A pathway for generation and processing of double-strand breaks during meiotic recombination in S. cerevisiae

Cell, Vol. 61, 1069-l

101, June

15, 1990, Copyright

0 1990 by Cell Press

A Pathway for Generation and Processing of Double-Strand Breaks during Meiotic Recombination in S. cerevisiae Liang Cao, Eric Alani, and Nancy Kleckner Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138

Summary We have identified and analyzed a meiotic reciprocal recombination hot spot in S. cerevisiae. We find that double-strand breaks occur at two specific sites associated with the hot spot and that occurrence of these breaks depends upon meiotic recombination functions RADSO and SPO77. Furthermore, these breaks occur in a processed form in wild-type cells and in a discrete, unprocessed form in certain nonnull fad50 mutants, rad50S, which block meiotic prophase at an intermediate stage. The breaks observed in wild-type cells are similar to those identified independently at another recombination hot spot, A RG4. We show here that the breaks at ARG4 also occur in discrete form in rad5OS mutants. Occurrence of breaks in rad5OS mutants is also dependent upon SPO77 function. These observations provide additional evidence that doublestrand breaks are a prominent feature of meiotic recombination in yeast. More importantly, these observations begin to define a pathway for the physical changes in DNA that lead to recombination and to define the roles of meiotic recombination functions in that pathway. Introduction Recombination is a prominent and essential feature of meiosis in most eukaryotic organisms. It ensures proper segregation of chromosomes at Meiosis I by providing a physical connection between homologs (Hawley, 1989). Also, it results in exchange and reassortment of information along the chromosomes. In Saccharomyces cerevisiae, meiotic recombination has long been the object of intensive investigation. A great deal of discussion has centered around the possibility that recombination initiates by a double-strand break (Resnick, 1976; Szostak et al., 1983; Thaler and Stahl, 1988). This view is supported by the fact that a common group of genes is required both for repair of double-strand breaks in vegetative cells and for recombination during meiosis; members of this group include RADW, -57, -52, -54, -55, and -57 (Resnick, 1987) and MRE77 (M. Ajimura and H. Ogawa, unpublished data). In addition, it is clear that double-strand breaks generated artificially during meiosis by HO endonuclease can stimulate recombination (Kolodkin et al., 1986). Genetic analysis of recombination does not distinguish unambiguously among possible recombination models. However, there is increasing enthusiasm for a model in which recombination initiates by

a double-strand break that is followed by resection of one strand at each end (Petes et al., 1990; Thaler and Stahl, 1988; Resnick, 1976). Finally, meiosis-specific doublestrand breaks have been observed by physical methods in three recent, independent investigations (Sun et al., 1989; Game et al., 1989; this work, see Discussion). Analysis of recombination in S. cerevisiae has also included the identification of a number of genes required for this process. Among the functions that play a role in both DNA repair and meiotic recombination, RADSO is required at a very early step in recombination, and MRE77 is probably very similar to RADSO in this regard; the other RAD genes in this group are required at later stages (Malone and Esposito, 1981; Resnick et al., 1986; Resnick, 1987). A number of additional recombination functions have been identified that are specific to meiosis. This latter class includes several genes that are similar to RADSO in that they are absolutely required for exchange (SPO77, MEl4, and MREI-4; Wagstaff et al., 1985; Klapholz et al., 1985; Menees and Roeder, 1989; M. Ajimura and H. Ogawa, unpublished data), at least one gene that is important but not absolutely essential for all types of recombination (MER7; Engebrecht and Roeder, 1989) and two genes in which mutations differentially affect different types of meiotic recombination (HOP7 and RED7; Hollingsworth and Byers, 1989; B. Rockmill and G. S. Roeder, personal communication). We describe here the identification of a strong hot spot for meiotic reciprocal recombination in S. cerevisiae. This hot spot was created by insertion of a 2.8 kb LEUP segment adjacent to the HIS4 locus in strain SKl. We describe the occurrence of meiotically induced doublestrand breaks at this site and the dependence of these breaks on certain meiotic recombination functions. Null mutations in the RADSO and SPO77 genes, which confer complete, early blocks in meiotic recombination, block the formation of breaks. In addition, certain nonnull rad50 mutations that block meiotic prophase at an intermediate stage (Alani et al., 1990) cause breaks to accumulate in a slightly different form whose properties suggest that it is a precursor to the type of break observed in wild-type. We also show that at the endogenous ARG4 locus, a hot spot for initiation of meiotic gene conversion shown recently to exhibit double-strand breaks (Sun et al., 1989) breaks exhibit the same unique functional dependence as breaks at the H&4-LEU2 hot spot. The results presented provide additional evidence that double-strand breaks play a direct role in meiotic recombination in S. cerevisiae. More importantly, they begin to define a pathway for the physical changes leading to recombination and to define the roles of meiotic recombination functions in that pathway. In this pathway, discrete double-strand breaks occur at specific sites, are rapidly processed to a nondiscrete form, and then are further processed, ultimately into mature recombination molecules. Temporal analysis of meiosis also raises the possibility that the early steps of recombination, i.e., formation

Cell 1090

STRAIN

A 4.5

I

I

Table 1. Effect of LEU2 Segment Flanking his4 and URA3 Markers Tetrad Dissection

kb

Number

URAJ X

his4.X

1

X I

Tetrad

HIS4

kb

PD T NPD GC

Probe 2 STRAIN

B I

I

1.4

11.8

I

Distance

4.5 c

URA3

LECJZ

kb 17.7 11.8 16.3 13.2

1.4

16 kb

-59

* ,I’+hi&X

B LEUZ

x x* O-5

I-WA hi&E

X I

B ’ B i LEU2

X kb

probe 2

Figure

1. Strains

Used

for Physical

and Genetic

Analysis

The structure of the 40 kb centromere-proximal region on the left arm of Chromosome Ill is diagrammed. Strains A and E: On the top chromosome of both strains, the his4-X allele was created by filling in the indicated Xhol restriction site within the HIS4 coding region (“), and the URA3 segment was inserted at a Xhol site downstream of the his4-X allele, eliminating that site. In Strain 9, both chromosomes carry a 2.8 kb LEUZ segment (hatched box) inserted immediately downstream of the HIS4 region. Arrows indicate the direction of transcription of the HIS4 and LEU.2 genes. The source of the LEUP coding sequence was YEP19 which contains a Pstl LEUP fragment cloned from strain X2180-la (Ratzkin and Carbon, 1977); the 2.6 kb insert is a Bglll subfragment of the Pstl fragment with a unique internal Xhol site “filled in”. The final configuration of.Xhol sites (X) is shown, as are the fragments resulting from Xhol digestion of parental (Par.) and recombinant (Rec.) chromosomes. Strain NKY641: This strain is essentially the same as Strain 6 except that it lacks the URA3 insertion on the top chromosome. The final configuration of Xhol (X) and Bglll (6) sites is shown, as are the fragments resulting from a Xhol + Bglll double digest. All three strains are heterothallic derivatives of SK1 that carry the following additional markers: ura3, lys2, ho::LYS2, and leu2::hisG. The leu2::hisG disruption lies midway between HIS4 and the centromere on Chromosome III (data not shown). Strain constructions are described in Experimental Procedures.

of breaks, are contemporaneous with chromosome pairing. The implications normal meiotic recombination hot spot a molecular construct are considered.

of Tetrads

A Strains

(4 Isolates)

B Strains

1 17 1 0 1

3 5 3 0 2

1 9 18 0 1

between

2 18 0 0 0

4 16 2 0 1

Total 66 5 0 4

2 15 12 0 1

(4 Isolates) 3 13 15 0 0

4 12 10 0 0

Total 49 55 0 2

his4X and URA3

kb

Genetic Physical kis4-X

NKY641

Type

on Recombination between as Determined by

the early steps of of the fact that a can be created by

3.5 CM 9.0 kb

26.4 CM 11.8 kb

Several independently constructed isolates of Strains A and B (Figure 1) were sporulated (Experimental Procedures). More than 90% of cells formed tetrad% and about 90% of the tetrads produced four viable spore colonies after dissection on YPD plates. The phenotypes of colonies from such tetrads were determined by replica plating onto selective plates lacking histidine, leucine, or uracil. Tetrads were catagorized with respect to their His and Ura phenotypes as parental ditypes (PD), tetratypes (T), nonparental ditypes (NPD), or gene conversions (GC). CG tetrads are those in which one of the two markers exhibited 3:l or 4:O segregation. The particular conversion events observed are listed in Experimental Procedures. Genetic distance (CM) was calculated according to the formula of Perkins (1949): 50 x [(T + GNPD)/(T + PD + NPD)]. GC tetrads were excluded from this calculation. The genetic distance observed in Strain B is significantly different from those observed in Strains A: by Fisher exact test, the probability of results being the same is <10e8. All spore clones of Strain B were Leu+, indicating that the leu2::hisG allele is not efficiently transferred into the LEU2 segment. In Strain B, reciprocal recombination exhibits positive chiasma interference. Based on the proportion of tetratypes observed, about 6 NPD tetrads (diagnostic of four strand double cross-avers) should have been seen (Papazian, 1952). In fact, none were observed. The probability of obtaining zero NPDs in the absence of interference as assessed by Chi square analysis is 0.005

Results Genetic Identification of a Meiotic Recombination Hot Spot

Reciprocal

A strong hot spot for meiotic reciprocal exchange is created by insertion of a 2.8 kb segment containing the LElJ2 region of strain X2180-la at a site adjacent to the HIS4 locus of the rapidly sporulating strain SK1 (Figure 1). In the absence of the insert, recombination between an appropriate pair of markers (his4-X and URA3) occurs at approximately the frequency expected for typical intervals in S. cerevisiae (1 CM per 3 kb; Strathern et al., 1979; Olson et al., 1988); the markers are separated by a genetic distance of 3.5 CM and a physical distance of 9 kb (Strain A: Figure 1, Table 1). An isogenic strain carrying the LEUP insert exhibits a 6-fold higher level of recombination between these markers, 26.4 CM (Strain B: Figure 1, Table 1). Thus, the presence of an additional 2.8 kb between the markers increased the frequency of recombination by 23 CM rather than the typical 1 CM.

Physical 1091

Strain

Pathway

for Yeast

A

Figure 2. The LEUP Insert binants

Meiotic

Ra

Increases

Recombination

B

Rb

the Level of Physical

Recom-

The appearance of physically recombinant DNA molecules during meiosis was monitored in Strains A and B in which parental and recombinant chromosomes give rise to Xhol restriction fragments of distinguishable lengths (Figure 1). Relatively synchronous meiotic cultures of the two strains were sampled at the indicated times after transfer to sporulation medium. DNA was extracted, digested with Xhol, run on a 0.6% agarose gel, and probed with HIS4 sequences (Probe 2, Figure 1). For each strain, the positions of recombinant fragments are indicated by analysis of control DNAs. Ra+ and Ra- for Strain A and Rb+ and Rb- for Strain B are DNAs extracted from His+ Ura+ and HisUra- reciprocal recombinants obtained from a single tetratype tetrad of each strain. The parental bands in Strain B are each about 2.8 kb larger than the corresponding bands in Strain A as expected from the presence of the LEUZ insert. At 6 hr, recombinant fragments in Strain B represent 21% of total DNA as determined by densitometer tracings of this autoradiogram; recombinants in Strain A represent 3% of total DNA. At 7 hr. the proportions of cells that had completed Meiosis I were 60% (Strain A) and 68% (Strain B). Analysis of similar cultures suggests that bulk DNA replication is essentially complete by 3 hr (e.g.. Figure 4).

Recombination at the Hot Spot Is Qualitatively and Temporally Normal Recombination in the presence of the insert exhibits two properties that are characteristic of meiotic recombination, reciprocality and interference. Reciprocality is indicated by the observation that 55 of 57 recombination tetrads from Strain 6 contain two parental and two reciprocally recombinant spores (tetratypes); only two arose via gene conversion (Table 1). interference is indicated by the observation that despite the high level of reciprocal exchange, none of the 104 tetrads examined exhibited a double recombination event involving both pairs of chromosomes (no NPD tetrads). In the absence of interference, about 6 NPD tetrads would have been expected (Table 1, legend). The presence of the LEU2 insert results in a high level of physically recombinant DNA molecules, commensurate with the high frequency of recombinants detected genetically. In both Strains A and B, exchange between the his4-X and MA3 markers generates diagnostic Xhol fragments that are cleanly separable from the two parental Xhol fragments (Figure 1). The proportion of DNA present in recombination fragments at the end of meiosis is m20% and 3% in Stains B and A, respectively, very similar to the percentages of recombinant chromosomes observed genetically (26% and 40/o, respectively [from Table 11).

Figure

3. Mapping

of Meiotic

Signal

Break

Sites

A map of relevant restriction sites in the vicinity of the H/S4-LEU2 region and the positions of the two double-strand break sates are shown. Distances are assigned relative to the Xhol site at the upstream end of HIS4 Three probes used for mapping fragments in Southern blots are indicated. Each horizontal line represents a wild-type meiotic sig nal identified by digesting NK641 (RADSO) DNA with a particular enzyme and hybridizing with an appropriate overlapping probe. Lines are drawn in such a way that one end lies at the appropriate restriction site and the other end lies at the distance from that site which corresponds to the average mobility of the signal fragment as given in Table 2. The two vertical broken lines ending in arrows indicate the locations of the discrete break sites observed in DNA from a rad5OS version of NKY641, positions 4.5 and 6.8, respectively (mappmg in Table 2 and text). Site I is more prominent than Site II. The maximal levels of breaks at Sites I and II relative to total parental DNA are 6% and l%, respectively, in wild-type, and 12% and 3% in rad5OS. The two ends of the Site I break in fad5OSK181 were mapped precisely as follows. For the downstream end, DNA was digested with Pstl and hybridized with Probe 1, and the length of the signal fragment was 3.9 kb and was 400 bp longer than a parental Pstl-Sal1 fragment run in an adjacent lane. For the upstream end, digestion with Kpnl and hybridization with Probe 4 revealed a 1.7 kb meiotic signal fragment that was 200 bp shorter than the parental Kpnl-Hindlll fragment and 400 bp longer than the parental Kpnl-Sal1 fragment.

Furthermore, in cultures undergoing relatively synchronous meiosis, the recombinant fragments appear at the same time in both Strains A and B and at approximately the stage expected from other analysis (Borts et al., 1984; Game et al., 1989). In both strains, recombinant fragments reach a maximum level relatively late in meiotic prophase, 5-6 hr after initiation of sporulation, 2 hr after DNA replication, and shortly before Meiosis I (Figures 2 and 4). Low levels of recombinant products are observed prior to 5 hr as a result of asynchrony in the culture (L. C. and Ft. Padmore, unpublished data). Meiosis-Specific Double-Strand Breaks Occur at Two Specific Sites Associated with the LEUP Insert Strains carrying the LEUP insert adjacent to HIS4 exhibit meiosis-specific double-strand breaks at two sites. Site I is located at the junction between the insert and HIS4distal sequences; Site II is located within the insert, about 500 bp from the junction with HIS4, and is less prominent than Site I (Figure 3). These breaks were identified and their endpoints were mapped by analysis of DNA extracted from sporulating cultures of a strain very similar to

Table 2. Size of Meiotic Signal (kb) upon Restriction of NKY641 DNA as Detected by Southern Blot

T 80 a, 3

60

Upstream

E

40

Site1

RAD50

rad50.S

Site I

RADBO

rad5OS

Pstl (-2.1) Xhol (0.0) Banll (0.5) Xhol (1.4) Kpnl (5.1)

8.7 6.5 6.2 5.1 -

8.9 6.8 6.4 5.4 1.7

Pstl (10.7) Xhol (13.4) BamHl (13.6)

3.7 6.4 6.7

3.9 6.7 6.9

5.9 8.8 9.0

6.1 9.0 9.2

* Recomb. (His+) n Meiosis I

$? 20 0 HS

0123456

Probe

Downstream

Digestion

Probe

7.6kb Site II cS.lkb

Pstl (-2.1) signal

3.0kb

cl :

Figure 4. Identification Course

:, . .

of a Meiotic

.6kb Recombinant

Signal

in a Sporulation

Time

Samples were taken from a sporulating culture of NKY641. Top: DNA replication (Rep.), commitment to recombination (His+), and occurrence of Meiosis I were analyzed. Replication was monitored by FACS analysis after staining of DNA with propidium iodide. Cells were sorted according to their levels of fluorescence, the distribution of cells having different fluorescence levels displayed. At time 0, the majority of cells were in a single peak (two copies of each chromosome, 2N); by 4 hr, more than 80% of cells were in a second peak of greater fluorescence (4N). The parameter plotted is the fraction of cells at each time point present at the position of the 4N peak. This parameter represents the fraction of cells that has completed bulk meiotic DNA replication. Commitment to recombination reflects the ability of cells to give His+ recombinants upon return-to-growth medium (Sherman and Roman, 1963; Esposito and Esposito, 1974) as determined by sampling cells at the indicated times and plating diluted aliquots of cells on plates lacking histidine; the final level of recombinants at the end of meiosis was about 8 x 10m3. Occurrence of the two meiotic divisions was determined by examining fixed, DAPI-stained cells in the fluorescence microscope and counting the proportion of cells containing one, two, or four staining bodies. The parameter plotted is the percentage of cells that has completed Meiosis I; many of these cells have also completed Meiosis II. Bottom: DNA was extracted, digested with Bglll and Xhol, and analyzed as in Figure 2 except that the blot was probed with sequences from between the his4 heteroalleles (Probe 2 of Figure 1). The 1.6 kb Xhol-Bglll fragment, diagnostic of recombination between the heteroalleles (Figure l), is faintly visible at both the 5 and 6 hr time points. The origin of the 5.1 kb meiotic signal observed in this experiment iS described in detail above (Table 2 and Figure 3).

B (NKY641; Figure 1). The same breaks are also observed in Strain B (data not shown). When DNA samples are appropriately digested and analyzed, nonparental fragments are observed that do not correspond to any predicted recombinant fragments. Furthermore, these fragments have two distinctive properties: they are diffuse and they are present only transiently during meiosis. The occurrence of one diffuse band is compared with other important meiotic events in Figure 4. In this culture, the 5.1 kb transient band is first detected at about 3 hr, just after DNA replication and “commitment” of cells to meiotic

Strain

Site II 6.3

6.6

Pstl (10.7) Xhol (13.4) BamHl (13.6)

DNA samples from NKY641 (RAD50) or NKY974 (NKY641 rad5OS K/87) were analyzed. The sizes of meiotic signals identified with various combinations of restriction enzymes, and probes are listed. Signals are grouped according to whether they were observed with a probe to sequences located upstream or downstream of the LEU2 insert and according to whether they correspond to breaks at Site I or Site II. The name of each restriction enzyme is given together with the location of the relevant site. The probes were: Probe 1 (downstream) and Probe 2 (upstream) except for the Kpnl digest of rad5OS DNA, which was analyzed with Probe 4. Break sites, restriction sites, and probes are shown in Figure 3. -, not examined.

recombination (see legend to Figure 4). The band disappears by 6 hr, concomitant with the appearance of fragments diagnostic of recombination. In the experiment presented, a faint 1.6 kb band corresponding to the product of recombination between two his4 markers is present at 5 and 6 hr; these recombinants probably occur by gene conversion (Fogel and Hurst, 1967). However, reciprocal recombination products also appear at about this same time, as shown by the experiment in Figure 2 and additional higher resolution temporal analysis to be presented elsewhere (L. C., Ft. Padmore, and N. K., unpublished data). Meiosis I follows the appearance of recombinant products. In the discussion below, we will refer to the transient diffuse bands as meiotic signals. The nature of the meiotic signals was further investigated by digesting meiotic DNA samples with a variety of restriction enzymes and probing with sequences located either upstream (HI%-proximal) or downstream (H/S4-distal) of the LEUP insert. This analysis demonstrates that each signal fragment corresponds to a segment extending from one of the two double-strand break sites to the next adjacent restriction site. The digests performed and fragments identified are summarized in Figure 3 and Table 2. Mapping of the two break sites from the downstream side is shown for three different restriction sites in the three panels of Figure 5 (0, 3, and 7 hr FfAD50 lanes of each panel). Each digest reveals two different meiotic signals, a shorter more prominent signal (solid line) and a longer less prominent signal (dotted line). Each signal is a diffuse band that is most prominent in the 3 hr sample and is absent or faint in the 0 and 7 hr samples. The observed length heterogeneity of any given signal is equivalent to about 200 bp of double-stranded DNA. In each

Physical 1093

Pathway

Hrs

for Yeast

Meiotic

Recombination

03733

03733

03733

kb I 23.1

6.6 Site

”[ 4.4

site

[ Psti

Figure

rad508,

’

5. Comparison of Meiotic and rad5OS Strains

Xhol Signals

’

BamHl

at Sites I and II in RAD5Os,

Metotic DNA samples were obtained from sporulating cultures of NKY641 (RAOSO; Figure 1) and two isogenic derivatives, NKY591 (rad5OA) and NKY974 (rad5OSKl87). DNAs were digested with Pstl, Xhol. or BamHl and hybridized with (downstream) Probe 1 of Figure 3. The meiotic signals corresponding to Sites I and II are indicated with solid and dotted lines, respectively. The sizes of all meiotic signals observed are listed in Table 2. Wild-type signals are most prominent at 3 hr and are diffuse, with a band width corresponding to about 200-300 bp. rad5OS singles are discrete and are the equivalent of about 200 bp larger than the corresponding wild-type signal. In rad5OA, no wild-type signal is observed (see also Figure 6). Wild-type and rad5OA samples also contain a faint meiotically induced band that migrates close to the position of the rad5OS signal, also seen in Figure 6; the relationship of this band to other signals is not yet established. Sands migrating more slowly than Site II signals have not been characterized. Among them, the most prominent bands in wild-type samples, which appear at 7 hr in meiosis, might reflect the reciprocal recombinant between the two LEUP loci on chromosome Ill (see legend to Figure 1). These fragments represent about 2% of the total DNA, about one-tenth the level of normal interchromosomal recombinants (Figure 2).

case, the average apparent length of the signal species corresponds to the distance from the relevant downstream restriction site to one of the two break sites. In the experiment presented, shorter signals map to Site I and longer signals map to Site II (Table 2 and Figure 3). The same two break sites are identified by mapping from the upstream side (Table 2 and Figure 3). At each of the two break sites, the meiotic signal endpoints all fall within a 200-300 bp region. Furthermore, there is a gap between the apparent endpoints of the upstream and downstream signals at each site; this feature is discussed further below. The level of breaks at either Site I or Site II is always rather low, but Site I breaks are always more prominent than Site II breaks (Figure 5 and data not shown). The maximal level of signal observed at

Site I at any particular time point is usually about 6% of the total DNA while that at Site II is about 1% (as determined by densitometric analysis of Figure 5). We assume that the observed double-strand breaks present in the extracted DNA are also present in vivo. Breaks are present even when DNA is extracted very rapidly; normal meiotic signals are observed when cells are converted into spheroplasts prior to initiation of sporulation, carried through meiosis without cell walls, rapidly pelleted, and lysed immediately by the addition of 0.3% SDS, 10 mM EDTA, and proteinase K at 65°C (L. C. and Ft. Padmore, unpublished data). However, it is difficult to exclude rigorously the possibility that the breaks result from extraction-induced disruption of important proteinDNA complexes. Sequence Requirements for Breaks at Sites I and Ii The occurrence of breaks at both Sites I and II appears to be the consequence of specific genetic determinants in or around each site. The appearance of meiotic signals is specific to the H/S4-LElJ2 construct. No comparable signals are detectable at the HIS4 locus in a wild-type SK1 strain (NKY278; data not shown). Because Site I maps to the junction between the insert and adjacent sequences (see also below), the occurrence of a signal at that site probably reflects some feature specific to the new construct. In contrast, Site II maps internal to the LEU2 segment, about 400 bp from the junction with HIS4. The occurrence of breaks at this site probably reflects the existence of specific determinants intrinsic to that region. Restriction analysis suggests that SK1 is completely different from X2180-la in the region upstream of the LEU2 coding region, which includes this site (M. Lichten, personal communication), and no breaks are observed at the SKI LEU2 locus. More precise mapping of breakpoints (below) suggests that Site II is probably near or within a delta sequence known to be present at this location in X2180-la but not in SK1 (Andreadis et al., 1982). Occurrence of breaks only requires that the LEUP insert be present on one of the two chromosomes. In a strain isogenie to Strains A and B but heterozygous for the insert, a signal was observed only from the chromosome carrying the insert, and the level of that signal was comparable to that observed for the same chromosome in Strain B (data not shown). Also, it seems unlikely that the occurrence of breaks is influenced significantly by the existence of a second (disrupted) copy of the LEU2 locus on Chromosome III in the strains analyzed (Figure 1). Genetic analysis did not reveal any frequent transfer of information from the disrupted endogenous LEU2 locus into the LEU2 insert adjacent to HIS4 (Table 1, legend), and DNA fragments diagnostic of intrachromosomal reciprocal exchange between the two loci are rare (see legend to Figure 5). lntrachromosomal interactions are clearly not required for generation of analogous breaks at ARG4 (Sun et al., 1989, and below). Formation of the Meiotic Signal Requires Meiotic Recombination Functions Formation of meiotic signals depends upon meiotic re-

Cell

rad5OA (del) Strain RADSO (wt) Hrs 0 2 3 4 5 61 0 2 3 4 6 6

kb l 23 ,

Figure

6. The Meiotic

Signal Is Dependent

upon RAD50

Closely related RAD50 and rad50A stains carrying the LEUP insert (NKY641, Figure 1, and NKY591) were sporulated and DNA samples were taken at the indicated times, extracted, and digested exactly as in Figure 4 (digestion with Xhol and Bglll; hybridization with the upstream Probe 2 of Figure 1). The 5.1 kb meiotic signal corresponding to Site I is apparent in the RADBO strain and absent in the rad5OA strain.

combination functions RADSO and SPOll. The RADSO and SPOll genes are absolutely required for both meiotic recombination and chromosome synapsis; a null mutation in either of these genes completely abolishes both processes (Game et al., 1980; Malone and Esposito, 1981; Alani et al., 1990; Giroux et al., 1989; Klapholz et al., 1985; Farnet et al., 1988; B. Byers, unpublished data). Presumably as a consequence of these defects, mutations in either of these genes result in aberrant chromosome segregation at Meiosis I. Neither mutation grossly alters other meiotic events, such as DNA replication or the occurrence of the two meiotic divisions. In fact, the meiotic inviability conferred by each of these mutations is relieved by the presence of an additional mutation that causes cells to bypass Meiosis I (~~073; Malone and Esposito, 1981; Klapholz et al., 1985; Klapholz and Esposito, 1980). A null mutation in either RADSO or SPO77 completely eliminates formation of meiotic signals. Figure 6 compares meiotic time courses of NKY641 (RADSO) and an derivative. Digestion of RADSO DNA with isogenic fad508 Xhol and analysis with an upstream probe reveals the 5.1 kb band characteristic of breaks at Site I. This 5.1 kb band is absent in DNA of the fad50 deletion mutant; the same result is obtained for an isogenic spo77A strain (NKY677; data not shown). Comparison of RAD50 and rad5OA DNAs with a downstream probe confirms the absence of meiotic signals at both Site I and Site II (Figure 5). Discrete Meiotic Breaks Are Observed in Certain Nonnull rad50 Mutants (rad5OS) The nature of meiotic double-strand breaks is altered by certain nonnull mutations in the RADSO gene, rad5OS. These mutations cause an intermediate block in meiotic chromosome metabolism (Alani et al., 1990). rad5OS mutants undergo DNA replication but undergo little or no commitment to meiotic recombination. In addition, they display a number of defects indicative of partial progres-

sion through meiotic prophase, the time when recombination and chromosome synapsis occur. Most notably, rad5OS mutants display an “asynaptic” phenotype: axial chromosome cores, structures that normally become the lateral elements of the synaptonemal complex, form but fail to pair. As a result of the various rad5OS defects, Meiosis II is delayed, spores form at very low levels, and the few spores formed are inviable. Moreover, the nature of the intermediate block is such that the failure of rad5OS mutants to form viable spores is not relieved by the spo73 mutant (above). mutation, in contrast to the fad5OA Analysis of meiotic DNA extracted from rad5OS mutants reveals that double-strand breaks occur at the same two sites and with the same relative abundance as observed in wild-type. However, the breaks are qualitatively different in a number of interesting ways, which suggests that they are precursors to the breaks observed in wild-type. First, the rad50S signals are meiotically induced at the same time as wild-type signals but are stable rather than transient. In the experiment shown in Figure 7, DNA samples were digested with Xhol and probed with upstream sequences (Probe 2, Figure 3). This protocol identifies individually the signals generated by each of the two parental chromosomes, which have different arrays of Xhol sites (as in NKY641, Figure 1). Two prominent bands are observed that correspond to signal breaks at Site I on each of the two chromosomes; breaks at Site II are also observed in the same DNA under appropriate gel electrophoresis conditions (data not shown). Both signals are first detected at 3 hr, increase in prominence for the next 2 hr, and remain at maximal level for the duration of the experiment. At 7 hr, the signal is still maximal, although more than 70% of cells have completed Meiosis I (see the legend to Figure 7). Additional features of rad5OS breaks are illustrated in and Figure 5, in which DNA samples from RAD50, rad50A, rad50S strains are compared directly using a downstream probe (Probe 1, Figure 3). First, the signal bands observed in rad5OS DNA are much sharper than those observed in wild-type DNA. Second, all of the mutant signals are not only much more discrete than the corresponding RAD50 signals, they are also of higher molecular weight (see also Table 2). The difference in apparent mobility between the rad5OS and average wild-type signal species is the equivalent of about 200 bp of double-stranded DNA. Importantly, the same mobility difference between wild-type and rad5OS signal fragments is also observed for signals identified with an upstream probe (Table 2). Third, the level of signal is higher in the rad5OS mutant than in wild-type: the rad5OS signals at Sites I and II represent 12% and 3% of total DNA, respectively, while the corresponding wild-type signals represent 6% and 1%. A similar estimate of the level of Site I rad50S signals is provided by Figure 7 (11%). The discrete nature of rad50S signals permits more accurate mapping of break points. Within the resolution of our analysis (250 bp), the two DNA ends generated in rad5OS at break Site I map to the same position, which is exactly the junction between the LEU2 segment and adjacent downstream sequences (Figure 3). These features are suggested by mapping data in Table 2 but are demon-

Physical 1095

Pathway

for Yeast

Strain Hrs

Figure 7. Identification in rad50S-K/81

Meiotic

Recombination

raLi5OS 0

1 2

3 4

of Discrete,

5

6 7

Meiotically

size (kb)

Induced,

Strains: LCYl%

(rad5OS-spoil)

Hrs

3

0

2

4

5

LCY198 60

2

(rad5OS-SPOII) 3

4

5

$ 6s

Stable Breaks

DNA was analyzed from a strain similar to NKY641 (Figure 1) but carrying the rad50SK187 allele (LCYl98). Samples were taken from a sporulating culture at the indicated times, and the DNA was extracted, digested with Xhol. and examined by hybridization with Probe 2 of Figures 1 and 3. The two parental chromosomes differ at the his4-Xsite (‘), and thus, the two parental bands differ in length by 1.4 kb (Figure 1). The two meiotic signals observed represent segments extending from each of these heteroallelic Xhol sites to the major break site (Site I) on the two chromosomes and thus also differ by 1.4 kb (6.8 and 5.4 kb). The meiotic signal from the minor break site (Site II) is too small to be seen on this gel; however, it has been observed in other mapping experiments using the same DNA samples (Table 2). These rad50S signals are first detectable at 3 hr and increase in intensity throughout at least the first 6 hr. The progress of meiosis in this culture was comparable to that in previous experiments; at 7 hr, more than 70% of cells had undergone Meiosis I. Densitometer tracing indicates that the amount of DNA in the two meiotic signals at 7 hr is about 11% of the total DNA at Site I. A slight decrease in the absolute level of signal at the latest time point in this experiment and in Figures 8 and 9 reflects decreases in the total amount of DNA in these samples as shown by densitometer analysis. At later points, cells become somewhat refractory to DNA isolation for reasons that we do not understand.

strated most precisely by mapping of break sites from both the upstream and downstream sides relative to two nearby flanking restriction sites, Sal1 and Hindlll (see the legend to Figure 3). The Site II break in ra&OS lies within the LEUP segment, -500 bp from the junction with upstream sequences (Figure 3) as determined from the distance between Sites I and II (2.3 kb k 100 bp, from Figure 5):Both upstream and downstream signal endpoints map to exactly the same location at this site as well (Table 2). Comparison of wild-type and rad5OS signals demonstrates unambiguously the presence of a gap between the apparent endpoints of the upstream and downstream wild-type signals at both Sites I and II as suggested above. The two ends at each rad5OS break map to the same location, and every wild-type signal, both upstream and downrad5OS stream, migrates faster than the corresponding signal by an amount corresponding to about 200 bp of double-stranded DNA. Thus, the length of the apparent gap corresponds to about 400 bp of double-stranded DNA. However, the precise structure of the wild-type signal ends, and hence the nature of this gap remains to be determined (see Discussion).

kb 423.1

-23

Figure

8. Discrete

Meiotic

Signals

in radSOS-K/81

Require

SPO77

lsogenic rad5OS strains similar to NKY641 (Figure 1) with and without a spoil disruption mutation (LCY196 and LCY198) were analyzed for the occurrence of meiotic signals at Sites I and II. DNA was digested with Pstl and hybridized by Probe 1 (Figure 3). In the SPO77 strain, the two most prominent bands correspond to breaks at Site I(3.8 kb) and Site II (6.2 kb); neither signal is observed in the spo77 strain. The nature of faint lower mobility bands has not been investigated. Genetically, the so077 mutation is epistatic to rad5OS for its meiotic defects. In a spo73 background where Meiosis I is bypassed, the ~7~077 mutation rescues both the lack of spore formation and the spore inviability characteristic of rad5OS mutants; in a rad5OS spo77 spo73 triple mutant, >80% of cells form spores, 80% of which are viable (Experimental Procedures; see also Alani et al., 1990).

We show elsewhere that the occurrence of discrete, stable breaks is a feature of a second rad5OS allele in addition to that examined here and of a different type of rad50 nonnull mutant, which also confers an intermediate block in meiotic prophase (Alani et al., 1990). Discrete Signals in rad5OS Mutants Require SPO77 A null mutation in the SPO77 gene spo77A eliminates the meiotic lethality of rad5OS mutations in a spo73 genetic background, presumably by blocking prophase chromosome metabolism at a very early stage prior to the rad5OS block (Figure 8, legend; Alani et al., 1990). Such a mutation also drastically reduces the formation of discrete breaks in rad5OS mutants. Fragments of 6.2 kb and 3.8 kb, corresponding to downstream fragments generated by breaks at Sites I and II, are prominent in rad5OS DNA digested with Pstl, while no such fragments are detectable at comparable levels in DNA from an isogenic rad5OS spo77A strain (Figure 8). However, upon longer exposure, faint bands corresponding to both breaks are visible at much reduced levels, suggesting that breaks do occur at a low level even in the complete absence of SPO77 function. Observation of Site-Specific Breaks at ARG4 While this work was in progress, Sun et al. (1989) reported the identification of meiosis-specific double-strand breaks at specific sites in theARG4 gene of S. cerevisiae. The oc-

Cdl 1096

Strains: LCY196

(rad5OS.spoil)

HIS

3

Figure

0

2

4

9. Meiotic-Specific

5

LCY198 60

Breaks

2

at ARG4

(radsOS-SPOll) 3

4

5

6

-

6.6 kb

‘I

4.5 kb

-

1.7 kb

Locus

The DNA samples used for the experiment shown on Figure 8 were digested with Bglll and probed with an ARG4 probe (for details, see Sun et al., 1989, and Experimental Procedures). Two meiotic-specific chromosomal breaks are observed at positions similar to those by Sun et al. (1989). The breaks at ARG4 exhibit the same timing, discreteness, and functional dependence on rad5OS and SPOli as breaks at the HIS4::LEUZ locus.

currence of polarized gene conversion at the ARG4 locus suggests that it is a naturally occurring hot spot for initiation of meiotic gene conversion (Nicolas et al., 1989; Fogel et al., 1981). Using our SKl-derived strain NKY278, Sun et al. observed breaks both in non-SK1 ARG4 sequences carried on a plasmid and at equivalent positions in the endogenous SK1 chromosomal ARG4 locus. The properties of theARG4 breaks suggest that they are equivalent to the breaks we observe at the HIS4-LEU2 locus in wild-type strains: they exhibit a similar temporal dependence and have heterogeneous ends. We therefore analyzed the ARG4 locus in a rad5OS mutant for the occurrence of breaks. We find that breaks occur at theARG4 locus at the same two positions reported by Sun et al. Furthermore, these breaks have exactly the same unique properties as those observed at the HIS4-LEU2 locus: they appear at the appropriate time in meiotic prophase, are discrete rather than heterogeneous, are stable and persist throughnull mutation out meiosis, and are abolished by the spoil (Figure 9). These results confirm and extend those of Sun et al. and provide strong support for the view that breaks at both loci are fundamentally similar.

Discussion Evidence That Double-Strand Breaks Are a Normal Feature of Meiotic Recombination in S. cerevisiae We observed site-specific double-strand breaks at a meiotic recombination hot spot created by inserting a 2.8 kb LEUP segment from strain X2180-la downstream of HIS4 in strain SKl. The occurrence of breaks is directly

implicated in stimulation of recombination, because no breaks are detectable at the HIS4 locus in the absence of the insert. We also showed that the occurrence of breaks depends upon meiotic recombination functions and that the effects of specific mutations on the breaks are consistent with the known genetic properties of those mutations. Breaks are abolished by rad50A and spo77A mutations, which are known to block recombination at a very early stage. Furthermore, in fad50S mutants, which are known to block meiotic prophase at an intermediate stage, the breaks exist in what appears to be a discrete precursor form (see below), and the occurrence of these precursor breaks is also dependent upon the SPO77 gene. Furthermore, we showed that breaks at the H/S4-LEU2 hot spot in wild-type strains exhibit kinetics of appearance and disappearance during meiosis expected for a recombination intermediate. They appear subsequent to DNA replication, at about the time that cells become committed to undergoing meiotic recombination, and they disappear prior to the appearance of mature recombinants. Finally, results presented here and by Sun et al. (1989) demonstrate that identical breaks occur at the endogenous SK1 ARG4 locus and at the same sites in the nonSK1 ARG4 locus characterized genetically as a hot spot for initiation of meiotic gene conversion. Thus, in two independent cases, there is a strong correlation between the detection of site-specific double-strand breaks and the existence of a meiotic recombination hot spot. The failure to observe breaks at other loci in SK1 is attributable to the sensitivity of the detection method. Breaks at ARG4 and within the LEU2 segment at Site II are only barely detectable in wild-type cells; breaks occurring at less favored sites would not be seen. Taken together, these observations provide additional evidence that double-strand breaks are a general feature of meiotic recombination in S. cerevisiae. An alternative interpretation, that the identification of site-specific breaks at these two sites is fortuitous and represents some atypical pathway for meiotic recombination, seems much less likely. While this possibility cannot be excluded, there is no reason to suppose that fortuitous recombination should exhibit interference, which is a hallmark of meiotic exchange, or should depend upon meiosis-specific recombination functions such as SPO77. The general occurrence of double-strand breaks during meiosis is supported by another recent physical study of SK1 DNA, published while this paper was in preparation. OFAGE gel analysis of an SK1 derivative carrying a circular Chromosome Ill revealed the appearance of linearized chromosomes with the same temporal and functional dependence observed for site-specific breaks (Game et al., 1989). Breaks were observed in this analysis at the low level expected from the transient nature and low steadystate level of breaks described here. In older studies that examined bulk SK1 DNA, breaks were detected in one study (Hogset and Oyen, 1984) but not in another (Resnick et al., 1984) presumably due to the difficulty of detecting rare, transient species.

Physical 1097

Pathway

for Yeast

Meiotic

Recombination

Occurrence of Discrete Breaks in radLOS Mutants Five observations suggest that the breaks observed in far&OS are precursors to the breaks observed in wild-type strains: First, the mutant breaks map to the same two sites and occur with the same relative abundance at the two sites in both mutant and wild-type strains. Second, the ends of wild-type breaks appear to be processed versions of the ends observed in the mutants. The mutant breaks have discrete ends that map to exactly the same position (250 bp); in contrast, wild-type breaks have heterogeneous ends that map 200-300 bp to either side of the mutant break site, leaving an apparent “gap” at the break site. The gap could reflect the absence of sequences from one or both strands at each end. Third, the mutant breaks are meiotically induced and appear at the same time in meiosis as wild-type breaks but are stable rather than transient. They accumulate during the period of time when the meiotic signal is normally present and reach a maximum level at about the time when reciprocal recombination products are normally observed. Fourth, the discrete breaks are observed specifically in mutants where meiotic prophase chromosome metabolism is blocked at an intermediate stage (Alani et al., 1990). And finally, occurrence of the mutant breaks in rad5OS mutants depends upon, and is thus downstream of, the early meiotic recombination function SPO77. Proposed Pathway for Meiotic Recombination The results presented above lead us to propose a simple pathway of physical events leading to meiotic recombination and to define the dependence of each step on meiotic recombination functions (Figure 10). The first step in the pathway is the occurrence of a discrete site-specific break of the type observed in rad5OS mutants. These breaks might be generated by a site-specific nuclease, by a topoisomerase, or by a general nuclease in response to specific changes in DNA or “chromatin” structure. This step requires both RADSO and SPO77 functions; it seems likely that other functions absolutely required for recombination (see Introduction) will also block at this step. The second step in the pathway is the symmetrical processing of those breaks to the heterogeneous form observed in wild-type. Whether resection occurs on one or both strands at each broken end remains to be determined. Wild-type breaks at ARG4 have substantial singlestranded tails (Sun et al., 1989). This second step probably follows immediately after the first, since discrete breaks are absent or rare in wild-type cells. RADSO function must act a second time to permit processing of the breaks; it may act simply to release the broken ends or may play some more active role. The breaks that accumulate in rad5OS mutants may be protected from cellular nucleases in some type of protein-DNA complex. Alternatively, the breaks may be stable because RADSO is a nuclease directly responsible for their processing. In subsequent steps, heterogeneous breaks are converted to mature recombination products, presumably via additional, as yet unidentified intermediates. The proposed pathway does not specify the relationship

v RA 050 SPOli

: v

site discrete

specific breaks

rad5OS

block

4 processed breaks

.. .

. ..=

v v v mature Figure nation

recombinants

10. Proposed

Steps in a Pathway

Leading

to Meiotic

Recombi-

of the observed events to other steps in recombination. It is attractive to suppose that discrete breaks are a relatively early event in recombination. This feature is supported by the time of appearance of the breaks (immediately after DNA replication), by their discrete nature (which would not necessarily be expected if they resulted from disruption of intermediates that had undergone strand invasion and extension), and by the fact that rad5OS mutants make such breaks but are completely defective in meiotic recombination, including gene conversion events that could still arise by abortive interruption of recombination even if reciprocal exchange were excluded (discussed in Carpenter, 1987). However, other alternatives are not excluded (Hastings, 1988; Borts and Haber, 1987). An important question for the future is whether the occurrence of breaks precedes or follows the interaction of two homologous molecules. Breaks occur at normal levels in strains heterozygous for the LEU2 insert, and we argued above that the presence of a second copy of LElJ2 on Chromosome III is not relevant. However, these results do not exclude recognition of homology prior to breakage, because interaction between homologous sister chromatids is still possible in all of these situations. It is interesting that the transient double-strand breaks occur during the period between DNA replication and formation of mature recombinants. It is generally assumed that recombination occurs at about the time that synaptonemal complexes are present. Thus, these results raise the possibility that double-strand breaks are concurrent with earlier steps in chromosome pairing and synapsis. Such a temporal relationship is consistent with the possibility that recombination and chromosome synapsis share common early steps (Alani et al., 1990). Creation of a Meiotic Recombination Hot Spot: Implications Most of the increase in meiotic recombination resulting from the H/S4-LEU2 insert can probably be attributed to the creation of a favored site for double-strand breaks at the junction between LEUP and adjacent H/S4-distal sequences (Site I). The extent of this increase, while signifi-

Cell 1098

cant, is within the range of variation observed in “natural” regions of the yeast genome. The insert results in a B-fold increase in recombination in the interval examined, and variations of &15-fold have been reported for other intervals of similar size (Symington and Petes, 1988; Petes et al., 1990; Kaback et al., 1989). Furthermore, in the presence of the insert, the ratio of genetic to physical distance is only about g-fold higher than the average ratio observed for all of Chromosome Ill and the rest of yeast, about 0.35 CM per kb (Strathern et al., 1979; Olson et al., 1986); this is about the same as the level observed at a naturally occurring hot spot on Chromosome I (1.5 CM per kb; Coleman et al., 1986; Kaback et al., 1989). It is impressive that recombination stimulated by this construct is qualitatively normal despite the artificial origin of the hot spot. Recombination under the influence of this site is temporally appropriate, depends upon appropriate recombination functions, is reciprocal, and exhibits chiasma interference. This latter feature is in sharp contrast to recombination stimulated artificially by HOmediated breaks during meiosis, which does not exhibit interference (Kolodkin et al., 1986). These observations suggest that initiation of meiotic recombination may not require a highly specific genetically determined machinery designed to select particular sites but rather that recombination can potentially occur at a very large number of places in the yeast genome, wherever local features of the DNA and/or chromatin are appropriate. A currently popular idea is that patterns of transcription and/or local supercoiling might be important (Liu and Wang, 1987; Fink, 1989; Christman et al., 1988; Voelkel-Meiman et al., 1987; Wallis et al., 1989). Experimental

Procedures

Strains All of the strains used in this study are isogenic derivatives of SK1 (Kane and Roth, 1974; Williamson et al., 1983) and are shown in Table 3. Genetic markers were introduced by gene transplacement (lithium acetate transformation; lto et al., 1983), and all integrations were confirmed by Southern blot analysis (Southern, 1975; Maniatis et al., 1982).

Table

3. Experimental

Strains

All of the SKI strains described in this study were derived from NKY278 (W. Raymond and L. C., unpublished data). The LEU2 gene in NKY278 was disrupted by gene transplacement methods that were described previously (Alani et al., 1987). In /eu2strains, the LEUP gene is interrupted by a 1.1 kb insertion of Salmonella hisG DNA. The hisG DNA was inserted at the EcoRl restriction site of LEUS, which is located in the middle of the LEU2 coding region. The LEUP sequences used for gene disruption are contained on a 2.8 kb Bglll fragment derived from YEP13 (strain X2180-la; refer to Sherman et al., 1983, for plasmid map). The rad5OA, spo73, and spo77A mutations were introduced into SK1 strains by the same disruption scheme used to disrupt LEU2 (Alani et al., 1987). The spol7A plasmid (pGB518) was kindly provided by Craig Giroux. The rad50A and spollh disruption plasmids result in a complete coding region deletion of each gene (Alani et al., 1989, 1990; C. Giroux, personal communication). Strain LCY185 and derivatives were constructed to examine the meiotic viability of a homozygous rad5OS spoil spol3 triple mutant. This strain displays mitotic phenotypes that are characteristic of rad5OS strains: normal vegetative growth and slight sensitivity to MMS. In meiosis, the LCY185 strain is indistinguishable from spoil spold strains. In LCY185, the meiotic divisions are not delayed as they are in rad5OS spo73 strains (Alani et al., 1990), and 80% of the LCY185 cells form dyads, the majority of which are viable (83 out of 80). Media Yeast was grown vegetatively in YPD (1% yeast extract, 2% BactoPeptone, 2% glucose), YPA (1% yeast extract, 2% Bacto-Peptone, 2% acetate), and SD (0.84% yeast nitrogen base without amino acids, 2% glucose) supplemented when necessary with 0.004% uracil, histidine, or other amino acids (Sherman et al., 1983). Yeast was sporulated in a synchronous meiosis as follows: Yeast cultures were pregrown in YPD to saturation at 30°C, diluted 200-fold into 100 ml of YPA, and grown to early stationary phase (about 5 x 10’ cells/ml). Cells were then washed with water and resuspended into 100 ml of SPM (sporulation medium consisting of 1% potassium acetate supplemented with .004% histidine and other amino acids when necessary). Sporulation was carried out at 30% under conditions that allowed for good aeration. Tetrad Dissection Aliquots of cells that had been sporulated for 24 hr were resuspended in spheroplast buffer (1 M sorbitol, 10 mM sodium phosphate [pH 7.01, 50 mM EDTA). Cells were incubated with zymolyase (final concentration of 50 pg/ml) for 30 min at 37%. Tetrads from strains LCY-A and LCY-B were analyzed for the presence of reciprocal recombination and gene conversion events. The results of the dissection experiments are presented in Table 1 and below. Gene conversion events were scored in both strains, as shown below:

of S. cerevisiae

Strains

Genotype

NKY278 NKY591

ala ho::LYS2/ho::LYSP lys2Nys2 ura3/ura3 ala ho::LYS2/ho::LYSP /ys2//ys2 his4-8-LEU2/his4-X-LEU2 leu2::hisGNeuP::hisG rad50A::hisG/rad50A::hisG spa 13::hisGkpo 13::hisG ura3/ura3 ala ho::LYS2/ho::LYSP lys2Nys2 his4-B-LELWhis4-X-LEU2 leuP::hisGNeu2::hisG ura3/ura3 SPOl3/spol3::hisG ala ho::LYS2/ho::LYS2 lys2Nys2 his4-B-LELWhis4-X-LEU2 leuP::hisGNeuP::hisG ura3/ura3 spol lA::hisG-Ura3-hisG/spol lA::hisG-URA3-hisG ala ho::LYS2/ho::LYS2 lys2/lys2 his4-B-LEU2/his4-X-LEU2 /eu2::hisG//eu2::hisG ura3/ura3 rad50-KI8l::URA3/rad5O-KI8l::URA3 spol3::hisG/spol3::hisG a/a ho::LYS?/ho::LYSP lys2Nys2 his4-X-URA3/HIS4 leuP::hisGNeuP::hisG ura3/ura3 ala ho::LYS2/hot:LYSP lys2/lys2 his4-X-LEU2-URA3/HIS4-LEU2 leu2::hisG/leu2::hisG ura3Iura3 ala ho::LYS2/ho::LYSP /ys2//ys2 leuP::hisG/leuP::hisG ura3/ura3 rad50-K181::URA3/rad50-K/81::URAB spol lA::hisG/spoll spol3::hisG/spol3::hisG ala ho::LYS2/ho::LYSP lys2Nys2 his4-B-LEM/his4-X-LEU2 leu2::hisG/leu2::hisG ura3/ura3 rad50-K/81::URA3/rad50-K/81::URA3 spoil A::hisGlspoll A::hisG ala ho::LYS2ho::LYSP /ys2//ys2 his4-B-LEU2/his4-X-LEU2 leu2::hisG/leu2::hisG ura3/ura3 rad50-KI8l-URA3/rad5O-KIS1-URA3 SPOl l/spoil A::hisG SPOl3/spol3::hisG

NKY841 NKY877

NKY974 LCY-A LCY-B LCY185 LCY196 LCY198

A::hisG

Physical 1099

strain A strain 6

Pathway

for Yeast

Meiotic

Recombination

3His4+: lHis4-

lHis4+: 3His4-

3Ura3+: IthaS-

lUra3+: 3Ura3

Other

2 1

1 -

-

1 1

None None

Return-to-Growth Experiments Commitment to meiotic recombination was determined by methods previously described (Sherman and Roman, 1963; Esposito and Esposito, 1974; Alani et al., 1990). Briefly, yeast cultures derived from strains heteroallelic for mutations at his4 were removed from SPM at times following meiotic induction and then plated onto mitotic growth medium. Cells were diluted in distilled water and plated onto YPD plates to measure total viable colony forming units (CFUs) and onto SD plates without histidine to measure His+ CFUs. Recombination frequency was determined by dividing the total number of His+ CFUs by the total number of viable cells. In NKY641, no obvious drop in cell viability was observed throughout meiosis. Plasmids Two plasmids, pNKY159 and pNKY160, were used to integrate the his4-LEU2 heteroalleles into SKI strains. These two plasmids were constructed in several steps. In the first step, a 4.0 kb Sphl fragment containing the HIS4 gene (Alani and Kleckner, 1987; Donahue et al., 1982) was inserted into the Sphl site of pBR322. In the resulting plasmid, a BamHI-Smal adaptor was inserted at a BstEll site located downstream of the HIS4 coding region to create pNKY156. A 2.8 kb Bglll fragment containing the LEUP gene was inserted at the BamHl linker site in pNKY156 to form pNKY158. The Xhol site normally present within the 2.8 kb LEU2 Bglll fragment was destroyed by a restriction site fill in (treatment with T4 DNA polymerase and dNTPs; Maniatis et al., 1982). To construct pNKY159 (his4-X heteroallele), pNKYl58 was digested with Xhol, fill-in treated, and then religated. To construct pNKYl60 (his4-B heteroallele), pNKYl58 was digested with Bglll, fill-in treated, and then religated. The Xhol and Bglll restriction sites map to the position of codons 34 and 563, respectively, in the HIS4 open reading frame. Each fill-in reaction created a 4 bp insertion within the HIS4 open reading frame. To integrate the his4 heteroalleles, ~10 pg of Sphldigested pNKYI59 and pNKY180 was introduced into the appropriate SK1 strain by lithium acetate transformation. Plasmid pNKY295 is an integrating vector that was used to construct strains LCY-A and LCY-B. Digestion of pNKY295 with EcoRl and BamHl followed by transformation into the appropriate SKI strain resulted in an insertion of the URA3 gene at a site 9 kb downstream of the HIS4 gene (refer to Figure 1 for chromosomal position). pNKY295 was constructed in several step. In the first step, YIP300 (Donahue et al., 1982) a plasmid that contains the HIS4 gene on a 24 kb BamHl fragment, was digested with EcoRl and BamHI, and a 2 kb BamHI-EcoRI fragment containing sequences downstream of HIS4 was isolated. A single Xhol site is located about 300 bp away from the BamHl site. This fragment was then inserted into the EcoRl and BamHl sites of the pNK1415 backbone (C. Jain and N. K., unpublished data). The resulting plasmid was digested with Xhol, filled in, and ligated in the presence of Hindlll linkers, A 1.1 kb Hindlll URA3 fragment, derived from YEP24, was inserted into the Hindlll linker site to create pNKY295. Probes used for Southern blots are derived from plasmids bearing HIS4 (YIP300; Donahue et al., 1982) LEU2 (YEPIJ; Sherman et al., 1983) and ARG4 (Ye.511; Beacham et al., 1984) sequences. DNA fragments used as templates for randomly primed probes were isolated by restriction digestion from the above parental plasmids and inserted into corresponding restriction sites in pSP6 vector backbones. pNKY290 (Probe 1) contains a 1 kb Pstl-Bglll fragment derived from sequences located downstream of HIS4. pNKY155 (Probe 2) contains a 1.6 kb Xhol-Bglll fragment derived from sequences located in the HIS4 coding region. pNKY243 (Probe 4) bears a 700 bp EcoRl-Sall fragment derived from sequences located within the HI.94 coding region. The map locations of Probes 1,2, and 4 are displayed in Figures 1 and 3. pNKY247 (ARG4 probe) contains a 2.2 kb Bglll-Hindlll fragment that includes almost the entire ARG4 gene. DNA Replication and Chromosomal Divisions DNA replication was measured by flow cytometry analysis (Hutter and Expel, 1979). Yeast culture aliquots were fixed in an equal volume of

100% ethanol. Following a wash in distilled water, cells were sonicated, resuspended in RNAase solution (0.1 mglml, RNAase, 0.05 M Tris-HCI [pH 7.51) and then incubated at JloC for 1 hr. After RNAase treatment, cells were pelleted, resuspended in pepsin solution (55 mM HCI, 0.05 mg/ml pepsin) for 15 min at room temperature, and then stained with propidium iodide solution (0.2 M Tris-HCI [pH 7.51, 0.2 M NaCI, 0.1 M MgCls, 50 mglml propidium iodide). Fluorescence was measured using an EPICS C model fluorescence-activated cell sorter (FACS). Usually, 50,000 cells were counted at each time point. Meiotic cultures were stained with DAPI and examined by fluorescence microscopy for 1,2 (Meiosis I), and 4 (Meiosis II) staining bodies. In meiotic time course experiments, 0.5 ml cell aliquots were taken at 1 hr intervals and then fixed in an equal volume of ethanol. Cells were usually fixed overnight at -20% after which 1 nl of 1 ngglml DAPI was added to each sample. Cells (100 to 200) were examined for meiotic division at each time point.

DNA Extraction and Southern Blot with Random-Primed Probes Aliquots (10 ml) of sporulating cultures were removed from SPM and fixed in ethanol at -20%. To extract chromosomal DNA, cells were resuspended in 0.5 ml of spheroplast buffer (1 M sorbitol, 10 mM sodium phosphate (pH 7.01, 50 mM EDTA) containing 3 nl of 8-mercap toethanol and 5 ~1 of 1 mglml zymolyase. After incubation at 3PC for 15 min, cells were concentrated and resuspended in 0.5 ml of 50 mM EDTA, 0.3% SDS. Five microliters of 20 mglml Protease K (Boehringer Mannheim Biochemicals) was added and cells were incubated at 65% for 30 min. After placing the tubes on ice, 0.2 ml of 5 M potassium acetate was added. The tubes were then inverted several times and incubated on ice for 20 min. Cell debris was removed by centrifugation, and the aqueous phase was extracted three times with a mixture of 49% phenol:49% chloroform:2% isoamyl alcohol. Phenol extraction was followed by ether extraction, ethanol precipitation, and a Sephadex G-50 column purification. Ten percent of total DNA was digested with restriction enzymes and run on 0.7% agarose gels. Southern transfer was performed as described (Maniatis et al., 1982) using GeneScreen (New England Nuclear Research Products). Randomly primed probes were made as described by Bio-Rad. Isotope a-3zP-dCTP (800 Cilmmol) was used to achieve at least 70% incorporation of label. Hybridizations were performed at 65°C in hybridization buffer (0.5 M sodium phosphate (pH 7.21, 7% SDS, 1 mM EDTA) for 20 hr (Church and Gilbert, 1984). Blots were exposed to X-AR5 film in the presence of an intensifying screen. Exposures lasted from as little as a few hours to several days, DNA fragments were quantitated on Southern blots after exposing film under conditions where the darkest bands on the film are within the linear range of densitometry measurement. Bands were traced with a densitometer, and individual peaks representing each of the bands were integrated and analyzed by computer. Using this analysis, we estimate our error in measuring band intensity ratios at *500/o.

Acknowledgments We are grateful to Michael Lichten and James Haber for providing information about the LEUP region in strain SK1 and for critical discussion, to Wendy Raymond, Doug Bishop, Masahiro Ajimura, and Jacques Mahillon for helpful comments of this manuscript, and to Wendy Raymond, particularly for her work on constructing many of the NK laboratory yeast strains. We thank C. Giroux and R. E. Esposito for providing the spo77A construct and the SfO73 gene, respectively, and David Johnson for his help on FACS analysis of DNA. L. C. and this research were supported by National Science Foundation grant DCB8711551. E. A. was funded in part by a National Institutes of Health genetics training grant awarded to the Department of Biochemistry and Molecular Biology and in part by the above NSF grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

December

26, 1989; revised

March

21, 1990.

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Alani. E., Cao. L.. and Kleckner, N. (1987). A method for gene disruption that allows repeated use of URA3 selection in the construction of multiple disrupted yeast strains. Genetics 176, 541-545.

Hollingsworth, N. M.. and Byers, B. (1989). HOPI: ing gene. Genetics 121, 445-462.

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