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, and tNCZ-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 the same cut plasmid. Recovery of 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 repair of chromosomal double-strand breaks.

T HE repair of double-strand breaks (DSBs) by homologous recombination requires both sides

of the break to be involved in the recombination process. Whether the two sides have similar roles in the recombination process has not been determined. The DSB-repair model as described by SZOSTAK et al. (1 983) incorporates a symmetric intermediate in which ends from both sides of the DSB have invaded the unbroken chromatid. It is possible that the rules for invasion of the two sides are not the same (CAMP- BELL 1984; ROTHSTEIN 1984; THALER and STAHL 1988). For example, it is possible that the invasion by the first side alters the uncut chromosome so that it is a better target for the strand(s) from the other side of the break. It has been suggested that the invasion of a region of homology by DNA from one side of the break sets up a replication fork that may open the DNA that is homologous to the other side of the break (SZOSTAK et al. 1983). In other words, invasion of one end facilitates invasion of the other end. This coop- erativity would minimize the formation of chromo- somal rearrangements that would result if the two DNAs from opposite sides of a DSB recombine with different targets.

To determine whether there is cooperativity be- tween the two sides of a DSB, we compared two different topologies of the DNA ends. We determined the efficiency of integration of a cut plasmid at a target where the ends point toward each other when paired with a region of homology (“ends-in”) as com- pared with the integration of that same cut plasmid a t a target where the ends point away from each other

Genetics 135: 973-980 (December, 1993)

when paired with the homologous target (“ends-out”). Because direct cooperation of ends seems difficult with the ends-out configuration, ends-out events might be expected to occur less efficiently than ends- in events. We report here that the two topologies yield similar frequencies of integration with a two- to three- fold bias favoring the ends-in configuration.

MATERIALS AND METHODS

Plasmids: We filled in the EcoRI site of pBR322 (BOLIVAR et al. 1977) and inserted a XhoI linker to create pBR322X. The URA? gene was inserted as a XhoI fragment into the XhoI-Sal1 sites of pBR322X to create pBRpU3A. A 2-kb fragment of chromosome ZZZ extending from the Hind111 site centromere proximal of MAT to within 50-bp of the MAT Y region was inserted into pBRpU3A as a XhoI frag- ment. The trpl gene (a 857-bp EcoRI to BglII fragment) and the his3 gene (a 1075-bp fragment extending from a BamHI site inserted 210 bases upstream of the ATG to an EcoRI site inserted 201 bases downstream of the HIS3 termination codon) were inserted at an EcoRI site in the MAT fragment. The resultant plasmid, pCM54, carries the trpl-488 allele (an in-frame UAG codon) and the his3-192 allele (a frame-shift caused by filling in an NdeI site) (MCGILL et al. 1990). A Sal1 plus XhoI partial digest of pGM54 was self-ligated to create pCMTRPl and pGMHIS3. SmaI XhoI fragments from these two plasmids were combined to create the inside out version of pCM54 designated pCM54EVT (for everted). These plasmids are diagramed in Figure 1.

Yeast strains: The yeast strains (Table 1) used as recipi- ents for transformation with pGM54 and pGM54EVT have the genotype MATa::[trpl-U89 his3-6211 lys2::[trpl-089 his% 6211 leu2-A1 tyr7-1 trpl-A1 his3-A200 ura3-52 and are re- lated to the previously described strain GRY558 (MCGILL et al. 1990) by the insertion of the trpl-his3 module at lys2. They have the trpl-089 and his5621 alleles inserted next

974 P. J. Hastings et al.

x XIS

pGM54 MATU + - - + MATVWX +-+ +

pGMTRPl

XISB R x XIS N K

pGMHIS3 A ~ D Id his3

+ - + M A T V W X +

- + M A T V W X M A T U + - + ++ FIGURE 1 .-Plasmids utilized in this study. pGM54 includes the

trpl and his3 genes inserted into an EcoRI site in a DNA fragment from the centromere proximal side of MAT. Note the unique BamHI site used to linearize pGM54. MATU is the 527 base chro- mosome 111 region centromere proximal to that EcoRl site. MATV is the 428 base region between the EcoRI site and the start of M A W . pGMHIS3 and pGMTRPl are SalI-XhoI deletion deriva- tives of pGM54 used in the construction of the everted version of this interval designated pGM54EVT. Note the unique Xhol site used to linearize pGM54EVT. B = EarnHI, Bg = EgllI, K = KpnI, R = EcoRI, Sa = SalI , Sp = SpeI , Xb = XbaI, X N = filled in NdeI X = Xhol, XIS = XhoI/SalI ligation.

to the MAT locus in the same orientation as the trpl and his3 genes in the plasmid pCM54 (Figure 2A). GRYl 150 has the trpl-089 and his3-621 alleles plus MAT DNA, de- rived from a plasmid (pCM6 13) similar to pCM54, inserted into the lys2 gene on chromosome I I . The 4-kb MAT::trpl- 089 his3-621 insertion was made as a substitution for 2.4-kb of lys2 DNA (FLEIG, PRIDMORE and PHILIPPSEN 1986) from a BglII site (at which a Xhol linker was inserted) to a XhoI site (Figure 2B). CRY 1 148 differs from GRYl 150 in that the MAT::trpl-089 his3-621 insertion at lys2 in GRYl 148 has the inverted structure similar to pCM54EVT (Figure 2C). The 4-kb BamHI insertion into lys2 in CRY 1 148 was made as a substitution for 2.8-kb of lys2 from a BgllI site to a BamHI site. The insertions into the lys2 DNA were made in a plasmid derivative of pDP6 (FLEIG, PRIDMORE and PHILIPPSEN 1986). The insertions at LYS2 were introduced into yeast by identifying a-amino adipate (aAA) resistant (Lys-) transformants of the LYS2 parent strain (CHATTOO et al. 1979). The structures diagramed in Figure 2 were con- firmed by restriction digestion and blotting analysis (data not shown). CRY 1 148 carries the chromosome III shown in Figure 2A and the chromosome II shown in Figure 2C, providing homologous targets with different topologies for the cut plasmids. GRYl 150 has chromosome ZZZ shown in Figure 2A and the chromosome IZ shown in Figure 2B, providing a control with both targets in the same relative configuration for the cut plasmids.

Iden t i f y ing the site of plasmid insertion: The site of insertion of the URA3-based plasmids was established by determining the linkage of the URA3 gene to the MAT and lys2 genes. Each transformant was mated to a LYS2 ura3 MATa strain (GRYl 163 or GRYl174). Tetrad analysis of

TABLE 1

Yeast s trains

Strain Genotype

GRY558 MATa::[frpl-O89 his3-6211 LYS2 leu2-AI 477-1 t rp l - A1 his3-A200 ura3-52

GRYl148 MATa::[frpl-089 his3-6211 lys2::[his3-621 trpl-O89/ 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 his3-A200 ura3-52

GRY 1 152 MATa lys2-801 ura3-52 GRY 1 153 MATa lys2-801 ura3-52 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 at lys2. The MAT and lys2 targets were chosen because they allow direct analysis of spore patches without tetrad dissection. This replica-plating protocol allowed the analysis of many more transformants than could have been handled by tetrad genetics. The protocol involved making patches of the Ura+ transformants onto SC-ura [synthetic complete medium (SHERMAN, FINK and HICKS 1986) minus uracil]. The patches were mated to a lawn of CRY 1 174 (or CRY 1 163) selecting on SD + his + trp + leu (minimal medium plus histidine, tryptophane and leucine). This medium selected diploids carrying the URA3 and ADEP alleles from the transformed strain and the 7YR7 and LYS2 alleles from CRY 1 174. After 2 days, the mating plate was replica-plated to SD + his + trp + leu to enrich for the diploids. After one additional day of growth, the patches were replica-plated to two SPOR plates (1.5% potassium acetate, 0.25% yeast extract, 0.1 % glucose, 2% agar plus nutritional supplements at one-fourth the concentration used in synthetic complete medium) and incubated for three days to induce meiosis and sporulation. The sporulated patches were mated to MATa ura3 lys2 and MATa ura3 lys2 strains (GRYl 152 and CRY 1 153, respectively) by replica-plating the patches onto lawns of the testers spread on YEPD. After 1 day, the spore- mating plates were replica-plated to selective plates. TO determine whether the URA3 plasmid was inserted at MATa, we compared the ability of the spore patches to mate and complement the Ura requirement of the a and a tester strains (CRY 1 152 and CRY 11 53). For insertions at MATa, there were far more a Ura+ spores than a Ura+ spores. In contrast, URA3 insertions at lys2 gave spores that were able to mate and complement the Ura defect of both mating type testers equivalently. T o ascertain whether the URA3 plasmid was inserted at lys2, we determined whether the diploids could produce spores that were simultaneously URA3 and LYSP by mating the spore patches to CRY 1 152 and CRY 1 153 and selecting Lys+ and Ura+. For insertions at lys2, URA3 and LYS2 are alleles and hence could not segre- gate into the same spore. In contrast, for URA3 insertions at MATa, about one-fourth of the spores were a Ura+ Lys+. We dissected over 80 randomly picked diploids to confirm that the results of the patch tests are an accurate reflection of the insertion site.

RESULTS

Linear DNA fragments introduced into yeast are readily inserted into the genome by homologous re-

Role of DNA Ends in Recombination 975

R XXb BpSaeN XK R

A. CEN3 E L 3 FIGURE 2.-Chromosome targets utilized in this study. (A) Chromosome III target; the trpl and his3 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 as in

C E N 2 Figure 1. (B) Chromosome 11 target with MATU trpl his3 and MATVWX inserted into the lys2 gene in

MATU - + +- M A N W X "--)

B g X R XXb BpSasN XK R X B

3' /ys2 MATU - + +- M A N W X 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.

C E N 2 Note that the chromosomal targets carry different alleles of the trpl and his3 genes than do the plas-

SSaBgXXb R x R X K N B

c. 7EL2 c r z z @ Z q 5 ' / Y S 2 1 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 that the 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 (ends-out or omega insertion), insertion of the plasmid allows substitution for target sequences (ROTHSTEIN 1983). The topology and resolution requirements of these two processes are different. To compare the efficiency of ends-in and ends-out recombination, we constructed a strain (GRY 1 148) in which we could monitor both kinds of events with the same plasmid. The plasmid used (pGM54; Figure 1) carries the URA3 gene and mutant alleles of the MAT::[trpZ his31 mod- ule used in homologous recombination studies as de- scribed previously (MCGILL et al. 1990; STRATHERN et al. 199 1). For these studies, we created a yeast strain that has two copies of the [trpl his31 module, one at MATa and the other inserted at lys2. The insertion at lys2 is everted relative to the insertion at M A T a (Fig- ure 2C).

We transformed BamHI-cut pGM54 DNA into strain GRY 1 148 and selected Ura+. Because the pGM54 plasmid does not have a yeast origin of repli- cation, Ura+ transformants result from integration of the plasmid into regions of homology. The two ends of BamHI-cut pGM54 can pair with the [trpl his31 sequences at M A T a in the ends-in configuration (Fig- ure 3A). Alternatively, the two ends can pair with the everted [trpl his31 sequences at lys2 in the ends-out configuration (Figure 3B). The two targets were care- fully constructed to have the same amount of homol- ogy to the plasmid and to have no nonhomologous bases at the ends of the DNA (see METHODS).

We determined whether the plasmid had integrated at the lys2 gene or at M A T a by the classical genetic

B. pGM54

E L 2 -+ + - C E N 2

7ys2 I f r p 1 MATU MATX W V +-

t- -+ -

FIGURE 3.-Ends-in us. ends-out recombination. (A) Alignment of BamHlcut pCM54 with the chromosome III target in the ends- in configuration. (B) Alignment of BamHltut pGM54 with the everted chromosome I I target in the ends-out configuration.

techniques described in METHODS. The results shown in Table 2 indicate that insertions at M A T a (ends-in events) and at lys2 (ends-out events) are not equally frequent (x2 values of 130 and 46). Ends-in events exceed ends-out events by a factor of about three.

T o determine how much of the observed difference between insertion at M A T a vs. lys2 was a result of the orientation of the ends as opposed to features intrinsic to MAT and lys2, we performed a similar transforma- tion into an isogenic strain (GRY 1 150) that had the same configuration of the MAT::[trpl his31 module (ends-in for pGM54) at both M A T a and lys2 (see Figure 2B). The results (Table 2) indicate that the M A T a and lys2 chromosomal locations were equally efficient targets (x2 values of 0.74 and 0.89) and suggest that the threefold difference in the experi- ment with pGM54 transformed into GRY 1148 does reflect the configuration of the ends.

To confirm that the observed difference was the 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 1 08d 9 3 46 CRY1148 pGM54 240 112 6 0 20

CRY1148 pCM54EVT 45 155 7 0 33 CRY 1 148 pGM54EVT 47 169 8 0 16

CRY 1 150 pCM54 62 73 0 0 5 CRY 1150 pGM54 102 117 9 0 12 CRY 1150 pCM54EVT 109 98 7 0 26 CRY 1 150 pGM54EVT 92 109 6 0 32 CRY1 148/1174 pGM54 190 85 1 1 4 30‘

(73% “ends-in”)

(78% “ends-in”)

(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

’ These include “petite” strains and triploid strains (very poor spore viability) reflecting formation of diploids of the target strains during or lys2.

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 that had the MAT::[trpl his31 sequences everted (des- ignated pGM54EVT; Figure 1). When cut with XhoI, pGM54EVT has the opposite orientation of the ends relative to the target sequences in GRY 1 148. In other words, it should pair with the target at MATa in the ends-out orientation and with the target at lys2 in the ends-in orientation. Again, the length of homology with the two targets is the same; however, there are a few bases derived from a XhoI linker that are present at the lys2 target, but not at MAT. The results of transformation of GRY 1148 with pGM54EVT cut with XhoI show a threefold bias favoring the lys2 target over the MATa target (Table 2). Again, the ends-in configuration was favored.

T o determine that the bias favoring the lys2 target for pGM54EVT transformed into GRY 1 148 reflected orientation (and not the few bases at the XhoI site that are not homologous to MAT) , we transformed the XhoI-cut pGM54EVT plasmid into GRY 1 150. In this strain, both the target at MATa and the target at lys2 were ends-out relative to XhoI cut pGM54EVT. In GRY 1 150 there were no nonhomologous bases at the lys2 target at the ends of XhoI-cut pGM54EVT. As expected from the other experiments, the MATa and lys2 targets were used with equal frequency (x2 values of 0.48 and 1.27). The equality of target use in this strain suggests that the few nonhomologous bases at the MAT target do not bias target use.

Plasmid-induced translocations: In several events, one end of the plasmid was found to be integrated at MAT, while the other was integrated at lys2. The result

was a chromosomal translocation, revealed as linkage of URA3 to both MATa and lys2, while MAT and lys2 showed linkage to each other. Further, these strains showed the pattern of inviability of meiotic products expected of a translocation heterozygote. Two expla- nations are available for the origin of these translo- cations. It may be that one end of the plasmid inter- acted with one target, while the other end interacted with the other target. Figure 4 shows that there are two ways in which interaction of a plasmid with the different targets can lead to translocations. It also shows that formation of a reciprocal translocation requires a secondary recombination event between the fragments produced by the primary reaction. Al- ternatively, the plasmid may have integrated at a single target, and this target, being recombinationally active, may then have recombined with the other target. Such trimolecular events have been described in yeast (BORTS and HABER 1987; RAY, MACHIN and STAHL 1989). HUGHES and ROTH (1985) have de- scribed a similar method for creating chromosomal rearrangements of bacterial genomes.

If the choice of target by the two ends were truly independent, it would be expected that half of the events would result in translocation. T o determine whether the relative rarity of translocation was due to the improbability of the primary reaction (finding two different targets) or to the difficulty of the secondary recombination, we performed similar experiments in a diploid strain (GRYl148/1174) in which nonreci- procal translocations might be expected to survive. The data in Table 2 show that translocations were no

Role of DNA Ends in Recombination 977

++ CEN3 -+ +- 7EL3

e MATU + -

pGM54 2 his3 x CEN2

+- + - + +

CEN3 +

- + MATu x E L 2

t- + -+ -

"-)"-)

R CEN3 -+ +- TEL3

pGM54 +

E L 2 X CEN2 . .

"Ji MATU MATXWV +- -+

4- t-

1-1 his3 137~s MATU MATX W V

V

E L 3 MATX W V

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 dissections demonstrated that the four translocations obtained as a/a cells were reciprocal (data not shown). There were five transformants that lost the ability to sporulate and mated like a cells. These could be the result of nonreciprocal translocations but were not further analyzed.

The fate of the trpl and his3 heteroalleles: The formation of tryptophan and histidine prototrophs by recombination between the trpl or his3 heteroalleles on the transforming DNA and the target sequences on the yeast chromosomes did not reveal any differ- ences between ends-in and ends-out integration. The data obtained with pGM54 in GRY 1148 show 122 Trp+ and 84 His+ transformants among 594 total at MAT (ends-in) us. 31 Trp+ and 32 His+ among 220 at lys2 (ends-out). This difference is not significant (2 X 2 contingency x* = 2.63). Control transformations with pGM54 in GRY 1150 (both targets ends-in) showed no difference between frequency of proto- troph formation at the two targets: 38 Trp+ and 21 His+ among 164 at MAT vs. 42 Trp+ and 29 His+ among 190 at lys2. The number of transformants that were simultaneously Trp+ and His+ was 39 among all the assignable pGM54 transformants (1 168). The

number expected if their formation at the two ends of the transforming DNA was independent was 34, implying that in any integration event, the same thing happens at both ends. In transformations with the plasmid pGM54EVT, a much lower frequency of pro- totroph formation was observed (1 2 Trp+ and 25 His+ among 824) as expected from the orientation of the alleles relative to the ends (in this case the plus allele is proximal to the end) and the greater distance from the end to the trpl or his3 sequences.

DISCUSSION

The integration of a plasmid into a chromosomal site in yeast can be stimulated by a DSB within a region of homology (ends-in) between the plasmid and the target (HICKS, HINNEN and FINK 1978, ORR- WEAVER, SZOSTAK and ROTHSTEIN 1981). This plas- mid integration has many features in common with DSB repair. Several models differing somewhat in the details of the repair mechanism have been proposed (RESNICK 1976; SZOSTAK et al. 1983; HASTINGS 1988). However, these models have in common the proposal that the association of one side of the break with a homologous template initiates replication on the tem- plate. The extension of the replication past the break

978 P. J. Hastings et al.

A. Splice Junction

5' * ..h PI

k 3' A I

HJ SE

B. Ends-out

A A HJI HJ2

C. Ends-in

. . . .A" b A

HJ1 HJ2

FIGURE 5.-Plasmid integration intermediates. (A) The splice junction intermediate. Plasmid DNA strands are shown in solid lines. The target sequence is in open lines. The dashed 5' end of the plasmid DNA indicates possible exonuclease digestio& The dotted 3' end of the plasmid DNA indicates possible DNA synthesis. The intermediate can be resolved as a crossover by making the three cleavages indicated by the solid arrows or the three cleavages indicated by the open arrows. HJ = Holliday junction, SE = strand exchange. (B) Ends-out intermediate. Integration of the plasmid requires crossover at both splice junctions (for example the six cleavages indicated by the solid arrows). (C) Ends-in intermediate. Integration requires one noncrossover and one crossover resolution of the two Holliday junctions.

site and into sequences homologous to the other end of the broken plasmid provides a mechanism to span the lesion and may facilitate the invasion of the second end of the plasmid. The selection for integration of the plasmid requires that both sides of the break be involved, similar to the requirement that both sides of a chromosomal break be involved in repair of a broken chromosome. In addition, recovery of the integrated plasmid requires that the process result in the equivalent of one crossover. The requirements for insertion of a cut plasmid in which the DNA pairs with a region of homology with the ends pointed away from each other (ends-out or omega integration) are rather different (THALER and STAHL 1988). There is no inherent requirement for DNA replication. Fur- ther, replication initiated from an invading end would proceed away from the sequences homologous to the other end. Finally, ends-out integration of the plasmid requires the equivalent of two crossovers.

The plasmids with ends-in and ends-out configura- tion relative to their target are both efficiently inte-

grated in yeast and are substrates for homologous targeting in mammalian cells. A side-by-side compar- ison of transformation with an ends-in and an ends- out plasmid into embryo-derived stem cells indicates a ninefold difference in frequency favoring the ends- in vector (HASTY et al. 1991). However, in that study the DNA ends of the ends-out vector were not ho- mologous to the target, and most of the ends-out integrations were not simple replacement events. On the other hand, an experiment (DENG and CAPECCHI 1992) found similar targeting frequencies for ends-in and ends-out vector. We developed an internally con- trolled experiment to compare these two processes by creating a single yeast strain with an ends-in target and an ends-out target for the same plasmid.

The key observations were: (1) there was a two- to threefold excess of ends-in us. ends-out transformants; (2) this bias was 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 recovered gave rise to chromosomal translocations; and (4) heteroalleles between the plasmid and target were treated similarly in ends-in and ends-out events and independently in any given event.

Our results suggest that the targets at lys2 and at MAT are equally accessible to the cut plasmid. In the control strain GRY 1150, in which the targets have the same configuration, the number of integrations were the same at the two sites for both the ends-in events (BamHI-cut plasmid pGM54) and the ends-out events (XhoI-cut pGM54EVT). The most obvious interpretation of the equal receptiveness of the two targets is that the initial interaction of a DNA end with a homologous sequence is random with respect to which target is contacted. On this basis, then, the excess of ends-in over ends-out integration must result from a differential recovery of ends-in integrants. The question to be addressed here is whether the observed higher recovery of ends-in integrants represents evi- dence of cooperation between DNA ends. When the ends mimic a DSB, the cooperativity could, for ex- ample, result from repair synthesis initiated from one invading end extending past the site homologous to the break and opening the region of the target ho- mologous to the other end. Alternatively, the second invasion of a target by the other DNA end could be, like the first, the result of a random collision. In this view, ends-in and ends-out events would be initiated with equal frequency, but some component of the resolution of an ends-out event would yield a lower rate of recovery.

The idea that the second end finds a target at random, (that is, without reference to where the first end is integrated) would predict that half of the inte- gration events seen in the system used in these exper- 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 as often as integration on a single chromosome. This should be possible because the length of the plasmid as B-form DNA exceeds the diameter of the nucleus. The recovery, at a significant frequency of transloca- tions, may demonstrate that the two ends can act independently. However, reciprocal translocations represented much less than half of the transformants. Translocations must be reciprocal to be viable in the transformed haploid cells. However, as discussed above, the formation of reciprocal translocations in this system involves two sequential steps. Transfor- mations were done into a diploid strain (GRY 1 148/ 1174) to remove the requirement that reciprocal translocations be formed. We recovered reciprocal translocations among the a/a cells as well as a few candidates for nonreciprocal translocations (cells that lost the MATa allele). Combined, these translocation events represented only 3% of the transformants, much less than the 50% predicted by random inter- action of the ends with the two targets. While these results suggest that the ends act nonrandomly to use the same target, it cannot be determined whether they act in concert as a result of cooperativity or as a result of some physical constraint on the spatial distribution of the ends of the plasmid.

We observed a two- to threefold bias favoring the ends-in target. This bias was clearly a function of the orientation of the ends, because when the plasmid was everted, the bias relative to the genetic target was reversed. The simplest view of these experiments is that they support the proposal that the invasion of a chromosome by one end of a cut plasmid opens the DNA in a fashion that facilitates the invasion of the second end for ends-in events. While this cooperativity could also facilitate the repair of chromosomal DSBs, our data suggest that it has only a modest role.

Alternatively, the excess of ends-in events may re- flect the requirements of resolution rather than the frequency of formation of recombination intermedi- ates. Models for the integration intermediates for the ends-in and ends-out events are shown in Figure 5 . The initial interaction of an end forms a splice junc- tion intermediate that includes a structure similar to a Holliday junction plus an additional point at which there is a change in chain pairing partners (Figure 5A). Both the ends-in and ends-out events may initiate with two such splice junctions. For the ends-out inter- mediate, resolution by strand cleavage requires six cuts (Figure 5B). In the case of the ends-in interme- diate, DNA synthesis can result in fusion of the two splice junctions to produce a simpler structure. For both intermediates there are two Holliday junctions indicated. At the time the intermediates are resolved, complete Holliday junctions may not have been pres- ent in the sense that single strand nicks or gaps could

be present that influence the nature of the resolution. If a Holliday junction is as likely to be resolved as a crossover or as a noncrossover and if both the ends- in and ends-out processes have intermediates with two Holliday junctions which will be resolved independ- ently, twice as many ends-in events as ends-out events will be seen. Integration of an ends-in plasmid requires that one Holliday junction be resolved as a crossover and the other as a noncrossover (two of the four possibilities). Integration of an ends-out plasmid re- quires that both Holliday junctions be resolved as crossovers (one of the four possibilities). This addi- tional constraint on the resolution of the ends-out intermediate could produce the observed bias favor- ing ends-in products.

Since the relative frequency of ends-in and ends- out integration is subject to an interpretation in which the two configurations differ only in the way in which the event is resolved, it may be postulated that the splice junction produced during ends-out integration does not differ from that during an ends-in reaction. This leads to the concept that the single splice junction is the basic unit of recombination at double-strand ends in yeast and that, therefore, recombination in yeast is in many ways analogous to that seen in recom- bination mediated by the Escherichia coli recBCD sys- tem (reviewed by ROSENBERG and HASTINCS 1991).

In summary, our observations support the postulate that in mitotic DSB repair, the repair consists of two independent invasions of homologous DNA sequences by the two broken ends of the molecule on the follow- ing assumptions: (1) each invasion produces a splice- junction that may be resolved as a crossover or as a noncrossover with equal probability, and (2) there is no cooperative interaction of nearby splice-junctions that favors the ends-in orientation.

Research sponsored in part by the National Cancer Institute, DHHS under contract no. N01-CO-74101 with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply en- dorsement by the U.S. Government.

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Communicating editor: P. J. PUKKILA