Copyright 0 1996 by the Genetics Society of America
Genetic Requirements for the Single-Strand Annealing Pathway of Double-Strand Break Repair in Saccharomyces cerezkiae Evgeny L. Ivanov,' Neal Sugawara, Jacqueline Fishman-Lobell' and James E. Haber Rosenstiel Basic Medical Sciences Research Centerand Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110 Manuscript received August 25, 1995 Accepted for publication November 14, 1995
ABSTRACT HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Eschm'chia coli lacZ genes are repaired eitherby gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred inplasmid or chromosomal DNA. In bothplasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two laczsequences were in invertedorientation. These results suggest that thereis a competition between SSA and geneconversion processes that favors SSA in the absence of RAD51, RAD54,RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type ( M A 7 ) switching.
E
NVIRONMENTAL stresses induce a wide variety of injuries to DNA, forcing cells to restore the integrity of the genomeby DNA repair. Among the different forms ofDNA damage, DNA double-strand breaks (DSBs) are considered as the most harmful, because unrepaired DSBs result in the loss of broken chromosomes, leading to the deathof haploid cells or to aneuploidy in diploids. Considerable evidence has accumulated during the past 20 years that in yeast the major cellular mechanism of DSB repair involves homologous recombination, culminating in the DSB repair model (SZOSTAK et al. 1983). In this model, a DSB or a gap is repaired by a gene conversion event involving broken ends thatinvade and copy DNA from an intact,homologous donor copy (Figure la). Further supporting the idea that DSB repair involves homologous recombination is a striking overlap in the genetic requirements for homologous recombination and for DNA repair after treatments known to induce DSBs (e.g., ionizing radiation). Mutations in R A D S I , RAD52, RAD54, RAD55, and RAD57 genes confer high levels of sensitivity to ionizing radiation; the very same mutations demonstrate defects in homologous recombination both invegetativecells and during meiosis Currespunding author: James E. Haber, Rosenstiel Center, Brandeis University, 415 South St., Waltham, MA 022549110. E-mail:
[email protected] Present address: Transkaryotic Therapies,Inc.,Cambridge, MA 02139. Present address: CenterforNeurologicDiseases, Brigham and Women's Hospital, Boston, MA 01801.
'
Genetics 142 699-704 (March,
1996)
(reviewed inPETESet al. 1991).More recently, the RFAI gene codingfor the large subunit of the single-stranded DNA-binding protein RPAwas shown to be required for bothDSB-induced gene conversion and DNA repair (FIRMENICH et al. 1995). During the last few years, it has become evident that another pathway exists for DSB repair via homologous recombination. This pathway, called single-strand annealing (SSA), relies on the presence of two homologous sequences in direct orientation flanking the site of a DSB (Figure l b ) . According to current schemes of SSA, bidirectional 5' to 3' degradation of the DSB ends leads to theexposure of complementary sequences, which then anneal.Subsequent removal ofnonhomologous 3'-ended tails followed by ligation completes the process. The outcome of this reaction is the creation of a deletion of one repeat along with the intervening sequence. The mechanism of SSAwas first postulated to explain some features of extrachromosomal recombination in mammalian cells (LIN et al. 1984, 1990). We have shown that the SSA process operates in yeast cells as well, representing an alternative, kinetically separable and independently modulated pathway of DSB repair (FISHMAN-LOBELL et al. 1992). SSA is a truly homologous recombination process, in that it displays essentially the same homology requirements between interacting sequences as does geneconversion ( SUGAWARA and HABER 1992; N. SUGAWARA, unpublished results). It is also evident that both SSA and gene conversion strongly depend on the 5' to 3' degradation of DSB ends ( C A O et al. 1990; WHITE and HABER
E. L. Ivanov et al.
694
/
/
\
Gene conversion without crossing-over
Gene conversion with crossing-over
0-Q / Deletion product
a 1990; SUN et a/. 1991; FISHMAN-LOBELL et al. 1992; SU1992). Moreover, the extent of this degradation was shown to be under similar genetic control in both pathways (WHITEand HABER 1990; SUGAWARA and HABER 1992; IVANOV et al. 1994). At the same time, SSAis clearly different from gene conversion in that it does not involve such molecular steps as strand invasion and the formation and resolution of Holliday structures. Thus one might expect that the genetic requirements for SSA would not be identical to those for gene conversion, although some overlap should exist. At the present time, our knowledge of the genetic control of the SSA process is not complete. Previous results from our lab have demonstrated thattwo nucleotide excision repair genes, R A D 1 and RADIO, are required for the successful completion of the SSA process, being involved in the removal of 3"ended regions of nonhomology that are created during annealing (Figure l b ) (FISHMAN-LOBELL and HABER 1992; IVANOVand HABER 1995). However, the formation of these nonhomologous ends is a characteristic feature of the SSA process and is not expectedto occur duringrecombination by gene convesion involving fully homologous sequences. In agreement with this assumption, rad1 and rad10 mutants show the wild-type proficiency in interchromosomal gene conversion and IMAT switching (SCHIESTL and P m H 1988, 1990; FISHMAN-LOBELL and HABER1992) and are only marginally sensitive to X-ray and gamma irradiation. Deletion of the RAD52 gene significantly reduces the extent of repair by the SSA mechanism (FISHMAN-LOBELL et al. 1992; SUGAWARA and HABER 1992). However, it seems that this reduction is not as severe as the effects G A W and ~ HABER
FIGURE1.-Two alternative pathways of DSB repair as adapted for the plasmid substrate employed inthiswork. (a) DSB repair by gene conversion could theoretically yield both conversions withoutcrossingover and with crossing-over (SZOSTAK et al. 1983). However, repair of a n HO-induced DSB occurring in direct repeats of the lncZ gene was shown to occur without the formation of the reciprocal crossoverproduct (RUDINand HABER 1988; FISHMAN-LOBEI.I. et al. 1992). (b) SSA pathway repairs a DSB leading to a deletion of one of the direct repeats. In this pathway, the small reciprocal circular Droduct is not exDected to be formed. For discussion of current schemes of SSA, see IVANOV and HABER (1995).
b of rad52 mutations on gene conversion (see, for example, RATTRAY and SYMINGTON 1994). Moreover, the requirement for RAD52 during SSA can be overcome by substantially increasing the amount ofhomology between recombining sequences ( OZENBERGER and ROEDER1991). The roles for other RAD genes belonging to the RAD52 epistasis group in the SSA process are not clear. Mutations in RAD51, RAD54,RAD55, and RAD57genes do notprevent deletion formation during spontaneous mitotic recombination between naturally occurring and artificially constructed direct repeats; on the contrary, deletion formation is stimulated by these mutations (FAN and KLEIN 1994; MCDONALDand ROTHSTEIN1994;AGUILERA 1995; LIEFSHITZ et al. 1995). However, the major problem with interpreting these data is that SSA isonly one of a few possible mechanisms of deletion formation during direct-repeat recombination(reviewedin KLEIN 1995), and the fraction of events being processed through the SSA pathway is unknown. In addition, the identity of the lesion initiating a recombination event (DSB us. single-stranded break) cannot be revealed in any system employing spontaneous recombination. To gain more insight into the genetic control of the SSA pathwayof DSB repair inyeast, we studied the effectsof RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, and XRS2 genes on the repair of a single, HO endonuclease-induced DSB, using two directly repeated lacZgenes ofEscherichia coli as a recombination substrate (RUDIN et al. 1989; FISHMAN-LOBEI,I~ and HABER 1992; FISHMAN-LOBELL et al. 1992; IVANOV and HABER 1995). The main advantage of this experimental system for studying the genetic control ofSSAis that >80% of
69.5
a
PSI I
!
Psfl
- PSf I
'
Pst I
Sma I
H~ndlll
Psf I
Psf I
conversion without crossover
Psf I
Sma I
Hindlll
A
Psf I
conversion without cross over
strand annealing
'-
Sma I
conversion
-
PSf I
Psfl'
PSf I
pIH6X3 (AIK)l~ssl.:KIIRr\ PI n/. 1992); IL11152, pSM'L0 (S(:blIl.l) /'/ N / . 1983);I W 1 5 4 , pI,s101 (IL\TrR.\Y and ~ W l N ( ; l O N1995);
IC4/)55, pSTLll ( L o v ~ nand MORTIMER 1987); RAD57, pSM.51 (S(:llll,D PI n/. 1983); XR72, pE140 (I\'AXO\J d n/. 1994). Strains and media: The strains of Snrrhommy-rs rm-misinr usrtl i n this s t u d y are listed i n Table 1. All of these strains arc MATa-inr isogenic derivatives of the previously described 1995); the MATa-inc mutastrain.JKM40 (I\:\sov and H.ARER tion prevents HO cutting. These strains also c a r y the HO cnclonrtclcase gene under control of thc inducible GAL promoter, thc whole construction bring integrated into chromoand ZAKIAN 1993). For galactosesomal AlK3 locr~s(SASI)IX.I. inductionexperiments, strains E1534 through El.356 were transfc)rnmed w i t h plasmids pJF6 or pIF5. Strain E1558 and its dcrivativcs bear the /tr%directrepeat integrated into the u f d 5210~11s o n chromosome V ( R ~ W NP/ nl. 1989; see also Figwe h ) made by integrati\ve transformation of EI.534 with plasmid pNR28. The rad mutations were introduced into E1534 and El.5.38 strains by the one-step gene disr~lption/replacement mcthotl (RO~IS'I'EIN1983) using the plasmids listed above. For details of strain constructions, see the corresponding references. All the strain constructions were verified by Southern blot analysis. The lithium acetate method ( I n ) P/ nl. 198.7) was u s e d for yeast transformation. YEPI) medium and synthetic complete media lacking Ieucine (SLcu)o r uracil (SGUra) were described previously (SIII~KMANP/ nl. 1986). YEPGly and YEPGal were 1 % yeast
696 phorlmager. The same filters were used to expose Kodak XOMAT films, these being used to make photographs presented on Figures 3, 5, 6, and 7 .
TABLE 1 Yeast strains Strain
Genotype ~~~~
E1534 E1548 E1549 E1550 E1551 E1552 E1553 E1556 E1558 E1559 E1560 E1561
MAE-inc adel udd::GAI,::HO leu2-3, 112 trpl u r d 5 2 R w ' EI534, but rad50::hisG::URA3::hisG E1534, but rud51::URA3 EI534, but ,rad54::UKA3 EI534, but rad55::LEUZ EI534, but rad57::LElD EI534, but xrs2::LEUZ EI534, but md52::LEUZ EI534, but with direct lac% duplication integrated at u r d - 5 2 EI558, but rad51::IJR43 EI558, but mdjZ::LEU2 EI558, but rad54::URM
extract-2% BdctO Peptone media supplemented with 3% (v/v) glycerin or 2% (w/v) galactose, respectively. Synthetic complete mediumcontaining X-Galwas prepared as described (RUBYet al. 1983). YEPD medium containing 0.015% (v/v) methyl methane sulfonate (MMS) was used to follow the Rad- phenotype. Cultures were incubated at 30", unless otherwise indicated. Induction of DSBs: Strains to be tested were streaked to single colonies on SCLeu/Ura (for strains bearing pJF6 or pJF5 plasmids) or YEPD (for strains bearingchromosomal integration of Zad) plates. Liquid precultures (5 ml) in the same medium were inoculated from single colonies, grown to midlog phase, and transferred to 50 ml of YEPGly for 810 hr. These cultures were used to inoculate 0.5 liter cultures in YEPGly followed by the growth overnight to a cell density of lo7 cells per ml.At the time 0, an aliquot of cells was removed, while the rest of the culture was harvested and suspended in the same volume of warmed YEPGal, and incubation was continued for30 min. The cells were harvested again and suspended in an equal volume of warmed YEPD, an aliquot of cells was removed, and incubation resumed with samples ofthe cells being withdrawn at the time points indicated. Appropriate dilutions of cells taken before induction (0 hr) and afterinduction(0.5 hr) were spread on YEPD plates, grown to colonies, and replica plated on SC-I,eu/Ura plates to measure loss of pJF6 or pJF5 (if applicable) and on X-Gal plates to score for the LacZ phenotype. In the case of HO induction on plates, 5-ml cultures in SCL,eu/Ura or YEPD media were grown to saturation, washed twice with water, and equal samples of cells were plated directly on YEPD and YEPGal plates. Colonies were counted after growth for 4-6 days at 23" and for 7-10 days at 30" as cells grew slower on YEPGal plates at 30". After counting, the colonies were analyzed as described above. Physicalanalysis of DSB repair: DNA from galactose-induced cells was prepared by the glass bead protocol ( R U D l N and H A B E K 1988), digested with the appropriate restriction enzyme(s),separated on 0.8% neutral agarose gels, and transferred to Biotrans (+) nylon membranes(ICN) in 0.4 kl NaOH. Southern blot hybridization was carried out by the method of CHURCII and GILBERT(1984), using a 1.7-kb Bsu36I-Snd fragment of' the IucZ gene as a probe (FISHMANLom1.1.et al. 1992). '%labeled probes for hybridization were prepared by the random-primer protocol (FEINBEKC; and VO (;EiSTEIN 1984). Densitometry of Southern blot filters was carried out by using a Molecular Dynamics Storage Phos-
RESULTS
Analysis of DSB repair by the SSA mechanism on a plasmid substrate: We first analyzed the ability of mutants belonging to the epistasis group RAD52 to repair a single, HO endonuclease-induced DSB on plasmid DNA. This DSB must be repaired before the onset of' the next round of DNA replication; otherwise, the plasmid would be lost. Thus the level of plasmid loss/retention in galactose-induced cells can be used as a measure of theefficiencyof DNA repair. Data presented in Table 2 show that rad51,rad54,md55, and rud57 mutant strains displayed essentially the wild-type level of plasmid retention. Moreover, after induction >BO% of all the cells became LacZ+, demonstrating successful completion of the repair event and arguing against the possibility that the high levels of plasmid retention in mutant cells weredue to the inefficient cleavage of plasmid DNA. Additionally, in agreement with our previous reet al. 1992), a pronounced plassults (FISHMAN-L,OKFLI. mid loss was seen in a md52 strain implying that HO induction was high inthis particular genetic background. The kinetics of DSB repair of the plasmid substrate were also analyzedby Southern blot hybridization using DNA samples prepared from HO-induced cells. In a wild-type strain (Figure Sa), expression of the HO endonuclease led to the cleavage of plasmid DNA asevidenced by the appearance of cut fragments. These disappeared rapidly as the deletion product accumulated. The formation of the deletion product was severely decreased in a rad52strain (Figure 3c);however, deletions were formed in rad51 (Figure 3b), rad54 (Figure 3d), and rad55 and rad57 (data not shown) mutant strains. A densitometric analysis (Figure 4) of the Southernblot data of Figure 3 shows that the kinetics of appearance and the amount of the deletion product formed were quite similar in the wild-type and all the rcId strains, except rad52 (Figure 4a and Table 2 ) . In Figure 4b, we also analyzed the kinetics of formation and disappearance of cut fragments that reflect the kinetics of processing of the DSB ends. The levels of DSB induction and the rate of their subsequent degradation were essentially similar for wild-type and the rad strains. We concludethat RAD51,RAD54, KAII55, and M I 5 7 genes are not required for DSB repair by the SSA mechanism on plasmid DNA.We note, however, thatthe efficiency of cutting seen in the m d 5 2 strain ( 5 3 0 % ) does not correspond quantitatively to the high level of plasmid loss (>go%). It could be that the amount of cut fragmentsseen in the rad52 strain is underestimated due to more extensive 5' to 3' degradationof one strand as observed previously in rad52 mutant cells (WHITE and HABEK 1990; SL~GAWARA and HXUER 1992).
Annealing Single-Strand Control of
697
TABLE 2 HO-induced recombination and plasmid loss in wild-type and mutant strains
Strains
Cells containing pJF6 after Fraction HO induction (%In
LacZ+ ofRelative efficiency Fraction ofgene of cells ( % ) ” formation‘ deletion conversion
80 83 81
1 1.26 0.02 1.07 0.79 0.96
17 0.2 ND 0.5 0.3
65 73
0.39 0.45
7.4 5.5
84 83 4 68 70 72
93
radl5A rad52A rad54A rad55A rad5 7 A rad50A xrs 2A
26 21
Wildtype
( %)
90 0
0.4
ND, not detectable. “Fraction of Leu+/Ura+colonies among all the colonies grown on YEPD plates after HO induction. Before induction, these values varied from 91% for the rad50A strain to98% for the rad54A strains. Colonies combined fromtwo to three independent ex eriments (n = 150 to 250) were routinely analyzed for each strain. ’Fraction of blue colonies on X-Gal plates among Leu+/Ura+colonies. ‘Densitometric analysis of Southern blot data shown in Figure 3. Intensities of all laczhybridizing bands seenat the 6 hr time point were integrated. The intensity of the deletion product band was normalized to the resulting sum and expressed as a fraction of overall DNA content, thevalue being 33.7 2 3.3% (mean 2 SD) for the wild-type strain. The fractions of the deletion two to five measurements product in mutant strains were related tothat of the wild-type strain. Presented are data averaged from for each strain. ‘Densitometric analysis of Southern blot data shown in Figure 5. Intensities of lacZ-hybridizing bands corresponding to the recombination product (deletions plus gene conversions) were integrated from Phosphorimager analysis and the percentage representing gene conversion eventswas calculated. Bands with as little as 0.5% of total DNA can be detected by this approach.
It is also possible that in the rad52 strain, expression of HO is not completely shut off after transferring thecells into glucose-containing medium resulting in additional cutting of plasmid DNA. In any case, successful recombination to produce Lac+ recombinants was nearly completely blocked in rad52 mutant strains. In plasmid pJF6,only -80% of HO-induced DSBs are repaired by SSA with the rest being repaired by et al. 1992). We degene conversion (FISHMAN-LOBELL cided to test whether the distribution of deletion us. gene conversion events is different in the rad strains. By using a triple HindIII/PstI/SmaI restriction digest of DNA prepared from galactose-induced cells, it is possible to follow the formationof both recombination products simultaneously. The results are presented in Figure 5. A densitometric analysis of these data showed that in the wild-type strain (Figure 5a) the whole recombination product(deletion plus gene conversion) represented almost 60% of the total ZacZhybridizing signal, ofwhich 83%were deletion and 17%were gene conversion events (Table 2). As expected, no visible recombination products were seen in the rad52 strain (Figure 5c). In therad51, rad54, rad55, and rad57strains (Figure 5, band d-f, respectively), although thetotal recombination product was formed at even higher than wild-type level (60-70% of the total lacZ-hybridizing signal), almost no gene conversion product was formed. More precisely, according to densitometric analysis, the efficiency of gene conversion was 30-80 times lower in rad51, rad54, rad55, and rad57 strains compared with the wild type (Table 2).
In a parallel series of experiments, we analyzed the structure of recombinant plasmids from individual LacZ+ colonies grown on YEPD plates after induction. Each colony contained a single LacZhybridizing band, eitherthe 7-kb bandcorresponding to thedeletion product or the 11-kb band corresponding to the gene conversion event (see Figures 2 and 3). The results of this analysis were in general agreement with the timecourse experiments. Among 38 LacZ+ clones derived from the wild-type strain, 31 (82%) were deletions and seven (18%)were gene convertants. For 39 lacZ+clones derived from the rad57 mutant, these figures were 38 (97%) and one (3%), respectively. All 20 LacZ+ clones analyzed for the rad51 mutant were deletions. Thus the proportion of gene convertants was greatly affected by rad51, rad54, rad55, and rad57 mutations within direct repeats of lacZ genes. This result was rather unexpected because previously we showed that rad51, rad54, rad55, rad57 mutants are proficient in HO-induced geneconversion between two MAT genes held on a plasmid (SUGAWARA et al. 1995). To test whether theabsence of gene conversion in these rad mutants was an intrinsic property of lad-containing plasmids, we used plasmid pJF5 in which the two lacZ genes are in inverted rather than in direct configuration. As shown in Figure 2b, an HO-induced DSBis repaired in this system exclusively by gene conversion events half of whichare expectedto be accompanied by crossing-over (RUDINet al. 1989). The gene conversion events accompanied by crossing-over can be detected by Southern blotanalysis of DNA prepared from galactose-
-
I . . " I . I
-. . "-
.,
I
- . .-. -. - -
F l c ; t ' w J."Southcrn 1)lot ;In;ll!sis of the kinetics o f dclcrion formation i n Ivilcl-type and n I I t t ; 1 1 1 1 stlxins. Expression o l ' thc MO cndonuclcasc \ \ x initiated hy t h r addition o f ga1;~tosra t t i n l r 0 . A t the time points intlicatctl. s ; ~ ~ n p lol'cclls cs ~ v c r ct;tkct~, ;Ind D N A \vas p r q m r c d and tligcstctl w i t h A / I . The DNA li-agnlcnts \vert srparatetl o n ncrltl-;d 0.8% ;~garoscgels ;Ind p r o l x d w i t h ;I /w%-spc*cilic prol,c ;IS tlcscrihrtl in JI:YII;.KL\I.S .\SI) \ I ~ : I I I O I ) SPositions . o f ' thc 1l.l-kl1 duplication I'I-;Ignwnt(dl), 8 . 7 and 2.441) C I I I 1'I;Ignwnts (el], and the i.0-kh tlclction protlIlct (tlcl) arc intlicatrtl. Thc o t h e r prod~tcto f ~~cconIl~in;ltio~l. ;I gc~lc convcl-sion cvent. c;Innot he srcn on these Sorlthcrn I,lots tlrlc t o ;I small tliffcrcnrc i n sizes o l ' the gene convcv.sion and the p;Ircnral IxInds. )\ DX)\ I x I n d a t the position o f 3.4 kt) seen on sonic ol' t h r blots is probably due. t o nonspccilic hyhidimtion I~cr;~usr it iss(*c11 i n nonintlucctl cc*lls. (a) \viltl typv; (1)) r d 5 1 A ; ( e ) rcrd52A; ((1) t d 5 4 A ; ( e ) r d 5 0 A : (I) st:t2A.
induced colls containing pJF.5. The results of'this analysis are shown in Figure 6. I n the wild-typc strain (Figure ha), l m d s chanlctcristic o f ' the crossover product started t o appear a s early a s 30-60 m i n after induction o f a DSB. I n m / 5 1 (Figure (ib). r d 5 - l (Figure 6tl) and r(rd55and rod57 (data n o t shown) strains, theformation of recombination product was indisting~~ishal,Ic fi-om that o f ' wild type both i n kinetics and i n thc fil1iil amount. RccomI,in;ltion was still alxrnt in the t d 5 2 strain (Figure 6c). All ofthc ahovc-lnentioIlcd experiments were carried out a t 30". Howc~cr,i t \vas sho\vn that i n t d 5 5 and ,nd57 mutants defects i n DNA repair and reconllination are exaggerated by inculx~tiona t a lower tclnpcx1ture ( L o \ w - r and M ~ K I I \ I I : . I1987; < H;\\:c; r / cd. 199.5; R,YI-IXAY and S Y S I I S C ~ 1995). I ~ O S A s the salnc r d 5 5 iI11tl md57 mutant strains show complete tleficicnc\fi n MAT switching when tested a t 30" (SL.(~~\\V;\R.\ r / n / . 1993), we f e l t i t unlikely t h a t the proficicncy o f the r d 5 5 ;Ind r d 5 7 mutants i n /ur% recombination was c l w t o the higher tcmpcl-attlrc. Howcvcr, to r d e o u t this possilility completcly, w e cat-rietl o u t H O induction on plates containing galactosc a t 23" or 30" iltltl then plasmid loss
w a s estirnatctl. N o incl-casc i n loss o f either pJF6or pJF3 a t 2:3" \vas s w n i n t h r r d 5 5 and m d 5 i mutants, tior i n thc wild-type, t d 5 1 or r d 5 4 mutants (data n o t s h o w ) . I n summaly, 1LW51, 1bW54, 1&\1)55, m d I M D 5 i genes arc n o t required for DSB 1-epairby t h e SSA mech-
anism when duplicated /rrr%gencsarc i n direct orientation. The very same genes arc not rc!quiretl Ihr HOintltlcetl gene conveI-sion bctwrcn / r r % sequences in inverted orientation. However, i n thecorresponding mutants, gene conversion is sevcrc.ly I-educed whcn the recombining /ur% sequences are i n direct orientation and SSA is ;I competing proccw. Analysis of DSB repair by the SSA mechanism on a chromosome: Prcxviously, we have shown that rud51. r d 5 4 , r d 5 5 , and r d 5 7 mutants :~reable t o p e r f o m AIA'I's\vitching 0 1 1 a plasmid, btlt not on the chromosome (SL,(;.\\\,.\K\ P/ {//. 1995). Thus we tlecitlrd t o test whether these genes arc reqrlircd for DSB repair by the SSA mechanism when rcwmhining sequences constitutc ;I part of a chromosome. A dil-ect tlllplication o f /rr%genes used hefore was integrated intochromosome l f a s tlescribetl i n \IATIN..\I.S A N I ) \ ~ ~ r l I o [ )Repair s. of' HO-induced DSBs was analyzrd for ;I wild-type strain
a
1
0
3
2
I
5
4
6
Time after HO induction, h
b
1 40
E-u E
30
2 r;
20 10 0
and three mdmutants:md51, r(m52, and rc1d54.In these strains, the chromosomal MA'l'cu-int locus is not cleaved by HO endonuclease s o that the only intact HO cut site in the genome is located in one of the duplicated lor% genes. We first analyzed the efrects of a shortinduction when the cdtures were incubated in YEPGal medium for 1 hr. The percentage of LacZ' cells after induction in the strains carrying the integration construction was significantly lower (45-64%) than i n strainscarrying Irc% genes on a plasmid (Table 2). We attributed the lower appearance o f L a d ' cells to an inefficient cutting o f HO cut site in this particular position and to the formation of competingdeletions between flanking zrm3-52 and LU?A?sequences ( R L ~ I PtS nl. 1989). Thus, we employed HO induction by growing the strains on YEPGal plates. Table 3 summarizes the results of these experiments. Sunrival of wild-type cells was not affected, butthesunival of md52 cells decreased -200-fold. There w a s some drop in viability o f md51 and rc1d54 mutant strains, which is probably due t o the cutting at silent H M loci under conditions of continuous expression of HO ( C O N N ~ I . Pt I Scrl. 1988). However, the levels of LacZ' were very high and equal for the wild-type, rod51 and rr(d54 strains demonstrating successful completion of DSR repair in all three strains. The kinetics of DSR repair were monitored at the DNA level. Data presented in Figure 7B and Table 3 show that among three rod mutanb tested, only the md52 mutant displayed a dramatic reduction in deletion formation. The md51 and rod54 mutants were not only proficient in deletion formation, but in fact demonstrated even higher than wild-type levels of the recombination product.
4
Pe
8 genc conversion-
U a
-
b +deletion
k
b
c
d
e
h
+gene conversion
F I G ~ I R5.-Sortthern E blot analysis of thc formation of deletion and gene conversion products o f HO-intluced recombin;ltion in wild-type and mutant strains. Samples of cclls werc taken 6 hr after expression of HO. DNA was prepared anddigested with H ~ ~ ~ ~ l I l l / ~ ' Thc ~ ~ l /DNA ~~~~~aI. fragments were separated o n neutral 0.3% agarose gels and probed with a lac%-sprcificprobc. Positions o f the :i.X-kb deletion product arc indicated. Also indicated are positions o f the 4.3-kb genc conversion product seen in wildtype (a), mr15OA (g), and xrs2A (11) strains; this product cannot be scen i n md51A ( b ) , rnd52A ( c ) ,r ~ d 5 4 A(d), rd55A ( e ) , and r(rd57A (f) strains. The two other fragments represent parental hands. The cut fragments created by HO cle>1\.21geof the 4.4-kh Hi?~dlIlS n d fragment cannot he scen on this blot.
E. L,. Ivanov
700 ""
00.5 1 1 . 5 2 3 4 5 6
0 0 . 5 1 1.5 2 3 4 5 6
d"--
n/.
Hours
Hnurs "
P/
Pf
" .
efco
L
I
~
4-co +co Pf cf
cf
v .-
b. rad5lA
a. wild type
Hours 0 0.5 1 1.5 2 3 4 5 6
Hours 0 0.5 I 1.5 2 3 4 5 6
~-
cf
1
'
I
I
-
Pf
4- co
4- co
g-co co
Pf
p('
cf
cf
FIGI'RF.6."Southern blot analysis of the kinetics of HO-inducedrecombination in wildtype and mutant strains using plasmid pJF.5 as ;I substrate. The timecourse experiment w a s carried out and the DNA was prepared andanalyzed a s described in the legend t o Figure 3. Positions of 7.4- and 6.0-kb parental fragments (pl), 4.4and 3.0-kb cut fragment., ( c Q , and i.O- and 6.5kb crossing-over products (co) arc shown. The i.Rkb hand corresponding to gene conversion withoutcrossingovercannotbe resolved on these Southern blots. A DNA ktnd at the position of -3.4 kb is probably due to nonspecific hybridization. (a) wild type; (h) rrrd5lA; (c) md52A; ( d ) md54A.
cf
cf
d. rad54A
c. rad52A
M'e then wanted to know whether the distribution of recombinationproducts(deletions vs. gene conversion) resembled that for plasmid substrate. Analysis of thc structure of individual LacZ' colonies showed that 37 of 37 clones derived from md51 strain and 40 of 40 clones drrivrd from mI54 strain were deletions; no genc conwrtants were found among these two sets of I x % + colonies. Among 40 LncZ' clones derived from the wild-typc strain, five (13%) were gene convertants and 33 (87%) were deletions, which is close to the distri-
TABLE 3 HO-induced recombination within direct lacZ duplication integrated into chromosome V
butionseenforthe plasmid substrate. We conclnde from these data thatas for theplasmid pJF6 recombinationsubstrate, RAD51 and RAD54 genes are not required to carry out SSA on a chromosome, but in the corresponding mutants, HO-induced gene conversion behveendirectlyrepeated k c % genes is severely repressed or being prevailed by SSA. Mutations in RAD50 and XRS2 genesdecreasethe efficiency of SSA on a plasmid: Although RAD50 and XRS2 genes belong to the I W I 5 2 epistasis group, these two genes have some properties making them very different from other members of this group [for detailed discussion of this point, see IVANO\' et nl. (1994)]. Although md50A and m 2 A deletion alleles do not prevent the completion of MATswitching, the kinetics of this process in mutant cells are markedly retarded. Molecular analysis of this phenomenon demonstrated that the slower kinetics of MAT switching in md50A and xrs2A mutants is at least partly due to the decreased 5' to 3' degradation of the ends of HO-induced DSBs (SLIGIWARA and HARER 1992; IVANOY P/ (11. 1994). As the SSA process is critically dependent on5' to 3' degradation of DSB ends (Figure 1b), we analyzed the effects of rnd50A and m.s2A mutations on the efficiency and kinetics of DSB repair in the pJF6 plasmid system. Data in Table 2 show that HO-induced plasmid loss was significantly increased in rad50 and xn2 mutants, implying that repair of HO-inducedDSBs was affected. However, the fraction of L a d ' cells after induction is not significantly lower in the mutant strains compared
70 1
Control of Single-Strand Annealing
A
HOcs
ura3-52
LEU2
uRA3
I
I
” LACZ LACZ
P ura3-52
P
-
.
P
uRA3
LEU2
Deletion
I
LA CZ
P
P
ura3-52
F.-
LEU2
uRA3
”
Gene conversion
LACZ
LACZ
P
P
B Hours 016
Hours
7 :: @
Hours 0 16
0 1 6
-
Hours 016
y7”-
nrr?lnm:
C-u df
del
cf
cf del
df cf del
cf
cf
~r*y df I
.=“
cf del
FIGURE 7.-Repair of an HO-induced DSB within the direct duplication of larZ genes integrated into the ura3-52 locus on chromosome V. (A) schematic representation of the structures of integration and its recomhinational derivatives. The positions of two relevant Pstl sites (P) are indicated. The sizes of parental and HO-induced AtI-fragments hybridizing to the 1arZ prohe nsed are the same as for the plasmid pJFG suhstrate (Figure 2a). (B) Southern blot analysis of deletion formation in wild-type and mutant strains. The timecourse experiment was carried out and the DNA was prepared and analyzed as described in the legend to Figure 3, except that only three timepoints (0, 1 and G hr) were analyzed. Positions of the 11.1-kh duplication fragment (do, 8.7- and 2.4-kh cut fragment5 (cf), and the 7.0-kh deletionproduct (del) are indicated. Theother product of recombination, a gene conversion event, cannot he seen on these Southern blots (a) wild type; (h) md5ZA; (c) md52A; (d) rud54A.
cf a. Wild type
b. radSlA
c. radS2A
d. mdS4A
with the wild-type and proficient rad mutants indicating successful completion of recombination events. One explanation for this apparent discrepancy could be that rad50 and xr.32 mutants do not prevent but markedly retard recombinational repair of HO-induced DSBs between direct repeats of l a d genes, as was shown to be the case for MATswitching (IVANOV d al. 1994). If so, a significant fraction of DSBcontaining plasmids would remain unrepaired at the next round of DNA replication leading to increased plasmid loss. At the same time among recovered plasmids, completion of recombination should have the high proportion ofLacZfcells seen in wild-tvpe cells. Analysis ofthe repairevents at theDNA level supports the obsewation that the efficiency ofDSB repair within the 1acZ duplication was substantially reduced in rad50 (Figure Se) and m32 (Figure Sf) mutants. By 6 hr after induction, only 40-45% of the wild-type level of deletion production was seen in the mutant strains (Figure 8a). Characteristically, theamount of persistent cut fragment was dramatically increased in mutant cells (Figure 8b). Disappearance of the cut fragments reflects the degradation of DSB ends. Thusaccumulation of this intermediate in mutant cells support5 the idea
that the extent of 5’ to 3’ degradation of DSBsis decreased in rad50 and ms2 cells. We then wanted to know whether both deletion and gene conversion formation were affected to the same extent by rad50 and ms2 mutations. Data presented in Figure 5, g and h, respectively, and in Table 2 show that the efficiency of gene conversion was decreased two- to threefold in rad50 and ms2 strains compared with wild type, according to a densitometric analysis of Southern blot data. To exclude the possibility that there was a bias in survival of LacZ’ colonies bearing a deletion or a gene conversion plasmid derivative, we analyzed individual LacZf clones derived from the u s 2 strain. For 20 clones analyzed, two (10%) were gene convertants and 18 (90%) were deletions, which is close to the results derived from timecourse experiments. We conclude thatrad5OA and xrs2A deletion mutations decreased HO-induced recombination in directly repeated lac% genes, both deletion and gene conversion events being affected approximately to the same extent. DISCUSSION
Genes belonging to the epistasis group RAD52 share many common effects on DNA repair and recombina-
702
E. L. Ivanov rt nl.
a 40 1
0
1
2
3
4
5
6
Time after HO induction, h
b 60
10 "T
0
1
2
3
4
5
6
Time after HO induction, h
.,
FIGURE 8.-Densitometric analysis of Southern blot data on the formation of deletions in rad50A (Figure 3e) and xrs2A (Figure 30 strains. The analysis was carried out as described in the legend to Figure 4. The data for the wild-type strain are the same as in Figure 4. wild type; 0, rad50A; , xrs2A. The datafor the rnd50A and xrs2A strains are from four independent experiments for each strain.
+
tion [for review, see PETESet al. (1991)l. Moreover, genetic and biochemical data suggest that the corresponding proteins interact and even form a multiprotein complex (SHINOHARA et al. 1992; MILNE and WEAVER 1993; HAYSet al. 1995). However, in last few years, it has become evident that different genes play different roles in DNA metabolism. For example, null mutations in RAD52 cause more severe defects in spontaneous mitotic recombination than do null mutations in RALl51, RAD54, RAD55, and RAD57 (RATTRAY and SYMINCTON 1994, 1995). In the case of HO-induced DSBs, the situation is even more complicated. Mutations in each of these five genes completely block, at some early step, MATswitching on a chromosome,however, only RAD52 is required for this gene conversion event when the donor sequence is simultaneously not silenced and located on a plasmid (SU(;AWARA et al. 1995). In this communication, we extend this finding to show that only the RAD52 gene is required for gene conversion on a plasmid containing an inverted repeat of lacZ genes whereas the RAD51, RAD54, RAD55 and RALl57genes are not (Figure 6 ) . An attractive interpretation of these data is that only the RAD52 gene is required for recombinationper se, whereas the otherRAD genes are only needed to gain access to otherwise inaccessible regions of chromatin (SUCAWARA et nl. 1995). Despite the finding that theRad51 protein has a strandexchange activity (SUNG1994), our data suggest that Rad51 mayplay adifferent role in vivo. One would
expect more generaldefects in recombination in rad51 mutants if this strand-exchange activity were the major cellular one. The main result of this work is that KAL)51, RALl54, W 5 5 ,and RAD57genes are also not required for HOinduced DSB repair by the SSA mechanism. SSA, either on a plasmid substrate (Figures 3 and 4 and Table 2) or on a chromosome (Figure 7 and Table 3 ) , is not affected by null mutations in these genes. It is important to note that SSA is greatly repressed, both on a plasmid and ona chromosome,by a rad52 mutation demonstrating again the difference between RAD52 and other genes of the RAD52 epistasis group with respect to HOinduced recombination. The absence of requirementsfor RAD51, W 5 4 , W 5 5 , and RAD57genes in the SSA pathway is complicated, however, by the negative effects ofcorresponding mutations on the gene conversion process in the same direct-duplication system. For the chromosomal substrate these effects could be explained by the requirements for these KAL, genes to promote recombination in the context of chromatin (SLJGAWARA et nl. 1995). At the same time, it is difficult to understand why gene conversion is repressed on plasmid DNA (Figure 5 and Table 2) given the essentially normal levelsof gene conversion between lac%sequences in inverted orientation. We explain this apparent contradiction in the fold lowing way. As was shown before (FISHMAN-LOBELL al. 1992),in the pJF6 plasmid system,deletion and gene conversion events represent two alternative, competing and independently modulated outcomes of repair of an HO-induced DSB. Factors decreasing the efficiency of deletion formation (for example, increasing the distance between two recombiningsequences) increase the fraction of gene conversion; conversely, factors preventing gene conversion increase the proportion of deet al. 1992). We propose that letions (FISHMAN-LOBELL in rad51, rad54, rad55, and rad57mutant cells, the gene conversion process on a plasmid has a modest defect. This defect cannot be seen when the recombining sequences are in inverted orientation (as in the case of plasmid pJF5) because gene conversion is the only way to repair an HO-induced DSB. However, in the direct repeat configuration,this defect is apparent, dueto the presence of an alternative, highly efficient SSA pathway that competeswith gene conversion. In agreement with this view are results obtained with plasmid pNR43 that contains a4.4kb insert between the direct repeats leading to a higher proportion of gene conversion in wildel al. 1992). In a rad51 mutype cells (FISHMAN-LOBELL. tant cells,thisalso leads to the appearance of some gene conversion product, albeit with slowerkinetics and at a lower level compared with wild type ( J . FISHMANLOBELL, unpublished results). Alternatively, one can imagine that gene conversion in ,rad51, rad54, md55, and md57cells is unaffected but instead the efficiency of the SSA process is increased
703
Control of Single-Strand Annealing
due, for example, to higher levels of single-stranded DNA formation as seen in the case of MAT switching (SUGAWARA et al. 1995). This idea, however, does not find supportwhen analyzing the kinetics of DSB repair on plasmid DNA. Indeed, if in these rad cellsDSB ends were degraded and processed into the nextrecombinational intermediate faster than in wild-type strains, this would cause an apparentdecrease in the amountof cut fragments on Southern blots (Figure 3 ) . However, a densitometric analysis ofthe Southernblot data (Figure 4) does not suggest that the stability of DSB ends is changed in these mutants. In any case, our results show that inrad51,rad54, rad55, and rad57 mutants DSB-induced direct-repeat recombinationproceedsalmost exclusivelyvia the SSA mechanism. This could serve as a basis to explain increased deletionformation seen in these mutants duringspontaneous mitotic recombination involving directly repeated sequences (FANand KLEIN 1994; MG DONALDand ROTHSTEIN 1994; AGUILERA 1995; LIEF% HITZ et al. 1995). In this communication, we report thatnull mutations in RAD50 and XRS2 genes affect the efficiency of SSA leading to increased plasmid loss. Our results confirm our previous observations that rud50A and xrs2A mutations decrease the extent of 5’ to 3’ degradation of the ends of HO-induced DSBs (SUGAWARA and HABER 1992; IVANOV et al. 1994). The observed decrease in the efficiency of deletion formation, however, is only two- to threefold (Table 2). This could explain why during spontaneousmitoticdirect-repeatrecombination, rad50 mutants show little or no effect (MCDONALD and ROTHSTEIN 1994). The effectsof rad50 and xrs2 mutations on MAT switching suggest that the rate of 5’ to 3‘ degradation of DSB ends is a rate-limiting factor for this specialized gene conversion event (IVANOV et al. 1994). In lac2 direct-duplication substrates, rad50and xrs2mutations decrease deletion and gene conversion events to approximately the same extent (Figure 5 and Table 2). This argues that degradation of HO-induced DSB ends is a rate-limiting factor for geneconversion not only during MAT switching but in other recombination systems as well. So far, only a handful of genes are known to significantly affect the SSA process. These include theRAD52 (FISHMAN-LOBELL et ul. 1992; SUGAWARA and HABER 1992), RADl, and RAD10 (FISHMAN-LOBELL and HABER 1992; IVANOV and HABER 1995) genes. Recently, a mutation in the CDCl gene was described that most likely affects the SSA pathway of HO-induced intrachromosomal recombination (HALBROOK and HOEKSTRA 1994). At the same time, many genes known to be essential for “classical” homologous recombination were shown here not to be required for SSA. This means that the genetic requirements of SSA are different from those of gene conversion. Thus additional genes controlling
SSA remain to be discovered. To this point, we have recently undertaken agenetic screen to isolate mutants defective in SSA. Among the mutations isolated, some are neither UV or MMS sensitive, making them different from mutations in the above-mentioned genes (S. SATO,A. MALKOVA and J. E. WER, unpublished results). We hope thatcharacterization of these mutations will allow us to identify new genes responsible for SSA in yeast and to use them as probes to detect their human counterparts. We are grateful to A. MALKOVA for continuous discussion of this work. This work was supported by National Institutes of Health Grant GM-20056 to J.E.H.
LITERATURE CITED ABOUSSEKHRA,A., R. CHANET,A.ADJIRI and F. FABRE, 1992 Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiaemap in theRAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol. Cell. Biol. 12: 3224-3234. A,, 1995Geneticevidence for different RAD52depenAGUILERA, dent intrachromosomal recombination pathways in Saccharomyces cerevisiae. Curr. Genet. 27: 298-305. ALANI, E., S. SUBBIAH and N. KLECKNER, 1989 The yeast RAD50 gene encodes a predicted 153kd protein containing a purine nucleotide binding domain and two large heptad-repeat regions. Genetics 122: 47-57. CAo,L., E. AIANI and N. KLECXNER, 1990 A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae. Cell 61: 1089-1101. CHURCH,G. M. and W. GILBERT,1984 Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991-1995. CONNOLLY, B., C. I. WHITEand J. E. HABER,1988 Physical monitoring of mating type switching in Saccharomyces cereuisiae. Mol. Cell. Biol. 8: 2342-2349. FAN,H., and H. L. KLEIN, 1994 Characterization of mutations that suppress the temperature-sensitive growth of the h p 1 D mutant of Saccharomyces cermisiae. Genetics 137: 945-956. A. P., and B. VOGELSTEIN, 1984 A technique for radiolaFEINBERG, beling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266-267. FIRMENICH, A. A,, M. ELW%ARNANZand P. BERG,1995 A novel allele of Saccharomyces cerevisiaeREA1 that is deficient in recombination and repair andsuppressible by RAD52 Mol. Cell. Biol. 1 5 16201631. 1992 Removal of nonhomoloFISHMAN-LOBEIL, J., and J. E. HABER, gous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene R A D I . Science 258: 480-484. FISHMAN-LOBEIL, J., N. RUDINandJ. E. HABER,1992 Two alternative pathways of double-strand break repair that are kinetically separable and independently modulated. Mol. Cell. Biol. 12: 12921303. HALBROOK, J., and M. F. HOEKSTRA, 1994 Mutations in the Saccharomyces cereuisiae CDCl gene affect double-strand-break-induced intrachromosomal recombination. Mol. Cell. Biol. 14: 8037-8050. HAYS, S. L., A. FIRMENICH and P. BERG,1995 Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc. Natl. Acad. Sci. USA 92: 69256929. ITO, H.,Y. FUKADA, K. MURATAand A. KIMURA,1983 Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168. IVANOV, E. L., and J. E. HABER,1995 RADl and RADIO, but not other excision repair genes, arerequiredfor double-strand break-inducedrecombination in Saccharomycescereuisiae. Mol. Cell. Biol. 15: 2245-2251. IVANOV, E. L., N. SUGAWARA, C. I. WHITE,F. FABRE and J. E. HABER, 1994 Mutations in XRSL, and RAD50 delay but do not prevent mating-type switching in Saccharomyces cmeuisiae. Mol. Cell. Biol. 1 4 3414-3425.
704
E. L. Ivanov et al.
H. L., 1995 Genetic control of intrachromosomal recombination. BioEssays 17: 147-159. LIEFSHITZ, B., A. P..UU(ET, R. MAYA and M. KUPIEC,1995 The role of DNA repair genes in recombination between repeated sequences in yeast. Genetics 140: 1199-1211. LIN, F.-L., K. SPERLEand N. STERNBERG, 1984 Model forhomologous recombination during transfer of DNA into mouse L cells: role for DNA ends in therecombination process. Mol. Cell. Biol. 4: 1020-1034. LIN,F.-L., K.SPERI.E and N. STERNBERG, 1990 Intramolecular recombination between DNAs introduced into mouse L cells by a nonconcervative pathway that leads to crossover products. Mol. Cell. Biol. 10: 103-112. Lovt-rr, S. T., and R. K. MORTIMER, 1987 Characterization of null mutants of the RAD55 gene of Saccharomyces cereuisim effects of temperature, osmotic strength and mating type. Genetics 116: 547-553. MCDONALD, J. P., and R. ROTHSTEIN,1994 Unrepaired hetercduplex DNA in Saccharomyces cereuisiae is decreased in R A D 1 RAD52-independent recombination. Genetics 137: 393-405. MII.NE,G. T., and D. T. WEAVER, 1993 Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52. Genes Dev. 7: 1755-1765. B., and G. S. ROEDER,1991 A unique pathway of douOZENBERGER, ble-strand break repair operates in tandemly repeated genes. Mol. Cell. Biol. 11: 1222-1231. PETES,T. D., R.E. MAI.ONE and L. S. SYMINGTON, 1991 Recombination in yeast, pp. 407-521 in The Molecular and Cellular Biology of the Yeast Saccharomyres, edited by J. BROACH, J. PRINGI.Eand E. JONES. Cold Spring HarborLaboratory, Cold Spring Harbor, NY. RhTrwx, A. J., and L. S. SYMINGTON, 1994 Use of a chromosomal inverted repeat to demonstrate that theRAD51 and RAD52genes of Saccharomyres cermisiae have different roles in mitotic recombination. Genetics 138: 587-595. RATTRAY, A. J., and L. S. SYMINCXON, 1995 Multiple pathways for homologous recombination in Saccharomyces cereuisiae. Genetics 139: 45-56. ROTHSTEIN, R., 1983 One step gene disruption in yeast. Methods Enzymol. 101: 202-211. RUBY,S. W., J. W. SXOSTAK and A. W. MURRAY, 1983 Cloning regulated yeast genes from a pool of ZacZ fusions. Methods Enzymol. 101: 253-269. KLTIIN, N., and J . E. HABER,1988 Efficient repair of HO-induced chromosomal breaks in Saccharomyces cereuisiaeby recombination KLEIN,
between flankinghomologoussequences. Mol. Cell. Biol. 8: 3918-3928. RUDIN,N., E. SUGARMANand J. E. WER, 1989 Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cereuisiae. Genetics 122: 519-534. SANDELL, L. L., and V. A. ZAIUAN, 1993 Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75: 729-739. SCHIESTL, R. H.,and S. PRAKASH, 1988 R A D I , an excision repair gene of Saccharomyces cereuisiae, is also involved in recombination. Mol. Cell. Biol. 8: 3619-3626. 1990 RADIO, an excision repair SCHIESTL, R. H., and S. PRAKASH, gene of Saccharomyces cereuisiae, is involved in the RAD1 pathway of mitotic recombination. Mol. Cell. Biol. 10: 2485-2491. SCHII.D,D., I. L. CALDERON, C. R. CONTOPOUI.OU and R. K MORTIMER, 1983Cloning of yeast recombinationrepairgenes and evidence that several are nonessentialgenes,pp. 417-427 in Cellular Responces to DNA Damage, edited by E. C. FRIEDBERG and B. A. BRIDGES. Alan R. Liss, New York. SHERMAN, F., G. R. FINK^^^ J. B. HICKS,1986 Methods zn Yeast &etics:Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. SHINOHARA, A,, H. OGAWA and T. OGAWA,1992 Rad51 protein involved in repair and recombination in S. cereuisiae is a RecA-like protein. Cell 6 9 457-470. SUGAWARA, N., and J. E. HABER,1992 Characterization of doublestrand break induced recombination: homology requirements and single-stranded DNA formation. Mol. Cell. Biol. 12: 563575. N., E. L. IVANOV, J. FISHMAN-LOBELL, B. L. RAY, X. Wu et SUGAWARA, al., 1995 DNA structuredependent requirements foryeast RAD genes in gene conversion. Nature 373: 84-86. 1991 Extensive 3"overhangSUN, H.,D. TRECOand J. W. SZOSTAK, ing, single-strandedDNA associated with meiosis-specificdoublestrand breaks at the ARM recombination initiation site. Cell 6 4 1155-1161. SUNG,P., 1994 Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265: 1241-1243. SZOSTAK, J. W., T. L. ORR-WEAVER, R. J. ROTHSTEIN and F. W. STAHI., 1983 The double-strand-break repair model for recombination. Cell 33: 25-35. WHITE,C. I., and J. E. HABER,1990 Intermediates of recombination during mating type switching in Saccharomycescereuisiae. EMBO J. 9: 663-673. Communicating editor: S. JINK~ROBERTSON
