at lys2 gave spores that were able PHILIPPSEN1986). The insertions at LYS2 were introduced to mate and complement the Ura defect of both mating type into yeast by identifying a-amino adipate (aAA) resistant testers equivalently. To ascertain whether theURA3 plasmid (Lys-) transformants of the LYS2 parent strain (CHATTOOet was inserted at lys2, we determined whether the diploids al. 1979). Thestructures diagramed in Figure 2 were con- could produce spores that were simultaneously URA3 and firmed by restriction digestion and blotting analysis (data LYSP by mating the spore patches to CRY 1 152 and not shown). CRY 1 148 carries the chromosome III shown in CRY 1 153 and selecting Lys+ and Ura+. For insertions at Figure 2A and the chromosome II shownin Figure 2C, lys2, URA3 and LYS2 are alleles and hence could not segre- providing homologous targets with different topologies for gate into the same spore. In contrast, for URA3 insertions the cut plasmids. GRYl 150 has chromosome ZZZ shown in at MATa, about one-fourthof the spores were a Ura+ Lys+. Figure 2A and the chromosome IZ shownin Figure 2B, We dissected over 80 randomly picked diploids to confirm providing a control with both targets in the same relative that the results of the patch tests are an accurate reflection configuration for the cut plasmids. of the insertion site. Identifying the site of plasmid insertion: The site of insertion of the URA3-basedplasmids was established by RESULTS determining the linkage of the URA3 gene to the MAT and lys2 genes. Each transformant was mated to a LYS2 ura3 Linear DNA fragments introduced into yeast are MATa strain (GRYl 163 or GRYl174). Tetrad analysis of readily inserted into the genome by homologous re- Role of DNA Ends in Recombination 975

R XXb BpSaeN XK R A. CEN3 EL3 FIGURE2.-Chromosome targets utilized in this study. (A) ChromosomeIII target; the trpl and his3 MATU - + +- MANWX genes inserted into an autochthonous EcoRl site "--) centromere proximal to MAT. CEN = centromere XXb = filled in Xbal site XK = filled in (Asp718 cut) Kpnl site TEL = telomere.Other symbols asin BgX R XXb BpSasN XK R XB CEN2 Figure 1. (B) Chromosome 11 target withMATU trpl 3' /ys2 his3 and MATVWX inserted into the lys2 gene in MATU - + +- MANWX the same relative orientation as the chromosome III " target and the pGM54 plasmid. (C) Everted chro- mosome I1 target with the orientations of MATU trpl his3 and MATVWX present on pGM54EVT. SSaBgXXb R x R XKN B CEN2 Note that the chromosomal targets carry different 7EL2 c. alleles of the trpl and his3 genes than do the plas- crzz@Zq5'/YS21 trpr 4- - MATU MATXWV "I- mids. 4- 1- combination. The orientation of the homology on the fragment relative to the target sequences can deter- mine whether it is inserted as a substitution or an addition. Cleavage within the region of homology so thatthe ends are pointed inward(ends-in) when aligned with the target sequence allows efficient inte- gration of the plasmid as an addition to the genome (ORR-WEAVER,SZOSTAK and ROTHSTEIN 198 1). When alignment of the cut plasmid with the target results in the ends pointing away from each other B. (ends-out or omega insertion), insertion of the plasmid pGM54 allows substitution for target sequences (ROTHSTEIN 1983). The topology and resolution requirements of these two processes are different. To compare the EL2 -+ +- CEN2 efficiency of ends-in and ends-out recombination, we 7ys2 I frp 1 MATU MATX W V constructed a strain (GRY 1 148) in which we could +- -+ monitor both kinds of events with the same plasmid. t- - The plasmid used (pGM54;Figure 1) carries the URA3 FIGURE3.-Ends-in us. ends-out recombination. (A) Alignment gene and mutant alleles of the MAT::[trpZ his31 mod- of BamHlcutpCM54 with the chromosome III target in the ends- ule used in homologous recombination studies as de- in configuration. (B) Alignment of BamHltut pGM54 with the scribed previously (MCGILL et al. 1990; STRATHERN everted chromosomeII target in the ends-out configuration. et al. 199 1). For these studies, we created a yeast strain techniques described in METHODS. The results shown that has two copies of the [trpl his31 module, one at in Table 2 indicate that insertions at MATa (ends-in MATa and the otherinserted at lys2. The insertion at events) and at lys2 (ends-out events) are not equally lys2 is everted relative to the insertion at MATa (Fig- frequent (x2 values of 130 and 46). Ends-in events ure 2C). exceed ends-out eventsby a factor of about three. We transformed BamHI-cutpGM54 DNA into To determine how much ofthe observed difference strain GRY 1 148 and selected Ura+. Because the between insertion at MATa vs. lys2 was a result of the pGM54 plasmid does not have a yeast origin of repli- orientation of the ends as opposedto features intrinsic cation, Ura+ transformants result from integration of to MAT and lys2, we performed a similar transforma- the plasmid into regions of homology. The two ends tion into an isogenic strain (GRY 1 150) that had the of BamHI-cut pGM54 can pair with the [trpl his31 same configuration of the MAT::[trpl his31 module sequences at MATa in the ends-in configuration (Fig- (ends-in for pGM54) at both MATa and lys2 (see ure 3A). Alternatively, the two ends can pair with the Figure 2B). The results (Table 2) indicate that the everted [trpl his31 sequences at lys2 in the ends-out MATa and lys2 chromosomal locations were equally configuration (Figure 3B). The two targets were care- efficient targets (x2 valuesof 0.74 and 0.89) and fully constructed to have the same amount of homol- suggest that the threefold difference in the experi- ogy to the plasmid and to have no nonhomologous ment with pGM54 transformed into GRY 1148 does bases at the ends of the DNA (see METHODS). reflect the configuration of the ends. We determined whether the plasmid hadintegrated To confirm that the observed difference was the at the lys2 gene or at MATa by the classical genetic consequence of the orientation of the ends of the 976 P. J. Hastings et al.

TABLE 2 Plasmid integration sites

Insertion site

Not Spore Strain Plasmid MAT lys2 assigned” Translocation Minusb

CRY 1148 pGM54 354c 108d 9 3 46 CRY1148 pGM54 240 112 6 0 20 (73% “ends-in”) CRY1148 pCM54EVT 45 155 7 0 33 CRY 1 148 pGM54EVT 47 169 8 0 16 (78% “ends-in”) CRY 1 150 pCM54 62 73 0 0 5 CRY 1150 pGM54117 102 9 0 12 CRY 1150 pCM54EVT98 109 7 0 26 CRY 1 150 pGM54EVT109 92 6 0 32 CRY1 148/1174 pGM54 190 85 11 4 30‘ (69%“ends-in”) These include strains with the plasmid integrated at both places as well as cases in which the URA3 gene is not an allele of either MAT or lys2. ’These include “petite” strains and triploid strains (very poor spore viability) reflecting formation of diploids of the target strains during the transformation protocol. Bold face numbers are ends-in events. Italicized numbers are ends-out events. These include five transformants that are no longer ala diploids and mate like a cells. plasmid, we constructed a plasmid related to pGM54 was a chromosomal translocation, revealed as linkage that had the MAT::[trpl his31 sequences everted (des- of URA3 to both MATa and lys2, while MAT and lys2 ignated pGM54EVT; Figure 1). When cut with XhoI, showed linkage to each other. Further, these strains pGM54EVT has the opposite orientation of the ends showed the pattern of inviability of meiotic products relative to the targetsequences in GRY1 148. In other expected of a translocation heterozygote. Two expla- words, it should pair with the target at MATa in the nations are available for the origin of these translo- ends-out orientation and with the target at lys2 in the cations. It may be that one end of the plasmid inter- ends-in orientation. Again, the length of homology acted with one target, while the other end interacted with the two targets is the same; however, there are a with the other target. Figure 4 shows that there are few bases derived from a XhoI linker that are present two ways in which interaction of a plasmid with the at the lys2 target,but not at MAT. The results of differenttargets can lead to translocations. It also transformation of GRY 1148 with pGM54EVT cut shows thatformation of a reciprocal translocation with XhoI show a threefoldbias favoring the lys2 target requiresa secondary recombination event between over the MATa target (Table 2). Again, the ends-in the fragments produced by the primary reaction. Al- configuration was favored. ternatively, the plasmid may have integratedat a To determine that the bias favoring the lys2 target single target, and this target, being recombinationally for pGM54EVT transformed intoGRY 1 148 reflected active, may then have recombined with theother orientation (and notthe few bases at theXhoI site that target. Such trimolecular eventshave been described are not homologous to MAT), we transformedthe in yeast (BORTSand HABER1987; RAY,MACHIN and XhoI-cut pGM54EVT plasmid into GRY 1 150. In this STAHL 1989). HUGHESand ROTH (1985) have de- strain, both the target atMATa and the target atlys2 scribed a similar method for creating chromosomal were ends-out relative to XhoI cut pGM54EVT. In rearrangements of bacterial genomes. GRY 1 150 therewere no nonhomologous bases at the If the choice of target by the two ends were truly lys2 target at the ends of XhoI-cut pGM54EVT. As independent, it would be expected that half of the expected from the other experiments, the MATa and events would result in translocation. To determine lys2 targets were used with equal frequency (x2values whether the relative rarity of translocation was due to of 0.48 and 1.27). The equality of target use in this the improbability of the primary reaction (findingtwo strain suggests that the few nonhomologous bases at different targets) or to thedifficulty of the secondary the MAT target do not bias target use. recombination, we performed similar experiments in Plasmid-induced translocations:In several events, a diploid strain (GRYl148/1174) in which nonreci- one end of the plasmid was found to be integrated at procal translocations might be expected to survive. MAT, while the otherwas integrated atlys2. The result The data in Table 2 show that translocations were no Role of DNA Ends in Recombination 977

++ "-)"-) CEN3 -+ +- 7EL3 R CEN3+- -+ TEL3

eMATU + - pGM54 +

pGM54 his3x CEN2 EL2 X.. CEN2 2 "Ji +-MATU MATXWV -+ +- -+ 4- + + t-

CEN3 +

his3 137~s 1-1 MATU1-1 MATX W V -+ MATu x EL2 V

EL3 MATX W V +t- --+

FIGURE 4.-Translocations. (A) The CEN2-pGM54-TEL3 and CEN3-TEL2 translocations. (B) The CEN3-pCM54-TEL2 and CEN2-TEL3 translocations. The top part of the panels show the primary interaction of the plasmid with the two separate targets. The bottom part of the panels show the secondary recombination events between the fragments produced by the primary recombinations with the plasmid. more common in diploids than in haploids. Tetrad number expected if their formation at the two ends dissections demonstrated that the four translocations of the transforming DNA was independent was 34, obtained as a/a cells were reciprocal (data notshown). implying that in any integration event, the same thing There were five transformants that lost the ability to happens atboth ends. In transformations with the sporulate and mated like a cells. These could be the plasmid pGM54EVT, amuch lower frequency of pro- result of nonreciprocal translocations but were not totroph formation was observed (1 Trp+2 and25 His+ further analyzed. among 824) as expected from the orientation of the The fate of the trpl and his3 heteroalleles: The alleles relative to the ends (in this case the plus allele formation of tryptophan andhistidine prototrophs by is proximal to the end) and the greaterdistance from recombination between the trpl or his3 heteroalleles the end to thetrpl or his3 sequences. on the transforming DNA and the target sequences on the yeast chromosomes did not reveal any differ- DISCUSSION ences between ends-in and ends-out integration. The The integration of a plasmid into a chromosomal data obtained with pGM54 in GRY 1148 show 122 site inyeast can be stimulated by a DSB within a Trp+ and 84His+ transformants among 594 total at region of homology (ends-in) between the plasmid and MAT (ends-in) us. 31 Trp+ and 32His+ among 220 at thetarget (HICKS, HINNENand FINK 1978, ORR- lys2 (ends-out). This difference is not significant (2 X WEAVER,SZOSTAK and ROTHSTEIN1981). This plas- 2 contingency x* = 2.63). Controltransformations mid integration has many features in common with with pGM54 in GRY 1150(both targets ends-in) DSB repair. Several models differing somewhat in the showed no difference between frequency of proto- details of the repair mechanism have been proposed troph formation at the two targets: 38 Trp+ and 21 (RESNICK 1976; SZOSTAKet al. 1983; HASTINGS1988). His+ among 164 at MAT vs. 42 Trp+ and 29 His+ However, these models have in common the proposal among 190at lys2. The number of transformants that that the association of one side of the break with a were simultaneously Trp+ and His+ was 39 amongall homologous template initiates replication on the tem- the assignable pGM54transformants (1 168). The plate. The extension of the replication past the break 978 P. J. Hastings et al.

A. Splice Junction grated inyeast and are substratesfor homologous targeting in mammalian cells. A side-by-side compar- 5' ison of transformation with an ends-in and an ends- *..h PI k out plasmid into embryo-derived stem cells indicates 3' AI a ninefold difference in frequency favoring the ends- HJ SE in vector (HASTYet al. 1991). However, in that study B. Ends-out the DNA ends of the ends-out vector were not ho- mologous tothe target, and most of the ends-out integrations were not simple replacement events. On the other hand, an experiment (DENGand CAPECCHI 1992) found similar targeting frequencies for ends-in and ends-out vector. We developed an internally con- trolled experiment to compare thesetwo processes by creating a single yeast strain with an ends-in target A A and an ends-out target for the same plasmid. HJI HJ2 The key observations were: (1) there was a two- to C. Ends-in threefold excess of ends-in us. ends-out transformants; (2) this biaswas a function of the topology of the plasmid relative to the site, not inherent to the target itself; (3) a few of the integrated molecules that were .. . .A" recovered gave rise to chromosomal translocations; b and (4) heteroalleles between the plasmid and target A were treated similarly in ends-in and ends-out events HJ1 HJ2 and independently in any given event. FIGURE5.-Plasmid integration intermediates. (A)The splice Our results suggest that the targets at lys2 and at junction intermediate. Plasmid DNA strands are shown in solid lines. The target sequence is in open lines. The dashed 5' end of MAT are equally accessible to the cut plasmid. In the the plasmid DNA indicates possible exonuclease digestio& The control strain GRY 1150, in which the targets have dotted 3' end of the plasmid DNA indicates possible DNA synthesis. the same configuration, the number of integrations The intermediate can be resolved as a crossover by making the were the same at the two sites for both the ends-in three cleavages indicated by the solid arrows or the three cleavages events (BamHI-cut plasmid pGM54) and the ends-out indicated by the open arrows. HJ = Holliday junction, SE = strand exchange. (B) Ends-out intermediate. Integration of the plasmid events (XhoI-cut pGM54EVT). The most obvious requires crossover at both splice junctions (for examplethe six interpretation of the equal receptiveness of the two cleavages indicated by the solid arrows). (C) Ends-in intermediate. targets is that the initial interaction of a DNA end Integration requires one noncrossover and one crossover resolution with a homologous sequence is random with respect of the two Holliday junctions. to which target is contacted. On this basis, then, the site and into sequences homologous to the other end excess of ends-in over ends-out integrationmust result of the broken plasmid provides a mechanism to span from a differentialrecovery of ends-in integrants. The the lesion and may facilitate the invasion of the second question to be addressed here is whether the observed end of the plasmid. The selection for integration of higher recovery of ends-in integrants represents evi- the plasmid requires that both sides of the break be dence of cooperation between DNA ends. When the involved, similar to the requirement that both sides ends mimic a DSB, the cooperativity could, for ex- of a chromosomal break be involved in repair of a ample, result from repair synthesis initiated from one brokenchromosome. In addition, recovery of the invading end extending past the site homologous to integrated plasmid requires that the process result in the break and opening the region of the target ho- the equivalent of one crossover. The requirements mologous to the other end. Alternatively, the second for insertion of a cut plasmid in which the DNA pairs invasion of a target by the other DNA end could be, with a regionof homology with the endspointed away like the first, the result of a random collision. In this from each other (ends-out or omega integration) are view, ends-in and ends-out events would be initiated rather different (THALERand STAHL1988). There is with equalfrequency, but some component of the no inherent requirement for DNA replication. Fur- resolution of an ends-out event would yield a lower ther, replication initiated from an invading end would rate of recovery. proceed away from the sequences homologous to the The idea thatthe second end finds atarget at other end.Finally, ends-out integrationof the plasmid random, (that is, without reference to where the first requires the equivalent of two crossovers. end is integrated) would predict that half of the inte- The plasmids with ends-in and ends-out configura- gration events seen in the system used in these exper- tion relative to their target are both efficiently inte- iments would use one target for one end and the other Role of DNA Ends in Recombination 979 target for the other end,thus producing translocations be present that influence the natureof the resolution. as often as integration on a single chromosome. This If a Holliday junction is as likely to be resolved as a should be possible because the length of the plasmid crossover or as a noncrossover and if both the ends- as B-form DNA exceeds the diameter of the nucleus. in and ends-out processes have intermediates with two The recovery, at a significant frequency of transloca- Holliday junctions which will be resolved independ- tions, may demonstratethat the two ends canact ently, twice as many ends-in events as ends-out events independently.However, reciprocal translocations will be seen. Integrationof an ends-in plasmid requires represented much less than half of the transformants. that one Holliday junction be resolved as a crossover Translocations must be reciprocal to be viable in the and the other as a noncrossover (two of thefour transformed haploid cells. However,as discussed possibilities). Integration of an ends-out plasmid re- above, the formation of reciprocal translocations in quiresthat both Holliday junctionsbe resolved as this systeminvolves two sequential steps. Transfor- crossovers (one of the four possibilities). This addi- mations were done into a diploid strain (GRY 1 148/ tional constraint on the resolution of the ends-out 1174) to remove therequirement that reciprocal intermediate could produce the observed bias favor- translocations beformed. We recovered reciprocal ing ends-in products. translocations among the a/a cells as well as a few Since the relative frequency of ends-in and ends- candidates for nonreciprocal translocations (cells that out integration is subject to an interpretationin which lost the MATa allele). Combined, these translocation the two configurations differ only in the way in which eventsrepresented only 3% of the transformants, the event is resolved, it may be postulated that the much less than the 50% predicted by random inter- splice junction produced during ends-out integration action of the ends with the two targets. While these does not differ from that during an ends-in reaction. results suggest that the ends act nonrandomly to use This leads to the concept thatthe single splicejunction the same target, it cannot be determined whether they is the basic unit of recombination at double-strand act in concert as a result of cooperativity or as a result ends inyeast and that, therefore, recombination in of some physical constraint on the spatial distribution yeast is in many ways analogous to thatseen in recom- of the ends of the plasmid. bination mediated by the Escherichia coli recBCD sys- We observed a two- to threefold bias favoring the tem (reviewed by ROSENBERGand HASTINCS1991). ends-in target. This bias was clearly a function of the In summary, our observations supportthe postulate orientation of the ends,because when the plasmid was that in mitotic DSB repair, the repair consists of two everted, the bias relative to the genetictarget was independent invasions of homologous DNA sequences reversed. The simplest view of these experiments is by the two broken endsof the molecule on thefollow- that they support the proposal that the invasion of a ing assumptions: (1) each invasion produces a splice- chromosome by one end of a cut plasmid opens the junction that may be resolved as a crossover or as a DNA in a fashion that facilitates the invasion of the noncrossover with equal probability, and (2) there is second end forends-in events. While this cooperativity no cooperative interaction of nearby splice-junctions could also facilitate the repair of chromosomal DSBs, that favors the ends-in orientation. our data suggest that it has only a modest role. Research sponsored inpart by the National Cancer Institute, Alternatively, the excess of ends-in events may re- DHHS under contract no. N01-CO-74101 with ABL. The contents flect the requirements of resolution rather than the of this publication do not necessarily reflect the views or policies of frequency of formation of recombination intermedi- the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply en- ates. Models for the integration intermediates for the dorsement by the U.S. Government. ends-in and ends-out events are shown in Figure 5. The initial interaction of an end formsa splice junc- LITERATURECITED tion intermediate that includes a structure similar to a Holliday junction plus an additional point at which BOLIVAR, F.,R. L. RODRIGUEZ,P. J. GREEN,M. C. BETLACH,H. L. HEYNEKER,et al., 1977 Construction and characterization of there is a change in chain pairing partners (Figure new cloning vehicles 11. A multipurpose cloning system. Gene 5A). Both the ends-in and ends-out eventsmay initiate 2: 95-1 13. with two such splice junctions. For theends-out inter- BORTS,R. H., and J. E. HABER,1987 Meiotic recombination in mediate,resolution by strand cleavage requires six yeast: alteration by multiple heterozygosities. Science 237: 658-667. cuts (Figure 5B). In the case of the ends-in interme- CAMPBELL,A., 1984 Types of recombination: common problems diate, DNA synthesis can result in fusion of the two andcommon strategies. Cold Spring Harbor Symp. Quant. splice junctions to produce a simpler structure. For Biol. 49 839-844. both intermediates there are two Holliday junctions CHATTOO,B. B., F. SHERMAN, D. A.ATUBALIS, T. A. FIELLSTEDT, indicated. At the time the intermediates areresolved, D. MEHNERTand M. OGUR,1979 Selection of lys2 mutants of the yeast S. cerevisiae by the utilization of a-aminoadipate. complete Holliday junctions may not have been pres- Genetics 93: 51-65. ent in the sense that single strand nicks or gaps could DENG,C., and M. R. CAPECCHI,1992 Reexamination of gene 980 P. J. Hastings et al.

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