Copyright 0 1922 by the Genetics Society of America
Plasmid Recombinationin a rad52 Mutant of Saccharomyces cerevisiae Kenneth J. Dornfeld and Dennis M. Livingston Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455 Manuscript received November 18, 1991 Accepted for publication February13, 1992
ABSTRACT Using plasmids capable ofundergoing intramolecular recombination,we have compared the rates and the molecular outcomesof recombination eventsin a wild-type anda rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-foldless frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wildtype cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeatplasmids. In addition, crossovers constitutea larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parentalhis3 heteroalleles.
T
HE R A D 5 2 gene of Saccharomyces cerevisiae plays
a crucial yet ill defined role in the molecular pathways of DNAdoublestrandbreakrepairand heteroallelicrecombination. RESNICK (1969)and STRIKE (1978) first identified mutants based on their sensitivity to ionizing radiation. Further work showed mutants to be deficient in the repair of double and single strand breaks in chromosomal DNA (RESNICK and MARTIN 1976; RESNICKet al. 1981). T h e inability of mutants to repair double strand DNA breaksis also evidenced by their inability to switch mating type, a process of gene conversion initiated by a site-specific doublestrandbreak (MALONEand ESPOSITO1980; WEIFFENBACH and HABER198 1; RAVEHet al. 1989; WHITEand HABER1990). Severalstudieshaveexamined how heteroalleles recombine in wild-type and mutantcells. JACKSON and FINK(198 1) used a chromosomal construction ofhis4 heteroalleles in a direct repeat separated by a bacterial plasmid sequence. They found that in wild-type cells most HIS4 prototrophs arose by gene conversion and few by crossing over. In contrast, the rad52 mutant produced virtually no prototrophsby gene conversion but did so by crossing over. T h e crossover rate was the same for wild type and mutantleading the authors to conclude that the mutation abolishes conversion but not crossing over. KLEIN (1988) also studied heteroallelic recombination of a chromosomal direct repeat in wild-type and mutantcells. She also found that prototrophs predominantly arose by gene conversion in wild type and by crossing over in mutants, and, in Genetics 131: 261-276 (June, 1992)
addition,described that many of the crossovers in both thewild type and the mutant included conversion of flanking markers. BecauseJACKSON and FINK(198 1) andKLEIN (1988) used direct repeat constructions, they did not distinguish crossover events which were fully reciprocal from those which were nonreciprocal. For a direct repeat in the chromosomea reciprocal crossover produces a linearDNA and a circular plasmid (Figure 1). Only one of these two products emerges from a nonreciprocal event. To maintain cell viability, the direct repeat studies demanded the emergenceof the linear chromosome product. T h e presence of the reciprocal plasmid product was not established in these studies because the plasmids would have been incapable of replication. T o examine the issue of reciprocity, HABER and HEARN(1985) examined his4 heteroallelic recombination between homologous chromosomesin wild-type and mutant cells. As expected, they found thatgeneconversion was greatlydecreased in the mutant. T h e crossovers which occurred in the mutant were notstraightforwardreciprocaleventsfor two reasons. First, the markers flanking the HIS4 allele often were not those expected for a crossover in the region between the heteroallelic markers. This result implied that conversion occurred to produce thewildtype copy. Second, many prototrophiccells were monosomic for the chromosome. This observation suggested that mutants were incapable of completing a reciprocal event in which two homologs emerge from the recombination event (Figure 1). An interpretation
K. J. Dornfeld and D. M. Livingston
262 inverted repeat direct repeat
-
interchromosomal parental
”
c
r
reciprwal crossover
nonreciprocal crossover
--
--
- +
-
r
-+ -
c
” -
0
J
I
FIGURE1 .-Reciprocal and nonr&iprocal crossovers. Three recombination substrates are pictured as well as their reciprocal and nonreciprocal crossover products. The difference between a recip rocal and a nonreciprocal crossover is the loss of one of the two products in the nonreciprocal crossover (represented by a broken line). For each example of nonreciprocal crossing over the chance that either product is lost is presumed to be equal. Only one of two possible outcomes is shown. In the case of inverted repeat recombination a reciprocal crossover reorients the segment (represented by the arrow within the interstitial box) between the repeated sequences. In this case a nonreciprocal crossover results in the loss of the recombining DNA because the event leaves this DNA fragmented. The crossing over shown for direct and inverted repeat recombination is applicable to plasmid recombination.
of this study was that many of the crossovers observed by JACKSON and FINK(1 98 1) and by KLEIN(1 988) had not resulted from the reciprocal crossover pathway which accompanies RAD52-dependent conversions but instead had occurred by a separate RAD52-independent, nonreciprocal pathway. A plausible separate pathway was given by OZENBERGER and ROEDER (1991) in their examination of doublestrandbreakrepair in tandemly repeated genes. While HO endonucleasecutting at MAT is lethal in rad52 mutant cells (MALONEand ESPOSITO 1980; WEIFFENBACH and HABER 1981), they found such cutting at multiply repeated genes is repairable in themutant cells. Furthermore, theyfoundthat several copies of the repeated genes were often lost during the repair process in mutant cells, while such loss was less frequent and less extensive in wild-type cells. They suggested a model to account for these results in which a double strand break is resected to produce single strand tails (Figure 2). Because the sequence is repeated, thetails are capable of annealing like staggeredrestrictionendonuclease cuts. When annealingoccursthe number of repeating units is reduced. The resultingproduct is the linear DNA diagrammed in Figure1fora crossover between directly repeated sequences. The event is nonreciprocal because one or more copies are lost during the event. The single strandannealingreaction which they proposed to occur in yeast has been extensively studied in mammalian recombination(LIN, SPERLE MARYON and CARROLL and STERNBERC 1984; 1991a,b). In our studies of heteroallelic recombination we have developed recombination reporter plasmids in which his3 heteroalleles have beenorientedas in-
. + L
r
r
I
I
..
+ -
--
-
a. direct repeat
+ -
- +
t
- +
+ -
b. double strand gap
+ +
A,,
” + +
c. single strand annealing
+ +
+ +
d. trimming and ligation
FIGURE2.-Single strand annealing between heteroallelic direct repeats. (a)Both strands of the DNA molecule are shown. The directly repeated sequences are represented as bold arrows. Sites of mutation are shown as minus signs. (b) A double strand gap eliminates the sequence between the repeats. (c) Single strand annealing couples the repeats leaving single strand extensions. (d) Exonucleolytic trimming of the single strand extensions followed by ligation leaves an intact duplex with the configuration of a crossover. If the order of mutations had been reversed, single strand annealing could result in a wild-type repeat copy but annealing would have to cover both mutated sites and mismatch correction would need to favor the wild-type sequence.
verted repetitions (EMBRETSON and LIVINGSTON 1984;AHNand LIVINGSTON1986). We have used these plasmids to examine conversion tract continuity, conversion tractlength and associated crossovers which occur during mitotic recombination. A major objective of this study was to learn more about the role of RAD52 by asking whether any of these parameters are alteredby a rad52 mutation. In addition, we have constructed plasmids where the same his3 heteroalleles are present as direct repeats. Although we were interested in determining whether sequence orientation of the recombining alleles affected the rate and the manner in which plasmids undergo recombination, we also constructed the direct repeatplasmids to examine the issue of reciprocity of crossovers in a rad52 mutant. Crossing over need not be fully reciprocal in directrepeat plasmids, whereas crossovers must be reciprocal in inverted repeat plasmids. This is because crossing over in a direct repeat plasmid leads to looping out of two smaller plasmids, one of which may be intact and capable of replication while
rad52 263Plasmid Recombination direct repeat inpBYA819D and pBYA918D and the inverted repeat in pKD8TB9I and pKD9TB81, MluI linkers (New England BioLabs) were placed at its ends after the EcoRI XbaI overhangs were first filled-in by polymerization. Because of the linker sequence, the EcoRI and Sal1 sites are destroyed in the process. The EcoRI and SalI sites of the pBR3.22 hid Ikb' vector sequencesinto which the 6.1-kb fragment was ligated were also modified to contain MluI linkers.In this case the FIGURE3.-Markerplacementin HIS3 andsurroundingsetwo vector sites were first fused with EcoRIand SalI adapters quences. A 6.1-kb EcoRI-Sal1 fragment anda smaller 4.4-kb EcoRI(New England BioLabs) which create a SmaI site, and MluI Xbal subfragment of chromosome XV containing HIS3 are shown linkers wereadded to blunt ended SmaI cut DNA. Because (STRUHL and DAVIS1980). Restriction sites have been placed within of the adapter sequences, both the EcoRI and SalI sites are the gene and flanking DNA using linkers and an adapter (New recreated. England BioLabs) to create an 8-bp and a 10-bp insertion, respecGenerating and identifying recombination products: tively. The open triangles are ClaI sites, the closed triangles are Yeastcells were transformed either by the spheroplast Sac1 sites and the diamond is a SmaI site created by an adapter method (BEGGS 1978) or an electroporation method placed in an XhoI site. These have been placed at sites within and (BECKER and GUARENTE 1991). Plasmid recombination rates flanking the open reading frame whichis represented by an arrow. weremeasured by fluctuation analyses(LURIA and DELA plus and a minus are usedto indicate the relative positionof the BRUCK 1943; LEAand COULSON 1949; AHNand LIVINGSTON mutations within the HIS3 coding region. The construction of these 1986). marked segments have been previously described (AHN and LIVTo analyzerecombinationevents, cells from a colony INGSTON 1986). From left to right, the markers are 1.6, 2.2, 2.9, grown in the presence of histidinewereplated to -His 3.1, 3.2, 3.3, 3.6 and 4.2 kb from the EcoRI end. media. DNA was extracted from independent HIS3 colonies by the spheroplast lysis method (BOEKEet al. 1985) or by the other may be damaged and incapable of replicaglass bead disruption (HOFFMAN and WINSTON1987). Plastion (Figure 1). Conversely, crossing over in inverted midDNAwas introduced into E.coli RR-1 hisBrecAby repeat plasmids reorients flanking sequences keeping electroporation. Electroporation competent cells were prethe size oftheproductthesameastheparental pared by washing a log phase culture three times with 10% molecule. If the crossover is not reciprocal, the plasglycerol and suspending the finalcellcollectioninthis solution with a volume of 1% of the original culture. The mid product will contain interruptionswhich prevent following electroporation conditionswereemployed: 1.4 its recovery (Figure 1). kV, 1000 Q, 25 pF, and 10% of the DNA preparation in 0.2-cm cuvettes using a Bio-Rad Gene PulserwithPulse Controller. Transformed cells were incubated at 37" for 1 MATERIALS AND METHODS hr before plating on L agar supplemented with 30 pg/ml ampicillin. T o identify E. coli transformants with a recomYeast and Escherichia coli strains: Saccharomyces cerevisbined His+ plasmid,a replica of the colonies was made onto iae strains SSL204A (MATa his3A200 trpl ura3 ade2 leu2) a histidine omission plate. Because yeast HIS3 complements and SSL212A (MATa his3A200 trplura3 ade2 leu2 E. coli hbB (STRUHL and DAVIS1977), cells with a recomrad52:LEUZ)have beendescribedpreviously(DORNFELD bined plasmid grow on the omission plates. Not all E. coli and LIVINGSTON 1991). his3A200 deletes the entire coding transformants are His+because the copy number of the region of the gene (FASULLO and DAVIS1987). The rad52 reporter plasmidsinyeastis not always one, and, consemutant SSL212A is the isogenic deletiondisruption of quently, some bacterialtransformants are His- because they SSL204A in which RAD52 has been deleted from the HpaII contain parental plasmids which havenot undergone recomsite immediately upstream ofthe initiating codon to the SphI site 15 bp upstream of the stop codon (ADZUMA, OGAWA bination. Plasmid DNA wasprepared from E. colitransformants by alkaline lysis (MANIATIS, FRITSCHand SAMBROOK and OGAWA1984) and replaced with LEU2. E. coli strain RR-1 hisBrecA has been described previously (EMBRETSON 1982)and analyzed by appropriate restriction enzyme digestions. Although allyeast plasmid preparations were used to and LIVINGSTON1984). transform E. coli, some were either unsuccessful or did not Recombination reporter plasmids: All plasmids contain yield His+ transformants. The majority of these failed bethe samepairof hb3 heteroalleles (Figure 3) either in cause the HIS3 recombinationproduct lacks ori and cannot inverted or direct orientation (Figure 4). One allele is within transform E. coli. When yeast plasmidpreparations failed to a 6.1-kb EcoRISalI fragment of chromosome XV and the transform or failed to yield His+ transformants, the yeast other is within a 4.4-kb EcoRI-XbaI subfragment of the same extract was analyzed by in situ hybridization of restriction region (STRUHLand DAVIS 1980). pBYA819I and enzyme digested DNA in dried agarose gels(TSAO,BRUNK pBYA9181 have been described previously (AHN and LIVand PEARLMAN 1983). INGSTON 1986). In addition to the twohis3 containing fragments, the BYA plasmids contain CEN3, TRPI, ARSI and pBR322 sequencesfrom the SalI site at position 651 to RESULTS an XbaI site we introduced at position 4340 (SUTCLIFFE 1978; EMBRETSON and LIVINGSTON 1984). These plasmids Heteroallelic plasmids: Eight plasmids were used are 16.4 kb in length. The pKD series contains all of the sequences except for CEN3 found in the pBYA series, and, in this study as recombination substrates (Figure 4). in addition, the SalI-EcoRI fragment of YCp50 (JOHNSTON T h e plasmids differ in three respects. First, to study and DAVIS1984) which includes ARSI, URA3 and CEN4 the effect of sequence orientation on recombination, within portions of pBR322. These plasmids are 23.4 kb in half of the plasmids contain the his3 heteroalleles as length. Besides the his3 heteroallele repeats, they contain an inverted repeat and the other half contain the inverted repeats formed by the ARSl and pBR322 sequences. T o reorient the EcoRISalI fragment to make the recombining sequences as a direct repeat (designated EcoRI
his3
SalI
a
264
K. J. Dornfeld and D. M. Livingston
FIGURE4.-The eight heteroallelic plasmids. The four inverted repeat plasmids areshownascircularmolecules. The direct repeat plasmids arethe same except for the sequences shownas a sector to the rightof each circularmolecule. RI-EcoRI sites. Note that in reorienting the 6.1-kb EcoRISolI fragment to create the pBYA direct repeats and the pKD of the invertedrepeats,thelocation EcoRI and Sal1 restriction sites are reversed.
I ’
I and D, respectively). Second, in half of the plasmids the 5’-his3 mutation and flanking markers are within a 6.1-kb EcoRI-Sal1 fragment originating from yeast chromosome XV, andthe 3”mutation is within a smaller 4.4-kb EcoRI-XbaI subfragment (plasmids with numerical designations beginning with 8). In the other half the his3 mutations and flanking markers are reversed (plasmids with numerical designations beginning with 9). This difference in allele placement controls for possible contributions of vector sequences in recombination such as an influence conferred by the centromere sequence. Third, half of the plasmids have one yeast and oneE. colireplication origin and contain the yeast TRPl gene (pBYA series). T h e other half have two yeast and two E. coli replication origins and both the yeast TRPl and URA3 genes (pKD series). Allplasmids containa single centromere sequence (CEN3 in the pBYA series and CEN4in the pKD series). The salient difference between the pBYA and pKD series is the presence in the KD plasmids of a yeast replication origin (ARSI) in each interstitial sequenceseparatingthe homologous sequences. The two replication origins ensure that all loopout products formed by direct repeat plasmids can propagate in yeast. The plasmids were transformed into wild-type and isogenic rad52 mutant cells. Transformed cells were grown in the presenceof histidine but underselection for tryptophan (pBYA series) or tryptophan anduracil (pKD series). Recombinants were selected onagar plates lacking histidine. Plasmid DNA recovered from independently arisingHis+ colonies was then analyzed by restriction enzyme digestions to place recombining markers.
.
TABLE 1 Rate of plasmid recombination Rate lo-’ events/cell/generation pBYA819 pBYA918 pKD8TB9 pKD9TB8 Genotype
INV
RAD52 rad52
44 51 12
1.0
DIR
9.1
INV DIR
2.7
9.8 1.6
INV
INV
DIR
4.5 0.26
7.9 5.8 8.5 1.1 0.36
DIR
1.2
Ratio RAD52/rad52
44
1.3 19
6.1
177.1 22 5.3
Rate of plasmid recombination: Initially, to characterize these plasmid substrates, we measured their recombinationrates in wild-type andmutant cells (Table 1). Inverted repeat plasmids recombine at rates events/cell/generation in between 4.5 and51 X wild-type cells. pBYA8191 and pBYA9181 have approximately 10-fold higher recombination rates than pKD8TB9I and pKDSTB81. The cause for this difference is unknown but could result from the closer proximity of the recombining alleles in the BYA plasmids (LICHTEN and HABER1989). Switching the allelic positions has little effect by itself. For example, pBYA9181 recombines only 16%morefrequently than pBYA8191. The rad52 mutation decreases the recombination rate of these inverted repeat plasmids from 17-fold (pKD8TBSI) to 44-fold (pBYA8191). Direct repeat plasmids recombine at rates between events/cell/generation inwild-type 5.8and 12 X cells. In comparison to the inverted repeat plasmids, the two directrepeat plasmids of the BYA series recombine to produce a HIS3 allele 4-5 times less
rad52 Plasmid Recombination
265
A. INVERTED REPEATPRODUCTS
a. CONVERSION WITHOUT
b. CROSSING-OVER CROSSING-OVER WITHOUT CONVERSION CROSSING-OVER
c. CONVERSION WITH
6. DIRECT REPEAT PRODUCTS
a. CONVERSION WITHOUT CROSSING-OVER
b. CROSSING-OVER C. CONVERSIONWITH WITHOUT CONVERSION CROSSING-OVER
FIGURE5.-Potential products of plasmid recombination. (A) pBYA8191 products: (a) This molecule is a representative conversion event without an associated reciprocal exchange. (b) This molecule is a simple crossover which reorients flanking sequences and creates a doubly mutant his3 allele. (c) This molecule is a representative conversion with an accompanying reciprocal exchange which reorients flanking sequences but does not create a doubly mutant his3 allele. (B) pBYA819D products: (a) This molecule is a representative conversion event without an associated crossover. (b) This molecule is a simple crossover. Although the HIS3 bearing product can replicate in yeast, it cannot replicate in E. coli. Such products were identified by hybridization of yeast DNA. In this particular plasmid, the reciprocal product containing the doubly mutant his3 allele cannot replicate in yeast cells and would be lost. (c) This molecule is a representative conversion with crossing over to yield a HIS3 allele with flanking markers and vector sequences opposite from those of a simple crossover. As noted, this HIS3 containing plasmid cannot be recovered because it cannot replicate in yeast. Some of the potential products of pBYA918D also cannot replicate in yeast or be recovered by transformation into E. coli. All potential products of pKD8TBSD and pKD9TB8D should propagate in yeast and be recoverable by transformation into E. coli.
frequently than the corresponding plasmids with inverted configuration. The two KD plasmids with direct repeats recombine at similar rates to the corresponding plasmids with inverted repeats. A comparison ofdirect repeat recombination rates between wildtype and mutant cells shows a drop of 1.2-7.1-fold. This is lessdrastic than the drop observed for inverted repeat plasmids. Classification of recombinationproducts: The next step in analyzing plasmid recombination was to determine the distribution of conversion and crossover events which occurred. From these distributions we wereable to observe the continuity of coconversion tracts and calculatecoconversion tract lengths. T o establish a distribution, the recombination products were grouped into categories based on the number and pattern of altered heterologies and on the arrangement of flanking sequences (Figures 5). The major difference between the two orientations is that crossing over ininverted repeats plasmids leads to reorientation of plasmid sequences and retention ofplasmid length, whereascrossing over in direct repeat plasmids leads to looping out of smaller plasmids. This difference is important because recombi-
nation products can be recovered after nonreciprocal crossing over of direct repeat plasmids but not after nonreciprocal events of inverted repeat plasmids (Figure 1). The KD plasmids with direct repeats were made so that either potential nonreciprocal crossover product could be recovered. We did not attempt to recover both products of a reciprocal crossover from direct repeat plasmids of the KD series becausewe could not ensure that both loopout plasmidswould segregate together during cell division. Furthermore, the KD plasmids contain repeats of certain vector sequences (MATERIALS AND METHODS), and this providesthe basis for additional products as will be noted below. We first Products of invertedrepeatplasmids: compared inverted repeat plasmid recombination events from wild-type cellsto those from mutant cells. The conversions found among inverted plasmid productswere further subdivided (Figure 6; AHN and LIVINGSTON 1986).The largest class ofrecombination products from wild type-cells are asymmetric continuous gene conversions (Table 2). These represent from 40% (pKD8TBSI) to 79% (pBYA8191) of the total products. This type of event also predominates
266
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K. J. Dornfeld and D.M. Livingston
his3
>h parental configuration
hw
HE33 continuous, asymmetric coconversion his3
Hz33 discontinuous. asymmetric coconversion his3
nT3 symmetric coconversion his3
I n33 reciprocal eraover
FIGURE6.-Recombination patterns in inverted repeat plasmids. The two allelic segments from Figure 3 are shown asblack and white rectangles. Coconversion events in which the changes occur on only one allelic segment-that containing the HIS? allele-are termed asymmetric. When the only change has been the conversion of one of the his? markers or when adjacent flanking markers have been coconverted without interruption, the events are labeled continuous and asymmetric. When coconversions occur with interruptions, theevents are labeled discontinuous and asymmetric. Changes on both of the allelic segments are termed symmetric. A reciprocal crossover between the HIS? heteroalleles is also shown.
among products from rad52 mutant cells, accounting of HIS3 for 52% (pKD8TBSI)to 75%(pBYA8 191) the products. As all classes ofevents recovered from wildtype cells are also recovered from mutant cells, the drastic drop in recombination rate found in the mutant reflects a nearly uniform drop in each product category. We have also determined the average conversion tract lengths among the products recovered from each cell type.To calculate the conversion tract lengths we used the frequencies of asymmetric continuous groups 1986) and (Figure 7, Table 3; AHN and LIVINGSTON the marker distances. The minimal meantract lengths range from 0.21 kb (pKD9TB8I) to 0.34 kb (pBYA8191) for wild-type events and from 0.35 kb (pKD9TB81) to 0.52 kb (pKD8TB9I) for mutant events. Although for all plasmids the mutant events average longer than the wild-type events, application of a two-tailed t-testto thesets ofdata (Table 3) shows that none are significantly different ( P > 0.17 in all cases). Only when all four sets of data are pooled is
there a significant difference between wild-type and mutant tract lengths ( P < 0.02). We interpret the significance which arises to be the statistical result of increasing the sample size and the scientific result of a small lengthening of conversion tracts in the mutant. Also, the percentage of events which include reciprocal recombination does not differ between wild type and mutant (Table 2). For the two pBYA substrates in both cell types the percentage of reciprocal recombination ranged from 17% (pBYA8 191 in the mutant) to 27% (pBYA8 191and pBYA9 181 inthe wild type), values which are within the range we have previously (AHN and LIVINGSTON observed inwild-typecells 1986). For the two KD plasmids in both cell types the percentage was lower, ranging from 4% (pKD8TBSI in the mutant) to 11% (pKD8TB9I in the wild type). The smaller percentage of crossovers in the KD plasmidslikelyresults from repeated vectorsequences (MATERIALS AND METHODS). Reciprocal recombination withinthese inverted repeat plasmidsplacesvector sequences into direct orientation, and these direct repeats maygiverise to loopout and other novel products during a subsequent round of recombination. Novel products both smaller and larger than the parental plasmids have been found after recombination of the KD plasmids (Table 2) but have not been extensively analyzed due to their heterogeneity and complexity. A feature of crossingover which is observed in both the wild-type and themutant cells is the association of reciprocal recombination with the longer asymmetric continuous coconversion tracts (Table 3; AHN and LIVINGSTON 1986). The minimal mean tract lengths among wild-type events of this class with and without an accompanying reciprocal exchange are 0.25 and 0.59 kb, respectively. For mutant events the values are 0.39 and 0.66 kb. Both sets of values are significantly different ( P < 0.05). One subtle difference we can discern between inverted repeat plasmid recombination in wild-typeand mutant cells is the preference with which each allele converts to wild type. We noted previously (AHNand LIVINGSTON 1986) that the allele within the shorter EcoRI-XbaI fragment is converted to wild typeapproximatelytwo-timesas often as the allele in the longer EcoRI-Sal1 fragment. This is irrespective of which his3 heteroallele (5’- or 3”) is within this fragment. For example, the results for the asymmetric, continuous events (Table 3) showsthat for pBYA9 181 in a wild-typecell the allelewithin the EcoRI-XbaI fragment converts three times as often as the allele in the otherfragment. Of the fourplasmids the only one which does not show this preference is pKD8TB9I. A comparison of events from wild-type and mutant cells shows that inall four cases there is a shift from conversions in the EcoRI-XbaI fragment to ones in the
Recombination rad52 Plasmid
267
TABLE 4 Analysis of recombination events which give rise to a wild-type HIS3 allele in inverted repeat plasmids No. of convertants Asymmetrica Symmetricb
No. of reciprocal recombinants'
No. of smaller productsd
10 (3)
10 (7) 10)
5 2
0 0
0 0
100 (27) 52 (9)
10 (5) 12 (4)
12 (6) 4 (2)
1 0
0 0
0 0
100 (27) 53 (14)
4 (0) 4 (0)
6 (1) 1 (0)
0 0
9 3
1(1)
1 (0) 0
0 0
9 5
Plasmid
Genotype
Continuous
Discontinuous
pBYA819I
RAD52f rad52
79 (13) 39 (3)
pBYA9181
RAD52f rad52
77 (15) 37 (8)
pKD8TB9I
RAD52 rad52
21 ( 5 ) 29 (2)
pKD9TB8I
RAD52 rad52
42 (4) 36 (4)
5 (0)
6 (2)
No. of larger products'
53 56
Total
13 19
(6) (2)
2 6
55 (5) 52 (4)
a Asymmetric conversions change markers along one allelic segment. If only the marker within the his3 coding region or adjacent markers are coconverted without interruption, the events are continuous (Figure 6). If two or more markers are converted but a marker between them is not converted, then the event is discontinuous (Figure 6). The numbers in parentheses are the number of events which include reciprocal recombination. Symmetric conversions change markers along both allelic segments (Figure 6). The numbers in parentheses are the number of events which include reciprocal recombination. Reciprocal recombinants exhibit sequence reorientation with one his3 copy becoming wild type and the otherdoubly mutant (Figure 6). Plasmids with one his3 copy. These plasmids have not been analyzed in greater detail. Although these plasmids have undergone crossing over, they were not counted as having undergone a reciprocal crossover. Plasmids with three or more his3 copies. As with plasmids with a single his3 copy, these plasmids were neither analyzed nor included among those which had undergone crossing over. f This data is from AHNand LIVINGSTON (1986).
EcoRI-Sal1 fragment. Taking pBYA9181 again as an example, the results show that in the mutant slightly more conversions occur in the EcoRI-Sal1 fragment than in the EcoRI-XbaI fragment. Both the cause of the preference in wild-type cells and the reason for its absence in mutant cells are unknown. Products of direct repeat plasmids: Next, we examined the products formed by direct repeat recombination to learn whether they were similar to those found for inverted repeat plasmids. T h e direct repeat plasmids were classified accordingtotheproducts shown in Figure5 (Table 4). Conversions yielding parental size products were a sizable fraction of the events, comprising 31% (pKD8TBSD) to 52% (pBYA918D) of the molecules from wild-type cells. T h e remainderwerenonparental insize and evidenced crossing over. These results indicate that although direct repeatplasmids, like the inverted repeat plasmids, are capable of undergoing conversions witho u t an accompanying crossover, they undergo crossing over at a significantly higher frequency. Comparisons between inverted and direct repeat plasmids, e.g., pBYA8191 and pBYA8 19D, shows that inall cases the direct repeat plasmid has a higher percentage of crossing over. x* comparisons for pBYA8 19D, pBYA918D and pKD9TB8D give values with P < 0.05, and the comparison for pKD8TBSD yields a value of P < 0.10. The shift away from gene conversions to crossovers is even more pronounced among the products from
mutant cells where products of parental size account for only 4% (pKDSTB8D) to 29% (pKD8TBSD) of the total. Except for plasmid pKD8TB9D, theincrease in crossovers in the mutant is significant ( P < 0.01). This selective change contrasts with the behavior of inverted repeat plasmids in mutant cells which shows an even decrease in all product classes. Within the convertant class giving rise to parental size molecules, asymmetric, continuous events comprise most of the eventsfromboth wild type and mutant. As for inverted repeat plasmid products, this class can be further divided based on the extent of coconversion (Figure 7, Table 5). The minimal mean tract lengths range from 0.23 to 0.58 kb for wild-type events and from 0.14 to 1.3 kb for mutant events. T h e differences are neither significant for each data set nor for the composite of all four data sets. For asymmetric, continuous conversion events without an accompanying crossover, direct and inverted repeat coconversion tracts are similar in length. For wildtype cells the mean tract lengths forthis class are 0.32 kb and 0.25 kb ( P > 0.3) for inverted and direct repeat plasmid events, respectively. In mutant cells the mean tract lengths for this class are 0.39 kb and directrepeat 0.40kb ( P > 0.8) forinvertedand plasmid events, respectively. The predicted loopout plasmids of both the simple crossover and theconversion with crossover type (Figure 5) were among the crossover products from both wild type and mutant. A wild-type HIS3 allele within
--
K. J. Dornfeld and D. M. Livingston
268
his3
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-v
I H33
FIGURE 7,"Extentof asymmetric, continuous coconversions. -1'he tnembers of the asymmetric, continuous class have been grouped by the extent of the coconversion. As in Figure 6, the extent of coconversion is represented by shading and duplication o r removal of markers. Groups I through V are conversions of the :%'-his3mutation and coconversions extending downstream past the :I'-end of the open readingframe.Groups I' through 111' are conversions of the 5'-his3 mutation and coconversions extending upstream past the 5'-end of the open reading frame. These groupings are irrespective of whether the 3'- or 5'-his3 mutation and its flanking markers arewithin the EcoRI-Sal1 fragment or the EcoRIXbal fragment.
a simple crossover product couldarise either by crossing over between the heteroallelic markers or by conversion followed by crossing over. An indication that conversion occurs in many of the events classified as simple crossovers is shown by a tally of the number of such events inwhich flanking heterologies have been converted (Table 4). Such conversions were not uncommon in either the wild type or the mutant. T h e small number observed for the simple crossovers of pBYA819D reflects our inability to analyze these events completely by hybridization due to the presence of parental molecules within the cells. An unexpected result was that a considerable fraction of the simple crossover products contained the HIS? segment as part of a plasmid larger than the parental molecule. Analysis of these plasmids showed that the sequences expected for the simple crossover (both H I S 3 and vector)appeared in plasmids with three his3 copies, the wild-type and the two heteroallelic mutant copies (Figure 8). They are referred to as three-copy plasmids. The most notable characteristic about these products was the constancy of their architecture. The repetition of the simple crossover sequence was always located in the same position relative
to the parental sequences. As with other simple crossover products, those within the three-copy plasmids often showed conversion of flanking markers (Table 4). The frequency of three-copy plasmids was higher among rad52 mutant events than among wild-type products. Characterization of the three-copy plasmids:The novel finding of recombinationproducts inwhich sequence additions had occurred prompted us to examine further therules governing three-copy plasmid formation. First, we noted that two plasmids (pBYA819D and pKD8TB9D) did not readily yield three-copy plasmids (Figure 4). Inthese cases the crossover wild-type HIS3 allele would be accompanied by vector sequences containing the CEN sequence, and the predicted three-copy plasmid would be an unstable dicentric (KOSHLANDet al. 1987). T o show that pBYA8 19D could form three-copy products if such products were not dicentric, we moved the CEN3 sequence from one of the interstitial vector sequences to the other toyield pBYA889D (Figure 9A). Among 24 products analyzed from wild-type cells 7 were three-copy plasmids of the predicted structure (Figure 9B) and among 22 products analyzed from mutant cells 14 were of the predicted structure (Table6). Not only do the three-copy plasmids form, but also they are more likely to form in the mutant. The formation of such plasmids from pBYA889D butnotfrom pBYA8 19D is consistent with the hypothesis that pBYA819D may give rise to unstable, dicentric plasmids which further break down. T o study the mechanism giving rise to the threecopy plasmids, we investigated whether intermolecular recombination contributed to their formation. Potentially, either a loopout plasmid recombining with a parental plasmid or two parental plasmids recombining to yield an unstable, dicentric plasmid with four his? copies could give rise to the three-copy plasmids. The latter could break down, by a mechanism like single strandannealing (OZENBERGER and ROEDER 1991), to yield a three-copy plasmid. T o determine the proficiency of intermolecular recombination, we constructed two autonomously replicating plasmids each with one of the two his3 heteroalleles (Figure 10). These werecotransformed into wild-type and mutant cells. After growth of multiple cultures under selection for both tryptophan and uracil, samples were analyzed by hybridization and by plating on -His plates. Among five cultures of wild-type cells the fraction of HIS3 cells ranged from 4.3 X 10" to 3.0 X Among five cultures of mutant cells we were unable to recover a single recombinant (l). ;I \ I t u t t l c * vc-cror Ixrwcl 0 1 1 pnK322 (]atiswas ; t n d I h v l s l!W4). Thr r m r i c t i o o s i t e ends of this lt.tgnwttt II;IVC.IWWI e w 11;mgcd;I\ dc.u rilwcl ~~nclc*r \IAWRIAIS AVI) \IF IIIOIK. I IN* OIIIC*I p h s n t i d r a r r i c - the Irrlcrnallelr n i r l r r 4.441) EroKIS h r r l \ul)lr;tgtl~rnt( lo~~ccl in ;I vcctor (ont;tining pIIKSL'2 ; a n d t l w .4/W/-T/Z/'/ II:I~III~III 01 v c t s r . Kc.ciprcxal c r o w i n g nwr w i t h i n t l t r lllS3 Iw.lting W ~ I I I WI)rcducc.\ I\ ;I onicp 6.I -kI) f . m K I fr;tgnwnt. Krciproc;d crossing over Iwtwc*rn honralqpts plIK122 vector sequences I)I'~M~IIC I*\ ;I u v i r t v 01 I ) ~ I M I I I ( , I \ clqwnding on t l w p s i r i o l l ol 11trrxc11;tngt~;111 nI wltic-I1 phce rltr t w o EroKl sitgrc-awr t h ; l n I4 kl) ;rp;lrt.
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I I I ~ ; I \ I I ~tlwir ~. ;tl)ilitv t o rrcotlll)itrc*.c ~ ~ t r ; ~ ~ ~ s l ow rr ~c .~ nnint;tinr-tl r ; ~ ~ ~ t s unclrr w l c c t i o t l lor Ir)rl~trvptoplr;tn ; I d u r a c i l ;ant1 prnviclrcl wirlr r c . p I ; t w t l t o u*lrc-tirc.nrc.tli;t w i t h ;trttl w i t h o u t l t i s t i d i n r t o t l r r c r r n i n r rlrr f m c t i o n of cells w i t h a //IS3 lri\ridittr. Portion\ ol IIW< c ~ l t ~ ~ twrt~ ) I . I \ I I I I ~ . l s o v r i m \ 111 111c.u. ( ultttrc-\ wrrr c o l l c ~ c - ~ c *;t Il w l I)\:\ c.utr;~c-~rclit1 orclrr t o p l l v k ; ~ l l vclrtret r r c o d ~ i n i n pkasnrids. g (It) I l v l ~ r i r i i ~ ~ t t i n n ;ln;ll\\i\ol I C ( o n t I ) i w d pl;wnid\.T o iwrc-;lw t l w l'txc-tion nl' p l a s m i d s w h i c h l r ; d Imclc-rgonritrrcrntolecul;w crossing over. ; t n o l t t g r o w t h t o c m t i c 11 lilr \II( 11 ~ I O ~I\I W;I\ I C lwrfornwd. 111 this otttgrowtlr ;I Imrtion of r l t r rlllturc-s c l c v tilwctl n i p r r h w r r e tlilutc4 in m r t l i a l a r k i n g t m c i l 1 w t I)ro\iding trvp1q)hmr (;IIIC~ lli\tiditrt*).I'rovicling t r v p t o l h t n slroulcl Ixwnil IIIC. t y i d lws 0 1 rlte T/tID/-ANS/ cont;tiningphsnrid ~ l ~ ~ ~ / ( ~ ~ ~ . ~ (:I.ARY t . t ~;IIIC~ I l (:ARROV . ~ ~ t s l!lX?: . ficXlllASl) cf nl. I!lX7) IIIII~Wi t i q st;rl)ili/t-d l)v i t s ;wnri;ttinn. i.r. crcnsing n w r . w i t h tl~r (.'l.;.\-