Vol. 11, No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1991, p. 2593-2608 0270-7306/91/052593-16$02.00/0 Copyright © 1991, American Society for Microbiology

Molecular and Genetic Analysis of the Gene Encoding the Saccharomyces cerevisiae Strand Exchange Protein Sepl DANIEL X. TISHKOFF, ARLEN W. JOHNSON, AND RICHARD D. KOLODNER* Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, 44 Binney Street,* and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts .02115 Received 7 December 1990/Accepted 29 January 1991

Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sepl), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sepl was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-M, protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sepl protein. The SEP] gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn1OLUK insertion mutations in the SEPI gene were characterized. sepi mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or y irradiation. Sporulation of sepilsepl diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sepilsepl diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sepilsepi his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His' recombinants than did isogenic SEPI/SEPI strains. sepilsepi mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sepi, radSO, and spol3 mutations suggested that SEP] acts in meiosis in a pathway that is parallel to the RADSO pathway.

purified from mitotic cells as a 132,000-Mr protein, although recent studies (37; see below) have shown that proteolysis during the original cell lysis step of the purification resulted in degradation of a portion of the C terminus. Intact purified Sepl has an approximate relative molecular weight of 160,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its biochemical properties are almost indistinguishable from those of the 132,000-Mr proteolyzed form (37; see below). Like the bacterial proteins, Sepl will promote homologous pairing and strand exchange of linear double-stranded and circular single-stranded DNA molecules as well as promote the renaturation of homologous single-stranded DNA (28, 45). These reactions require high ratios of Sepl to singlestranded DNA and do not require ATP or other nucleotide cofactors. Recently, two S. cerevisiae proteins which are similar to Sepl have been reported (11, 25). The first of these is biochemically similar to Sepl and has a molecular weight of 120,000 (25) but does not appear to be encoded by the same gene as Sepl (52). The second of these, referred to as STP,B, appears to be identical to Sepl based on similar biochemical properties (11), reactivity with a number of monoclonal antibodies specific for Sepl (11, 37), and sequence comparisons of the genes encoding these proteins (12). S. cerevisiae also appears to contain a third distinct protein that can promote homologous pairing. This protein, called strand transfer protein a (STPa), has a molecular weight of 34,000 and was identified as a homologous pairing activity that was induced during meiosis (26, 71). In the presence of a stimulatory factor, STPa promotes homologous pairing reactions including linear double-stranded ver-

Joint DNA molecules containing regions of heteroduplex DNA are a central intermediate in genetic recombination (19, 57). In bacterial and bacteriophage systems, the formation ofjoint DNA molecules is promoted by proteins such as the Escherichia coli RecA and bacteriophage T4 UvsX proteins. These proteins and the mechanism by which they promote homologous pairing and strand exchange have been extensively characterized (10, 20, 24, 59, 60, 76). The reaction is thought to initially involve the formation of an active single-stranded DNA-protein filament. This filament then scans duplex DNA for regions of homology, and once such regions are located, the final heteroduplex joint is formed and extended by branch migration (60). The bacterial proteins require ATP hydrolysis to promote homologous pairing. However, recent results with RecA show that joint molecules can be formed in the absence of ATP hydrolysis, suggesting that the likely role of ATP hydrolysis is to promote turnover of the homologous pairing protein (53). Eukaryotes also contain proteins that promote homologous pairing reactions. The first such protein described, the Ustilago Recl protein, resembled the bacterial proteins in that it required ATP hydrolysis to promote homologous pairing (42). Recent evidence indicates that eukaryotes contain a class of homologous pairing proteins that differ from the bacterial and Ustilago proteins in that they promote homologous pairing in the absence of a nucleotide cofactor (25, 45, 47, 54, 71). The first such protein to be purified to near homogeneity was the Saccharomyces cerevisiae Strand Exchange Protein 1 (Sepl) (45). This protein was first *

Corresponding author. 2593

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Strain

RKY1103 .............. RKY1301 .............. RKY1336 ..............

RKY1302 .............. RKY1337 .............. RKY1303 .............. RKY1263 ..............

RKY1308 .............. RKY1309 ..............

RKY1310 .............. RKY1307 ..............

RKY1304 .............. RKY1300 (NKY592) ..............

RKY1305 .............. RKY1333 ..............

RKY1334 ..............

TABLE 1. List of S. cerevisiae strains used in this worka Genotype

MATa/MATa ura3/ura3 MATa/MATa ura3/ura3 MATa/MATa ura3/urO3 MATa/MATa ura3/ura3 MATa/MATa ura3/ura3 MATa/MATa ura3lura3 MATa/MATa ura3/ura3

Iys2/1ys2 LEU211eu2::hisG ho::hisGlho::LYS2

lys2/lys2 LEU21/eu2::hisG ho::hisGlho::LYS2 sepl::TnlOLUK11-1/SEPI Iys2/lys2 LEU211eu2::hisG ho: :hisG/ho::LYS2 sepl::TnlOLUK42-3/SEPI Iys2/lys2 LEU211eu2::hisG ho::hisGlho::LYS2 sepl::TnlOLUK64-1/SEPl lys2/lys2 LEU211eu2::hisG ho::hisG/ho::LYS2 sepi::TnlOLUK75-6/SEPI lys2/1ys2 LEU211eu2::hisG ho::hisG/ho::LYS2 sepi: :TnlOLUK79-2/SEPJ Iys2/Iys2 LEU21/eu2::hisG ho::hisGlho::LYS2 HIS41his4B sepl::TnlOLUK64-1/SEPl MATa/MATa ura3/ura3 Iys2/lys2 leu2::hisGlleu2::hisG ho::LYS21ho::LYS2 his4X/his4B MATa/MATa ura31ura3 Iys2/lys2 leu2::hisGlleu2::hisG ho::LYS2/ho::LYS2 his4X/his4B sepl::TnJOLUK79-2/sepl ::TnlOLUK79-2 MATa/MATa ura31ura3 Iys2/1ys2 leu2::hisGlleu2: :hisG ho::LYS21ho::LYS2 his4X/his4B sepl::Tn]OLUK11-1/sepl::Tn]OLUK11-1 MATa/MATa ura3/ura3 lys211ys2 Ieu2::hisGlleu2::hisG ho::LYS21ho::LYS2 his4X: :LEU2/his4B::LEU2 spol3::hisGlspol3::hisG MATa/MATa ura31ura3 lys2l1ys2 1eu2::hisG/Ieu2::hisG ho::LYS21ho::LYS2 his4X::LEU2/his4B::LEU2 spol3::hisG/spol3::hisG sepl::TnlOLUK79-2/sepl::Tn]OLUK79-2 MATa/MATa ura31ura3 Iys2/lys2 leu2::hisG/Ieu2::hisG ho::LYS21ho::LYS2 his4X::LEU21his4B: :LEU2 spol3::hisG/spol3::hisG radSOD::hisG/radSOD::hisG sepl::TnlOLUK79-2/sepl::TnlOLUK79-2 MA Ta/MA Ta ura31ura3 lys2l1ys2 leu2::hisG/leu2: :hisG ho::LYS21ho::LYS2 his4X::LEU21his4B::LEU2 spol3::hisGlspol3::hisG radSOD::hisG/radSOD: :hisG sepl: :TnlOLUK79-2/sepl::TnJOLUK79-2 MATa/MATa ura31ura3 lys2l1ys2 LEU211eu2::hisG ho: :hisGlho::LYS2 sepl::TnlOLUK11-1/sepl::Tn]OLUK11-1 MATa/MATa ura3/ura3 lys21lys2 LEU21leu2::hisG ho::hisGlho::LYS2

sepl::Tn]OLUK64-1/sepl::Tn]OLUK64-1

RKY1335 .............. RKY1348 .............. RKY1349 ..............

RKY1107 .............. RKY1228 .............. RKY1311 .............. RKY1164 .............. RKY1316 .............. RKY1319 .............. RKY1144 (NKY858) .............. RKY1147 (NKY861) ..............

MATa/MATa ura3/ura3 lys2/lys2 LEU211eu2::hisG ho::hisGlho::LYS2 sepl::TnlOLUK79-2/sepl::Tn]OLUK79-2 MATa/MATa ura3/ura3 lys2-lys2 LEU2-leu2::hisG ho::hisGlho::LYS2 HIS41his4X MATa/MATa ura31ura3 lys2/lys2 LEU211eu2::hisG ho::hisG/ho::LYS2 HIS41his4X sepl::TnlOLUK79-2/sepl::Tn]OLUK79-2 MATa ura3 Iys2 LEU2 ho::hisG MATa ura3 lys2 LEU2 ho::hisG sepl::TnJOLUK79-2 MATa ura3 lys2 leu2::hisG ho::LYS2

MATa ura3 Iys2 leu2::hisG ho::LYS2 sepl::TnJOLUK11-1 MATa ura3 lys2 LEU2 ho::LYS2 sepl::TnJOLUK64-1 MATa ura3 Iys2 LEU2 ho::hisG sepl::TnlOLUK79-2 MATa ura3 Iys2 leu2::hisG ho::LYS2 his4X MATa ura3 lys2 leu2::hisG ho::LYS2 his4B MATa ura3 lys2 leu2::hisG ho::LYS2 his4X::LEU2 spol3::hisG RKY1280 (NKY567) .............. MATa ura3 lys2 leu2::hisG ho::LYS2 his4B::LEU2 spol3::hisG RKY1281 (NKY575) .............. MATa ura3 lys2 leu2::hisG ho::LYS2 his4X::LEU2 spol3::hisG radSOD::hisG RKY1282 (NKY585) .............. MATa ura3 Iys2 leu2::hisG ho::LYS2 his4B::LEU2 spol3::hisG radSOD::hisG RKY1283 (NKY590) .............. MATa ura3 lys2 leu2::hisG ho::LYS2 his4X::LEU2 spol3::hisG sepl::TnlOLUK79-2 RKY1284 .............. MATa ura3 lys2 leu2::hisG ho::LYS2 his4B::LEU2 spol3::hisG sepl::TnlOLUK79-2 RKY1286 .............. MATa ura3 Iys2 leu2::hisG ho::hisG his4X sepl::TnJOLUK79-2 RKY1226 .............. MATa ura3 lys2 LEU2 ho::hisG his4B sepl::TnJO LUK79-2 RKY1240 .............. a All strains used in this study were isogenic derivatives of SK-1. They were derived from SK-1 either by transformation of SK-1 or by crossing with an SK-1 derivative that was previously derived by transformation. Strains listed with NKY numbers in parentheses were the gift of Nancy Kleckner.

sus single-stranded circular DNA pairing and D-loop formation in reactions that do not require nucleotide cofactors. STPa appears to be distinct from both Sepl and the DNApairing activity described by Halbrook and McEntee in that it does not appear to be encoded by the genes encoding these activities (9, 12, 52). Higher eukaryotes also contain homologous pairing proteins that are similar to the S. cerevisiae Sepl and STPa proteins in that they promote homologous pairing in the absence of an obvious nucleotide cofactor (47, 54). Such activities were probably responsible for the homologous pairing activities first detected in partially fractionated extracts of mammalian cells (6, 18, 23, 33). A human protein that was originally detected as a Z-DNA-binding protein has recently been purified to near homogeneity and shown to

have a molecular weight of 140,000 (54). This protein can promote a number of homologous pairing reactions in the absence of a nucleotide cofactor. A homologous pairing protein has also been purified from Drosophila melanogaster and shown to have a molecular weight of 120,000 (47). The relationship of these proteins to the S. cerevisiae proteins is unclear at present. Although the involvement of the prokaryotic enzymes (RecA and UvsX) in genetic recombination has been demonstrated by genetic evidence, so far the role of eukaryotic homologous pairing proteins in vivo can only be inferred by their biochemical similarity to the RecA and UvsX proteins. To better understand the role that Sepl plays in the cell, our laboratory has initiated a genetic analysis of the gene encoding S. cerevisiae Sepl. Many recombination-deficient mu-

TABLE 2. Protein sequences and oligonucleotide probes Peptide or Sequenceb derived

A

K

adh4 SEPI rad54

100 KB

N terminus ......

Trp-1 ...... V8-1 ......

?IP?FFRYISERWPMILQ?IE?TQ TGIA(GA)(GAT)ATCATIGG GG(GA)AA(GATC)CC(GATC)A(GA)CAT

B

V

HX C B

LGAEMLAGFPT

YITIPLDS?EI

a The oligonucleotides are listed below the protein sequence from which they were derived. Chy, Chymotrypsin-derived fragment; Trp, trypsin-derived fragment; V8, S. aureus V8 protease-derived fragment. b Standard one-letter protein sequence abbreviations are used. Nucleotides in parentheses refer to multiple bases at one position. I is inosine. Note that the Trp-1 fragment overlaps the Chy-1 fragment.

tants of S. cerevisiae have been isolated, and usually the

mutations also affect other cellular processes such as repair of DNA damage and sporulation (62, 65). In this communication, we describe the cloning of SEP] and demonstrate that sepi mutants have several unusual phenotypes: they sporulate poorly but show intermediate spore viability, and they show increased levels of both gene conversion and reciprocal exchange during meiosis. We also demonstrate that mitotic growth, response to DNA damage, and the character of mitotic and meiotic recombination events are all altered in sepi mutants. The relationship between SEP] and other recombination and repair functions in S. cerevisiae is discussed. MATERIALS AND METHODS Strains. The S. cerevisiae strains used in this study are all isogenic heterothallic SK-1 derivatives and are listed in Table 1. sepl::TnJOLUK or sepl::TnlOLUKx-y denote a transposon insertion in the SEP] gene, where x is the allele number of the transposon (see Fig. 1C for the position of the transposon alleles in the SEP] open reading frame ORF) and y is the isolate number. In some cases we use sepl-x as an abbreviated form of sepi::TnJOLUKx-y. E. coli HB101 (2) was the host for the YCp5O library, pRDK251, and pRDK252. E. coli and phage lambda strains for the TnJOLUK mutagenesis were described previously (34). Media. Rich (YPD and YPG), minimal (SD), and synthetic complete (SC) media were prepared as described previously (69). YPA consists of 1% potassium acetate, 2% peptone, and 2% yeast extract, and SPM consists of 3% potassium acetate and 0.02% raffinose. Genetic methods. Mating, sporulation, and tetrad analysis were performed essentially as described previously (69). In all experiments yeast strains were prepared by streaking out frozen cultures on YPD plates and incubating them at 30°C for 2 days. Diploid cells were sporulated in liquid SPM after growth in YPA to a titer of ca. 2 x 107 cells per ml as previously described (1). Microcolony analysis of return to growth after UV irradiation was performed essentially as described previously (27, 73), except that standard YPD agar plates were used instead of thin slabs of YPD agar (27, 73). S. cerevisiae cells were transformed by standard methods

S

B

B C H

x

S

/

2 KB

/ /

GCIA(GA)CAT(TC)TC(GATC)GCICC

TC(GA)TT(GA)TA(GAT)AT(GATC)GT(TC)TT

vii

/

GLLPNAKLGAEMLAGFP KTIYNE?FE?WK?E

suci

.l

/

probea

Oligo 937 ...... Oligo 928 ...... Chy-1 ...... Oligo 934 ...... V8-2 ...... Oligo 970 ......

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VOL. 11, 1991

4-

1,

C

IV .5 KB

S3

S

F--

A TG

*

,79 4

I

404- -041

--

1,75 64,42

B l

I

I

B C a

H.S H H S3 ,,1

r,

TAA

FIG. 1. Structure of the cloned and genomic regions of SEP]. (A) Position of SEP] on chromosome VII. The dashed lines indicate that the orientation of SEPI and radS4 relative to the centromere is unknown. (B) Restriction map of the chromosomal region containing SEP]. (C) Restriction map of pRDK252. Symbols: L, SEP] ORF, where ATG and TAA indicate the putative start and stop codons, respectively; -*. positions of TnIOLUK insertions with the allele number over them; * . +, orientation of the TnlOLUK insertion, with rightward-pointing arrows having the orientation LUK. Abbreviations: B, BglII; C, ClaI; H, HindIII; S, SacI; S3, Sau3A; X, XbaI.

(36), and E. coli cells were transformed by the calcium chloride method (50). Transposon TnlOLUK insertion mutagenesis was performed exactly as described previously (34). pRDK252: :TnlOLUKx-y indicates pRDK252 containing transposon TnJOLUK inserted at various positions in the SEP] ORF (see Fig. 1C and 2). pRDK252::TnlOLUK insertion mutations were transplaced into the diploid S. cerevisiae RKY1103 by using either the 1.6-kbp BgII restriction fragment (for transposon alleles 11, 42, 64, and 75) or the 2.4-kbp BgllI-XbaI restriction fragment (for transposon allele 79). Proper integration of transposon alleles into S. cerevisiae was verified by Southern analysis of genomic restriction digests or by a polymerase chain reaction method to be described elsewhere (61). sepi spol3 double-mutant strains (RKY1284 and RKY1286) were constructed by integrating the sepl-79 allele into MATa and MATa haploid spol3 strains (RKY1280 and RKY1281) by using the 3.3-kbp SnaBI restriction fragment of pRDK252: :TnJOLUK79-2. sepi spol3 rad5O triple-mutant strains were constructed by mating sepl strains (RKY1226 and RKY1240) to spol3 rad5O strains (RKY1282 and RKY1283). The URA+ MMSS (sepi radSO) progeny of this cross were analyzed for the presence of the spol3 mutation by backcrossing them to an appropriate spol3 radSO strain, sporulating the resultant diploids, and scoring for the production of viable spores. Only diploids which were homozygous for spol3 were rescued from the rad5O sporulation defect and produced viable spores resistant to treatment with Zymolyase (1).

Return-to-growth experiments were performed exactly as previously described (1), except that Nonidet P-40 was not added to the SPM. Frequencies of mitotic recombination at the HIS4 locus were determined by using fluctuation analysis (46), with the following modifications. Stationary cultures of the appropriate strains were serially diluted, plated onto YPD, and incubated at 30°C for 2 to 3 days. Individual colonies were resuspended in 1 ml of H20, sonicated, serially diluted, and plated onto SC media and SC media lacking histidine. Eleven colonies were analyzed for each strain. Recombination rates were determined as previously described (46). This fluctuation analysis was performed once

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GATCAGTAACTATTCTCACGATTAATGGTCTTTTTCAACTACAfTTTATTGAGAAAGCTGTAATACATTCCGTGTTGAGTGATATATACA ACCTCCTTTGCTCGTCTTTTCGCCACCGCAGAGCAAGTAACAACAGAGACAAACAAGAAGAGGTTAGAAAGCAATTTAAGGAGTAGTTTA

ACGAGTACTGAAAACGTCAAACTAAGTACTTACGACTTCCAATTACCGGTTGTAAAGTTTATCTACGCAAAZJLeTCAGTTGCAGCTTGC TTTTTTTTTCAGCAGCTCTTCCGTTATIALATCTGTTTGGCGTTCGCACAAGCGAACCGATTCTTTTATTATTTTCTCTCTGCCTTT TATTTCCGTTCCTGTTAATAGTTTATTTTCTAAAGGATACTGTCTTCTTCCGTACTZAIAACGGGTTCACAATAAGCAATTGACTAATC CTAGGACGATTCGTGTACTATAAGGAGAAAAAAAATCAACACTTGTAACAACAGCAGCAACAAATATATATCAGTACGGTATGGGTATTC M

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CAAAATTTTTCAGGTACATCTCAGAAAGATGGCCCATGATTTTACAGCTTATTGAGGGAACACAGATTCCTGAGTTTGATAACTTATACC

631

TGGATATGAATTCGATTTTACATAATTGTACGCATGGTAACGACGATGATGTAACCAAGCGATTAACTGAAGAAGAGGTTTTTGCAAAAA

721

TCTGTACGTATATCGATCACCTTTTTCAAACAATCAAGCCCAAGAAGATTTTCTACATGGCTATTGATGGTGTGGCCCCTCGTGCCAAGA

811

TGAATCAACAAAGAGCTCGTAGATTCAGAACCGCTATGGATGCAGAAAAAGCCTTGAAGAAGGCTATTGAGAATGGTGACGAGATTCCTA

901

AGGGTGAGCCATTTGATTCGAATTCTATTACTCCAGGTACGGAGTTTATGGCCAAATTGACCAAAAACTTACAATATTTTATTCACGACA

991

AGATTTCTAACGATTCCAAATGGAGGGAAGTGCAAATCATATTTTCTGGCCATGAAGTTCCAGGTGAAGGTGAACACAAGATCATGAACT

1081

TTATAAGGCATTTAAAATCCCAAAAGGATTTCAACCAGAATACGAGACATTGTATTTACGGTCTTGACGCAGATTTGATTATGCTGGGTT

1171

TGTCTACTCATGGGCCACATTTTGCGTTATTGAGAGAAGAAGTGACATTTGGTAGAAGAAATAGTGAAAAAAAATCGCTTGAACATCAAA

1261 1351

ATTTCTACTTATTACATCTTTCTTTATTAAGAGAATACATGGAGTTGGAATTCAAAGAAATTGCCGATGAAATGCAATTTGAATACAATT F Y L L H L S L L R E Y M E L E F K E I A D E M Q F E Y N F TTGAACGTATTTTGGATGATTTTATTCTTGTCATGTTCGTCATTGGTAATGATTTCTTGCCCAATTTGCCAGATTTGCACCTTAACAAAG

1441

GAGCATTTCCCGTTTTGTTACAAACGTTCAAAGAAGCTCTTTTACATACTGATGGCTACATTAATGAACATGGTAAAATAAATTTAAAGA

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GATTAGGTGTCTGTTAAATTATCTGTCTCAATTTGAGTTATTAAATTTCGAAAAGGATGATATAGACGTTGAGTGGTTCAACAAGCAAT L G V W L N Y L S Q F E L L N F E K D D I D V E W F N K Q L TAGAGAATATTTCTTTGGAGGGTGAAAGGAAAAGACAGAGGGTTGGTAAAAAATTACTGGTAAAACAACAGAAGAAATTAATTGGAAGTA E N I S L E G E R K R Q R V G K K L L V K Q Q K K L I G S I TAAAACCATGGTTGATGGAGCAATTACAGGAAAAATTATCGCCTGATTTACCAGATGAAGAAATTCCAACTTTAGAGTTACCTAAGGACT

1801

TAGACATGAAAGATCATTTAGAATTTTTAAAAGAATTCGCTTTTGATTTGGGTCTTTTTATAACGCATTCCAAATCCAAAGGTAGTTATT

1891

CGCTAAAAATGGATCTTGATTCTATTAATCCTGATGAAACAGAAGAAGAATTTCAAAATCGTGTTAATTCTATCAGGAAAACAATAAAAA L K M D L D S I N P D E T E E E F Q N R V N S I R K T I K K AATATCAAAATGCTATCATCGTGGAGGACAAAGAAGAATTGGAAACTGAAAAAACGATTTATAATGAAAGGTTTGAACGTTGGAAGCATG

1531 1621

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2071 2161 2251

2341

2521

2611 2701

2791 2881

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AGTATTATCACGACAAGTTAAAATTTACGACAGACAGTGAAGAAAAAGTGAGAGATCTTGCTAAAGACTACGTTGAAGGTTTACAATGGG Y Y H D K L K F T T D S E E K V R D L A K D Y V E G L Q W V TTCTATATTATTATTATAGAGGATGTCCATCTTGGTCGTGGTACTATCCGCACCATTATGCACCAAGAATCTCCGACTTAGCCAAGGGTT L Y Y Y R G C P S W S W Y Y P H H Y A P R I S D L A K G L TAGATCAAGACATTGAATTTGATTTGAGCAAACCATTTACTCCATTCCAGCAACTAATGGCAGTTTTACCGGAAAGGTCCAAAAATCTGA D Q D I E F D L S K P F T P F Q Q L M A V L P E R S K N L I TACCTCCCGCCTTTAGGCCATTAATGTACGATGAACAGTCGCCAATCCACGATTTCTATCCCGCTGAGGTTCAACTTGATAAAAACGGCA P

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K N G

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ATTTGCGCAAGTTAT AGACAGCTGATTGGGAAGCTGTGGTTTTGATATCGTTTGTAGATGAAAAAAGGTTGA=2&GGCTATG T A D W E A V V L I S F V D E K R L I E A M Q P Y L R K L S CACCTGAAGAAAAAACGAGAAATCAATTTGGCAAGGACTTGATATATTCCTTTAATCCTCAAGTTGATAACCTTTATAAGAGTCCGTTGG P E E K T R N Q F G K D L I Y S F N P Q V D N L Y K S P L G GCGGCATTTTTTCTGATATTGAACACAATCATTGTGTCGAAAAAGAGTACATCACCATCCCATTGGACAGCTCCGAGATTCGGTATGGTT G I F S D I E H N H C V E K E Y T T I P L D S SE ITR Y C L TATTACCTAATGCTAAACTCGGTGCCGAAATGCTGGCGGGTTTCCCCACGTTATTGTCTTTACCATTTACTAGTTCACTGGAGTACAATG L P N A K L G A E M T. A C F P T L L S L P F T S S L E Y N E AGACAATGGTTTTCCAACAACCTTCTAAACAACAATCAATGGTCTTACAAATAACTGACATATACAAAACGAATAATGTTACTTTGGAGG T M V F Q Q P S K Q Q S M V L Q I T D I Y K T N N V T L E D ACTTTTCCAAGAGGCATTTAAACAAAGTGATTTATACAAGATGGCCATATTTAAGAGAATCCAAATTGGTCTCTTTAACGGATGGTAAGA F S K R H L N K V

I

Y

T R W

P

Y

L R E

S

K L V

S

L T D

G K

T

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VOL. 11, 1991

CTATCTATGAATATCAGGAGTCCAATGATAAGAAAAAGTTCGGATTCATAACGAAGCCTGCGGAAACCCAGGACAAAAAACTTTTCAATA I

Y

E

Y

Q E 64'

S

N D '42

K

K

K

F

G

F

I

T

K

P

A

E

T

Q

D

K

K

L

F

N

S

3061

GTTTGAAGAAITIAATGCTAAGGATGIGMGCTAAACAGAAAGCTGTTAAAATAGGACCTATGGAAGCCATTGCTACCGTCTTTCCAGTGA

3151

CTGGTTTGGTGAGAGACTCTGATGGTGGTTATATTAAGACCTTTAGCCCTACCCCAGATTACTATCCATTGCAACTGGTTGTTGAATCTG

3241

TTGTCAACGAGGATGAAAGATATAAAGAAAGAGGACCCATTCCTATTGAAGAGGAATTTCCATTGAATTCAAAAGTTATTTTCTTAGGTG

3331

ATTATGCCTATGGTGGTGAAACTACTATTGACGGTTACAGCAGTGACCGCAGACTAAAAATTACTGTAGAAAAGAAGTTTTTGGATAGTG

3421

AGCCCACCATCGGCAAAGAAAGGTTACAAATGGATCATCAAGCCGTTAAATATTATCCGTCTTATATTGTGTCCAAGAACATGCACTTAC

3511

ACCCCTTGTTTTTGTCTAAGATTACTTCCAAGTTCATGATTACTGACGCTACTGGGAAGCATATCAATGTTGGTATCCCGGTTAAGTTCG

3601

AAGCTAGACACCAAAAGGTTTTAGGTTACGCGAGGAGGAACCCTAGGGGCTGGGAATACTCAAATTTAACTCTAAATTTACTGAAAGAGT

3691

ATAGACAAACTTTCCCAGATTTTTTTTTCAGGTTGTCCAAGGTAGGTAATGATATCCCAGTTTTGGAAGATCTTTTCCCCGATACTTCCA

3781

CTAAGGATGCCATGAATTTATTAGATGGTATCAAACAATGGCTAAAGTATGTCTCATCGAAGTTTATCGCGGTATCTTTGGAGTCTGACT

3871

CCTTAACTAAGACATCGATTGCTGCCGTGGAAGATCATATCATGAAATACGCAGCTAACATCGAAGGTCATGAAAGAAAACAGTTAGCCA

3961

AAGTTCCTCGTGAGGCTGTTTTGAATCCAAGATCATCATTTGCACTCTTACGTAGTCAAAAGTTCGATTTGGGTGACCGTGTTGTTTATA

4051

TCCAAGATTCTGGTAAGGTACCAATTTTCTCAAAGGGTACAGTTGTTGGCTATACTACTCTCAGTTCATCATTATCAATTCAGGTCTTAT

4141

TTGATCATGAAATCGTGGCCGGTAATAATTTTGGCGGCAGGTTGCGCACGAATAGAGGCTTAGGGCTTGATGCCTCTTTCTTATTGAATA

4231

TTACTAACAGGCAGTTCATTTATCACTCCAAGGCTTCCAAAAAGGCTTTGGAAAAGAAAAAGCAATCTAACAATAGGAACAATAATACCA

4321

AAACTGCTCACAAGACTCCTTCAAAGCAACAATCTGAAGAAAAACTGAGAAAAGAAAGGGCACATGATTTATTGAATTTTATCAAAAAGG

4411

ATACCAATGAAAAGAATTCTGAAAGTGTAGACAACAAGAGCATGGGATCGCAAAAAGATTCCAAACCCGCAAAGAAAGTTTTGTTAAAAA

4501

GACCAGCTCAGAAAAGCAGTGAAAACGTGCAAGTTGATTTGGCCAATTTTGAAAAAGCACCGCTTGATAATCCAACTGTTGCTGGATCTA

4591

TTTTCAATGCCGTTGCAAATCAATATTCTGATGGTATAGGCAGTAATTTGAATATCCCAACTCCACCTCACCCAATGAATGTGGTTGGGG

4681

GTCCTATTCCTGGAGCGAATGATGTTGCAGATGTTGGTTTGCCGTACAATATTCCCCCAGGTTTTATGACGCATCCTAATGGTCTTCACC

4771

CATTACACCCTCACCAGATGCCTTACCCTAATATGAATGGAATGTCTATTCCGCCACCAGCACCACATGGGTTTGGACAACCGATTTCCT

4861

TCCCACCTCCACCTCCTATGACAAATGTTTCAGATCAAGGAAGTCGTATTGTTGTCAATGAAAAGGAAAGCCAAGATTTGAAAAAATTCA

4951

TTAATGGTAAACAGCACAGCAATGGTTCAACTATTGGGGGAGAAACAAAGAACAGTAGGAAAGGCGAGATTAAACCTTCTTCTGGCACAA

5041

ACTCTACTGAATGTCAATCGCCAAAGTCACAAAGCAATGCTGCTGACCGTGATAATAAAAAAGACGAATCTACTTAGAACATACGACTAA

5131 5221 5311

AAACGAAGTATATTCGAGGTTACTTTAATAGTATATCTGAGACCTATATAAAGAGGGGTATTTGTATTTAATCGTTGATCTTGTTAAGAA AAAGCTTATAACAAATGGGGATTGTCAAAGGGTATTTTTTACACAAAGCTTTCCGCATAGTTATATATTATCTCATTTTCAATAATCCTC

L

G V

Y P P

A R K L V

Q D

T T T P

F P L P

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L N

A T L R

Q D T P D H

N A N A

N I H P

G T

N

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Y I F H T A K R S E

R H E

Q A P P P

K E

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R D

G G L

Q F M T E G I

Q K K K

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Q C

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D E G

K S K P N S A K V F T N S A A

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E K V D L I V

V A I P S S N N M M S S

R

D Y T

R I L F L A L

P G Y S E E

Q D P T N P

M

G K T

L T G F D A N

I N H K S N Y V Y N G K

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Q S Y F G V P

F N S

Q V V S A P V S S

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M K A R I E R

S F K

Q D

Q D D N S T

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K G A S N V G V M D I S

Q K P

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K E S L G L G G G A

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P V L M A

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K K M A S P M S E A

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D V D

R G K K L

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A L G N N Y S

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G N Y Y L

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W D V A R

Y N

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L Y G

E I S A S

T R

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Y P S N

Q T G

K R D A

P P

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M Y P I S H S V K I K

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T G

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E Y L T Y I N L F E F

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Q H K L

P F

P

K G D

A P N V I N L E I G D S L

S D P D

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E E E

I L S E V

V T D A H L S D

N L A N

H T

G S I S

A

Q K K S G L L V E G L

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N L K P P H

F

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T L V K K I N F S R D S

S R N K T M P

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V V I F N P L P L K R I

F N F V V N N

Q L S

F V F L M

V L D E

Q V

Q L N I

L A V G

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P E L D H

K K T S L V V

L N K L G V L I

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F E S D A Y L

N T K K S G H

S F T

T V D E H E Y T S K I F I K D R I G P F I N

T

CTGTAGGGTGTAGCTTTGCACTTTCTTCTCATTGATC

FIG. 2. DNA sequence of the SEP] region of chromosome VII. The sequence of a 5,637-bp region containing the SEP] gene is shown. The 5'-most ATG of the ORF located at nucleotide 530 matches the N terminus of the purified protein and is indicated as the start codon. The first stop codon in this ORF, TAG, is located at nucleotide 5135. The six underlined DNA sequences located between nucleotides 1 and 530 indicate potential TATA boxes in the 5' region of the gene. The underlined regions of protein sequence indicate the positions of the sequenced peptides listed in Table 2. The five regions of five underlined nucleotides in the coding sequence indicate the positions of the sepl::TnlOLUK insertions; the number above each insertion location indicates the Sepl allele number, and this number is located above the first nucleotide of the SEP] sequence read from the TnlOLUK-specific sequencing primer followed by the next four underlined nucleotides.

to three times on each strain. Meiotic frequencies of recombination in the HIS4-LEU2 and LEU2-MAT intervals were determined by using random spore analysis and tetrad analysis as described in Table 7, footnote a. Plasmids, enzymes, and related methods. Plasmids were

constructed by standard procedures. Restriction endonucleases were purchased from New England BioLabs, Beverly, Mass., and used as recommended by the manufacturer. T4 DNA ligase was purified in this laboratory by using the procedure of Kolodner (43).

2598

TISHKOFF ET AL.

Southern hybridizations were performed as described previously (8, 31), with the purified 3.1-kbp SacI-ClaI fragment of pRDK252 as a probe. Northern (RNA) hybridization was performed as described previously to a Northern blot already used in this laboratory (30), which had been previously hybridized to radioactive probes specific for RPAI, HIS4, and CYH2 mRNAs, allowing an estimate of the relative size of SEP] mRNA. For Western immunoblotting analysis, cultures were grown in 1.5 ml of YPD medium and the cells were harvested and lysed with glass beads by using standard methods. The proteins present were then analyzed by Western blotting with a ProtoBlot kit (Promega, Madison, Wis.) as specified in the instructions provided by the manufacturer. Incubation with the primary antibody was carried out overnight with a mixture of eight monoclonal antibodies specific for Sepl to be described elsewhere (29), each of which was diluted 10' in 10 mM Tris HCl (pH 8.0)-150 mM NaCl-0.05% Tween 20. Cloning of the SEP] gene. A homogeneous preparation of the Mr-132,000 Sepl protein was purified by W.-D. Heyer of this laboratory as described previously (45). Proteolysis of 100 ,ug of Sepl was performed in 9-p,l volumes of 50 mM Tris (pH 7.6) by incubating the Sepl with an optimized amount of protease (100 ng of trypsin, 1 ptg of chymotrypsin, or 1 ,ug of Staphylococcus aureus V8 protease) for 40 min at 37°C. The Mr-132,000 protein and various proteolytic products were then sequenced as described previously (51) on an Applied Biosystems 470A protein sequencer. Fully degenerate oligonucleotides were designed from amino acid sequence of several proteolytic fragments (Table 2). The hybridization characteristics of the oligonucleotides were evaluated by Southern hybridization to chromosomal DNA, and oligonucleotide # 970 was chosen for the initial screen. Oligonucleotide 970 was 5' 32P labeled (63) and used to screen the YCp50 library of cloned S. cerevisiae chromosomal DNA fragments (66) by using the method of Wood et al. (75). The positive clones from the initial screening were rescreened by using oligonucleotides 970, 928, 934, and 937; this resulted in the identification of two plasmids (pRDK251 and pRDK252) containing the SEP] gene; pRDK252 contains a 7.5-kbp Sau3A fragment of S. cerevisiae chromosomal DNA in the BamHI site of YCpSO, and pRDK251 is a similar plasmid containing an overlapping 12-kbp Sau3A fragment. DNA sequence analysis. DNA sequencing was performed on CsCl purified plasmid DNA by using a Sequenase kit from U.S. Biochemicals. The SEP] region of pRDK252 was sequenced on both strands by using appropriate oligonucleotide primers. Correspondence between cloned DNA sequences and genomic sequences was checked by using pairs of sequencing primers to amplify both cloned and genomic DNA by the polymerase chain reaction to show that the two DNAs yielded identical fragments. TnJOLUK insertion points were determined by using the oligonucleotide 5'CAAGATGTGTATCCACC-3' that hybridizes to the lacZ end of TnJOLUK and directs DNA synthesis outward. The DNA and translated protein sequences were analyzed by using the Pearson-Lipman algorithm (58) available on the Eugene program package (LARK Sequencing Technologies, Ltd., Houston, Tex.) for homology searches against the PIR protein sequence data base (release no. 23.0) and against the GenBank DNA sequence data base (release no. 63.0). The translated protein sequence was searched against a library of 152 protein signature sequences by using the Prosite search program (Molecular Biology Computer Research Resource, Dana Farber Cancer Institute).

MOL. CELL. BIOL.

FACS analysis. DNA replication was measured by flow cytometry analysis (35). Aliquots taken from mitotic or meiotic yeast cultures were fixed and stained with propidium iodide exactly as previously described (4). Fluorescence was measured by using a Coulter EPICS 750 series fluorescenceactivated cell sorter (FACS). Biochemical analysis of SEP]. Overproduction and purification of intact Sepl will be described elsewhere (38). SDS-PAGE analysis of the purified protein and assays for strand exchange activity with linear double-stranded M13mpl9 DNA and circular single-stranded M13mpl9 DNA as substrates were as previously described (45), except that rATP and the rATP regenerating system were omitted. Nucleotide sequence accession number. The DNA sequence reported in this communication has been submitted to GenBank and is listed under accession number M58367.

RESULTS Cloning and structural analysis of SEPI. SEP] was cloned by screening a plasmid library of S. cerevisiae DNA fragments with degenerate oligonucleotide hybridization probes designed by using protein sequence information derived from Sepl protein. Two different plasmids (pRDK251 and pRDK252) were identified by hybridization with four independent probes designed from protein sequence information obtained from three different regions of the protein. The two plasmids were shown to have common restriction fragments, and pRDK252 was chosen for further study. Comparison of the genomic restriction map with the map of the cloned region indicated exact identity in the region of the SEP] coding sequence. There appeared to be a short region of DNA present in the cloned fragment upstream of the N terminus of SEP] that was not present in the genomic copy. Polymerase chain reaction experiments demonstrated exact identity between the cloned and genomic regions starting at a Sau3A site 530 bp upstream of the ATG and extending to the end of the cloned fragment containing the C terminus of SEP] present in pRDK252 (data not shown). A map of this region is shown in Fig. 1C, and the DNA sequence of this region is presented in Fig. 2. SEP] was mapped to chromosome VII by Southern hybridization to filters containing S. cerevisiae chromosomes from strain AB972 separated by orthogonal field alternating

gel electrophoresis (data not shown) (5). Higher-resolution mapping was performed by L. Riles and M. Olson, Washington University, St. Louis, Mo., with a hybridization probe derived from pRDK240, which contains a XbaIHindIIl fragment of pRDK252 containing SEP] cloned into pUC18, to probe a library of ordered cloned S. cerevisiae chromosomal DNA fragments. By using this method, SEP] was shown to map to within 20 kbp of RAD54, but was distinct from RAD54 on the basis of both hybridization pattern and DNA sequence (data not shown). Linkage analysis of sepi-Ji and sepl-79 relative to MET13 is consistent with this map location (44). The order of RAD54 and SEP] relative to the centromere is not known. DNA sequence analysis of the SEP] region showed that it contained a 4,584-bp ORF capable of encoding a 1,528amino-acid protein with molecular weight of 175,000 (Fig. 2). Northern blotting experiments with probes derived from this ORF detected a 5.1-kbp mRNA species consistent with the coding capacity of the ORF (data not shown). Four lines of evidence indicate that this ORF encodes Sepl. (i) Five segments of independent protein sequence from four regions of Sepl matched the translated protein sequence. The N-ter-

B

A

RKY1301

RKY1302

RKY1303 ---

Ura -

U ra 1

+ +

+ +

+

+

2

A

kDa

1. ... 4m _~ _4

200

C.) 0

kb

.-

I16 97

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VOL . 1 l, 1991

-°

E

6 0i

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c

66

4 5

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0

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micrograms of SEP1 FIG. 3. Size and activity of Sepl overproduced from the cloned SEP] gene expressed under GALIO control. (A) Proteins were analyzed by SDS-PAGE as described in Materials and Methods, and the resulting gel was stained with Coomassie blue. Lanes: 1, molecular mass markers: 2, 0.2 ,ug of Sepl. The arrow marks the position of Sepl. (B) The activity of the indicated amounts of Sepl was determined in a strand exchange assay that measures the pairing of double-stranded linear M13mp19 DNA and single-stranded circular M13mpl9 DNA (see Materials and Methods).

minal protein sequence matched the translated sequence at the first potential translational initiation site, indicating that the purified protein had an intact N terminus. (ii) Western blotting and protein purification experiments under appropriate cell lysis conditions have demonstrated that Sepl present in crude extracts of mitotic cells can be obtained as a 160,000-Mr form (Mr determined by SDS-PAGE), which is consistent with the coding potential of the ORF (31b; see below). (iii) Western blotting and protein purification experiments with sepi mutants have demonstrated that sepi mutants lack Sepl protein and appear to lack strand exchange activity (39; see below). (iv) The cloned SEP] gene has been used to overproduce the SEP] protein in a GALIO expression system, and the overproduced protein has been purified to homogeneity (38). The purified protein has an Mr of 160,000 and has homologous pairing and strand exchange activity (Fig. 3). The SEP] DNA sequence has been compared with the GenBank DNA data base, and the translated protein sequence has been compared with both the PIR data base and a library of 152 protein signature sequences (by using the Prosite search program). These analyses did not reveal any significant matches to known protein or DNA sequences. Codon usage analysis revealed a codon adaptation index of 0.195, which is consistent with the notion that Sepl is a moderately expressed protein (67). Analysis of the 530-bp region upstream of the SEP] ORF indicates the presence of a number of potentially significant features, including (i) six potential TATA boxes (7, 74); (ii) three repeats of sequences matching the consensus sequence TTTTATTTT starting at positions 326, 358, and 381 respectively; and (iii) a run of nine T residues starting at position 271. The function of these sequences in vivo is unknown, but their presence suggests that the SEP] gene may have complex transcriptional regulation. Isolation and analysis of sepi mutations. We have undertaken a genetic analysis of SEP] to determine whether SEPJ plays a role in recombination and/or DNA repair in S. cerevisiae. TnJOLUK mutagenesis was used to obtain inser-

B

200

.1 16 97 -

66

FIG. 4. Southern blotting and Western blotting analysis of dissected heterozygous diploids containing sepl::TnlOLUK disruptions. One tetrad of each of the indicated strains was sporulated, and the resulting spore clones were propagated. The presence (Ura+) or absence (Ura-) of the sepi mutation in each clone was determined by genetic methods and is indicated above each lane by + or -. DNA and protein samples were prepared from each culture as described in Materials and Methods and analyzed for the presence and absence of the intact SEP] coding sequence and expression of intact Sepl protein by Southern blotting and Western blotting, respectively. (A) Southern analysis of a BgII digest of chromosomal DNA from the indicated strains by using the SacI-ClaI probe (see Fig. 1C and Materials and Methods). In spores derived from RKY1301 and RKY1302, the presence of a sepl allele is indicated by the loss of the 1.6-kbp fragment and the presence of the 7.7-kbp fragment. In spores derived from RKY1303, the presence of a sepl allele is indicated by the loss of the 3.7-kbp fragment and the presence of the 9.8-kbp fragment. The sizes of relevant DNA fragments are indicated on the right of the figure. (B) Western analysis of the presence of the 160,000-Mr form of Sepl in the cell extracts by using anti-Sepl monoclonal antibodies. The positions of molecular size markers run on the same gel are indicated on the right.

tion mutations in the SEP] gene. The exact locations of the five sepl: :TnlOLUK mutations selected for further study are indicated in Fig. 1 and 2. The sepl::TnlOLUK mutations were used to disrupt one copy of the SEP] gene of a diploid SK-1 strain to generate heterozygous SEPl/sepi diploids. Three of the heterozygotes (RKY1301, RKY1302, and RKY1303), containing mutant alleles 11, 64, and 79, were further characterized, and the mutant haploid derivatives of these strains all showed similar phenotypes. Sporulation of these heterozygotes (RKY1301, RKY1302, and RKY1303) and subsequent dissection of the spores showed a 2+:2segregation of sepl ::TnlOLUK with a small colony phenotype (data not shown), indicating that sepl mutations conferred a slow-growth phenotype. Southern blotting and Western blotting analysis of such segregants showed that the URA+ phenotype of these mutations cosegregated with both the presence of the disruption mutation in the SEP] gene and the complete absence of expression of full-length Sepl

2600

MOL. CELL. BIOL.

TISHKOFF ET AL. TABLE 3. Summary of mitotic effect of sepl mutations Strain

Doubling time (h)a

RKY1308 (SEPI/SEPI) RKY1310 (sepl-/llsepl-1J) RKY1309 (sepl-791sepl-79)

1.30 2.01 1.74

Small t budsa t Unbudded%

40.0 62.2 52.6

18.5 22.8 25.2

%budsa Large

Rate of His' recombinantsb

41.6 27.7 22.0

2.4 x 10-5 4.9 X 10-6 1.3 x 10-5

a Cultures of the indicated strains were grown in YPD with shaking at 30°C. At different times after they reached logorithmic phase, samples were removed and examined by light microscopy to determine the proportion of the cells that either were unbudded or had a small or large bud. b The rate of formation of His' recombinants was determined as described in Materials and Methods.

protein (Fig. 4). Two of the mutant strains, containing either the sepl-1i or sepl-64 mutation, appeared to produce very low levels of truncated derivatives of Sepl (Fig. 4). The lack of expression of full-length Sepl protein and the identical phenotypes of haploid sepi mutants containing either the sepl-11, sepl-64, or sepl-79 allele suggest that these insertion mutations are likely to be null mutations. The small-colony phenotype associated with sepi mutations suggested that sepi mutations cause a slow-growth phenotype. Growth experiments with sepi haploids and sepllsepi diploids showed that the mutant strains had growth rates that were reduced by about 25 to 35% compared with those of wild-type strains (Table 3). Cell-counting experiments performed with log-phase cultures of diploid wild-type and mutant strains showed that the mutant strains had higher levels of unbudded and small budded cells than did wild-type strains (Table 3). FACS analysis of these cultures to determine the proportion of cells which had replicated their DNA showed that the mutants had slightly more Gl-phase (unreplicated DNA) cells than did the corresponding wild-type strains (data not shown). This analysis suggests that sepi mutants are delayed in entering the S phase. Cell-counting experiments with haploid cells did not show a conclusive difference between sepi mutant and wild-type cells, even though haploid sepi cells also grow more slowly than wild-type cells. Effect of sepl mutations on repair of DNA damage. Because mutations in the genes encoding the bacterial homologous pairing proteins result in defective DNA repair phenotypes TABLE 4. Effect of UV and y irradiation on growth of sepl mutants Colony area relative to

Expt

Incubation

time (h)

Strain

U

zero

damagea; (50

U

(75J)

(100J)

kRads)

0.32 0.18 0.26 0.18

0.19 0.18 0.32 0.14

NDb

38

RKY1308 (SEPJ/SEPI) RKY1309 (sepllsepl) RKY1308 (SEPI/SEPI) RKY1309 (sepllsepl)

ND 0.52 0.41

2

24.5

RKY1107 (SEPI) RKY1228 (sepl)

0.41 0.13

0.18 0.09

0.60 0.26

3

40

RKY1107 (SEPJ) RKY1228 (sepl)

0.77 0.09

ND ND

0.75 0.20

1

26

a Late-log-phase cells were plated onto YPD plates and irradiated with the indicated dose of either UV irradiation or y irradiation. After the indicated incubation at 30°C, the diameter of 50 to 100 colonies was measured by using a dissecting microscope and the average colony area was calculated. The values reported were calculated by dividing the average area of the irradiated colonies by the average area of the unirradiated colonies for each strain. b ND, Not determined.

(10), the effects of sepi mutations on recovery from treatments that damage DNA have been analyzed. Initial experiments suggested that sepi and sepllsepi cells were not significantly more sensitive to killing by -y irradiation or methyl methanesulfonate (MMS) than wild-type cells (data not shown). Experiments to determine the sensitivity of sepi mutants to UV irradiation over a dose range of 0 to 200 J m-2 gave variable results. They suggested that sepi and sepll sepi mutants were 2- to 10-fold more sensitive to killing by UV irradiation than wild-type strains were over the dose range tested (data not shown). Although only moderately sensitive to DNA damage, sepi mutants appear to be delayed in their ability to recover from DNA damage. Table 4 contains the results of experiments in which wild-type and sepi mutant strains were treated with either UV irradiation or -y irradiation and then the area of the resulting colonies was determined during the growth of the survivors and normalized to the area of colonies of unirradiated controls. The results showed that sepi haploid strains had decreased colony sizes after treatment with either UV irradiation or -y irradiation. sepllsepi diploids had moderately decreased colony sizes after treatment with UV irradiation and possibly no significant decrease in colony size after treatment with y irradiation. Small colonies showed normal growth when restreaked on both YPD and YPG plates. To determine whether the small colony size observed was due to slow return to growth after irradiation, we treated sepl haploids with UV irradiation and analyzed them by using the microcolony assay described by Weinert and Hartwell (27, 73). The results (Fig. 5) show that the surviving sepi haploids have a long lag time before resuming growth than do similarly treated SEP] haploids after receiving a 75-J m-2 dose of UV irradiation. At 9 h after UV irradiation, none of the sepi cells had resumed growth, as evidenced by the lack of change in the distribution of cells per microcolony. In fact, only at 18 h after UV irradiation of the sepi cells did the number of microcolonies with one cell decrease from ca. 60% at time zero to ca. 40% along with an additional 15% of the cells being present as microcolonies with four or more cells per microcolony as compared with the time zero distribution (data not shown). In contrast, many SEP] cells had resumed growth by 3 h and almost all of the surviving SEP] cells had resumed growth by 9 h, as evidenced by the continuous decrease in the proportion of microcolonies having one cell. Similar results were obtained at a 50-J m-2 dose of UV irradiation, although the difference between the sepi and SEP] strains was not as pronounced (data not shown). These results suggest that sepi mutations cause an altered response to treatments that damage DNA. It is not known whether this is due to a decreased rate of repair of DNA damage or to an alteration in cell cycle regulation in response to DNA damage (27, 73).

VOL.

11, 1991

GENE FOR DNA STRAND EXCHANGE PROTEIN 1

cn

1

0

2

3

4

2601

5-9 10-14>14

E

0 C.) U

100 -

C

0 C C)

80 -

6-

0

60 40 -

20 0 1

2

3

4

5-9 10-14 >14

# Cells

1

2

3

4

5-9 10-14 >14

Microcolony FIG. 5. Microcolony analysis of return to growth after treatment of sepi and SEPI strains with UV irradiation. Cells from late-log-phase cultures of RKY1107 (SEPI) and RKY1228 (sepi) (Table 4) were plated on YPD agar plates and were irradiated as indicated with a 75-J m-2 of UV dose irradiation. After incubation at 30°C for the indicated time, the number of cells per microcolony for 150 to 200 microcolonies was determined as described previously (73). The plating efficiency for unirradiated cells was greater than 98% for RKY1107 and 85% for RKY1228, and the survival after treatment with UV irradiation was ca. 30%o for RKY1107 and ca. 15% for RKY1228, as indicated by the recovery of microcolonies containing more than four cells after 18 h of incubation. (A) Unirradiated RKY1107 (SEPI). (B) Irradiated RKY1107 (SEPI). (C) Unirradiated RKY1228 (sepl). (D) Irradiated RKY1228 (sepl). Symbols: O, no incubation; O, 3-h incubation; 0, 6-h incubation; A, 9-h incubation.

sepl mutants have a meiotic defect. Mutations in genes required for recombination and/or repair are often known to cause a defect in sporulation (21, 62). The ability of sepll sepi diploids to sporulate was studied to determine whether sepi mutations cause a meiotic defect. When sepllsepl diploids were synchronously sporulated at 30°C, they sporulated poorly compared with SEPJISEPI diploids (Table 5). Typically, seplisepi diploids showed 0 to 10o ascus formation from experiment to experiment, whereas SEPIISEPI diploids showed about 90% ascus formation. Four-spored asci formed during sporulation of sepilsepi diploids showed reduced spore viability compared with SEPI/SEPI diploids (Table 5). This was reflected by both a reduced number of viable spores and an increased proportion of four-spored asci with at least one inviable spore (Table 5). The sepilsepi cells that failed to sporulate remained viable, even after 48 h in sporulation media (50%o or greater viability [data not shown]). Similar results were observed when sporulation experiments were performed at 23°C, except that sporulation levels as high as 20% were observed (data not shown). The behavior of seplisepi diploids during sporulation experiments suggested that they arrest at some point during

per

the meiotic cell cycle. This possibility was investigated by monitoring DNA synthesis, spore wall formation, commitment to recombination of his4 heteroalleles, and nuclear division during meiosis. FACS analysis performed with propidium iodide-stained cells to monitor their relative DNA content (Fig. 6) showed that the kinetics of premeiotic DNA synthesis in both sepilsepi diploids and SEPJISEPI diploids was similar. In the example presented in Fig. 6, the midpoint of DNA replication was observed at approximately 5 h after transfer of the cells to sporulation media. FACS analysis also indicated that sepilsepi mutants did not form spore walls normally. Spore walls stain intensely with propidium iodide, resulting in a characteristic FACS analysis pattern in yeast cultures undergoing meiosis. When about 50% of a culture has formed spore walls, the resulting increase in background fluorescence begins to mask the fluorescence of propidium iodide-stained nuclei, leading to loss of the 2N and 4N fluorescence peaks. In SEPJ/SEPI cultures, this flattening occurs at 10 h after transfer to sporulation media, whereas in sepilsepi cultures the 4N peak remains visible up through at least 24 h (Fig. 6). This indicates that sepilsepi mutants undergo premeiotic DNA synthesis but arrest be-

2602

4,~ J$}h\_~

TISHKOFF ET AL.

MOL. CELL. BIOL.

TABLE 5. Effect of sepi mutations on sporulation in different genetic backgrounds'

Sporulation

Strain

Strain

Spore viability (%) 97.1e

(%)

Asci with all four spores viable (%)

His' recombinants (fold induction)

RKY1308 (SEPI/SEPI RAD501RAD50 SP0131SP013) 84.3b 88.4e 278.4b RKY1309 (sepllsepi RADSO/RAD50 SP0131SP013) 1.8b 62.le 21.8e 1,754b RKY1307 (SEPI/SEPI RAD501RADS0 spol31spol3) 64.6c 43.9e NAf 339 RKY1304 (sepllsepi RAD501RAD50 spol31spo13) L.oc 15.3e NA 255.79 RKY1300 (SEPI/SEPI radSO/radSO spol31spol3) 64 .9d 72.5d NA 1.9d RKY1305 (sepilsepl rad501rad5O spol31spol3) 4.9d 16.3d NA 0.9d a The indicated strains were synchronously sporulated as described in Materials and Methods. The levels of sporulation and spore viability were determined

by counting cultures after 48 h and subsequently dissecting at least 40 asci. Induction of His' recombinants was determined at 24 h as described in Materials and Methods. b Average of five experiments. c Average of three experiments. d Average of two experiments. ' Cumulative data from at least three experiments. f NA, Not applicable. Dyads are formed in spol3 mutants. 9 Data from one experiment.

fore spore wall formation. Return-to-growth experiments in which recombination between his4 heterolleles in two different isogenic sepilsepi diploids and one isogenic SEPI1 SEP] diploid was monitored (Fig. 7) showed that induction of recombination occurred with similar kinetics in both HOURS

B

A

"I

WJ,

2

.v 4

6

8

10

1 2

mutant and wild-type cells. If anything, the sepllsepi mutant cells showed a higher level of meiotic induction of gene conversion than the wild-type cells did (Table 5; also see Table 6). When the cells were stained with propidium iodide and examined by fluorescence microscopy to determine the number of nuclei per cell, SEPJISEPI diploids showed normal transitions from cells with one nucleus to cells with two and four nuclei and finally asci, indicative of normal progression during meiosis (Fig. 8). Similar analysis of two different isogenic sepllsepi diploids showed that most of the cells had one nucleus up through 10 h (Fig. 8). Some cells progressed further through meiosis (Fig. 8), but most of these cells had abnormal nuclei that appeared dispersed (data not shown). These results indicate that sepllsepl mutants arrest late in meiosis after premeiotic DNA synthesis and commitment to recombination but prior to the meiosis I cell division.

1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

'A

'A,* Jo-l

800 cn a

WI

1vf

600 E 0 0

03

0

400

c

0 0

200

U.

0

24

FIG. 6. FACS analysis of premeiotic DNA synthesis in synchronously sporulated cultures of sepilsepi and SEPI/SEPI diploids. Cultures of RKY1308 and RKY1309 were synchronously sporulated as described in Materials and Methods. At the indicated times in SPM, samples were removed, fixed, stained with propidium iodide, and analyzed for their relative DNA content by FACS. In each panel, the two peaks from left to right represent the relative proportions of G1 (2N) and G2 (4N) cells, respectively, and the region between the peaks represents the proportion of cells in the S phase. (A) RKY1308 (SEPI/SEPI); (B) RKY1309 (sepl-791sepl-79).

0

2

4

6

8

Hours in SPM FIG. 7. Kinetics of induction of his4 recombinants in synchronously sporulated cultures of sepllsepi and SEPI/SEPI diploids. Cultures of RKY1308 (SEPI/SEPI), RKY1310 (sepl-ll/sepi-ll), and RKY1309 (sepl-791sepl-79) were synchronously sporulated essentially as described in the legend to Fig. 6, except that after the indicated times in SPM the proportion of His' cells present was determined. Symbols: O, SEPI/SEPI; A, sepi-Illsepi-lI; 0, sepl-

791sepl-79.

GENE FOR DNA STRAND EXCHANGE PROTEIN 1

VOL. 11, 1991

TABLE 6. Meiotic induction of recombination between his4 heteroalleles in sepi mutants'

120 100

Frequency of His'

80-

Strain

60 -

RKY1308 (SEPI/SEPI)

RKY1309 (sepilsepi) 5

0

10

120

0)

B

1 rin

0

-

Fold induction

cells at:

Culture

Oh

24h

x 10-2 x 10-2 x 10-2

1 2 3 4 5

8.2 5.1 4.8 4.6 4.1

x 10-5 2.3 x 10-5 1.2 x 10-5 1.8 x 10-5 7.9 x 10-5 1.4

1 2 3 4 5

5.2 5.0 3.0 2.7 1.7

x 10-5 3.9 x x 10-5 7.4 x x 10-5 2.0 x x 10-5 7.5 x x 10-5 5.2 x

x

10-3

x

10-2

10-2 10-2

10-2 10-2 10-2

278 242 370 172 330 745 1,490 680 2,788

3,067

aFive independent cultures of each strain were synchronously sporulated as described in Materials and Methods. After the indicated time in SPM medium, cells were plated to determine the frequency of His' cells present. b The average fold induction of His' recombinants for each set of five cultures is 278 for RKY1308 and 1,754 for RKY1309.

U

0

2603

80 60 -

L-

0

cJ

0)

40 -

20 0

5

10

120 -

C

100 ^ 80 -

60 40 20 n

.9.

--mommonow-

_

5

0

10

Hours in SPM FIG. 8. Nuclear morphology of cells present in synchronously sporulated cultures of sepilsepi and SEPI/SEPI diploids. Cultures of RKY1308 (SEPI/SEPI), RKY1310 (sepi-ll/sepl-ll), and RKY1309 (sepl-791sepl-79) were synchronously sporulated, fixed, and stained essentially as described in the legend to Fig. 6, except that the cells were examined by fluorescence microscopy to determine the number of nuclei per cell. (A) SEPJ/SEPI; (B) sepl-Ill sep-1l; (C) sepl-791sepl-79. Symbols: O, cells with one nucleus; A, cells with two nuclei; cells with four nuclei; 0, spores. K,

Effect of sepi mutations on recombination. Because the bacterial homologous pairing proteins have been shown to function in recombination (10), we have performed a preliminary analysis of the effects of sepi mutations on recombination in S. cerevisiae. The effect of sepi mutations on the rate of mitotic gene conversion between his4 heteroalleles has been determined by using standard fluctuation tests to determine the rate of generation of His' recombinants during mitotic growth. The results showed a modest decrease (two- to fivefold) in the rate of His' recombinants formed per generation in the sepi mutants relative to their wild-type counterparts (Table 3). In contrast, initial results of experiments measuring meiotic induction of recombinants

between his4 heteroalleles suggested that there was greater induction of His' recombinants in sepi mutants than in the wild-type strain (Fig. 7). This was verified in an experiment in which the level of induction of His' recombinants was measured after 24 h of sporulation (Table 6). The results showed a five- to sixfold-greater average induction of His4 recombinants during meiosis in sepilsepi diploids than in SEPJ/SEPI diploids. It is not clear whether this hyper-rec phenotype is due to increased rates of recombination or an altered recombination mechanism or occurs because sepi mutants are arrested during a stage where cells are competent for recombination (3, 70). The effect of sepi mutations on meiotic crossing over has also been examined by measuring the linkage between HIS4, LEU2, and MAT on chromosome III, using random spore analysis of the limited number of viable spores that are formed during sporulation of sepllsepi strains. sepilsepi mutants showed an increase in crossing over between HIS4 and LEU2 and between LEU2 and MAT relative to the wild-type strain (Table 7). An increased frequency of apparent double recombinants (spores in which a recombination event in both the HIS4-LEU2 and LEU2-MAT intervals occurred) was also observed in sepilsepi diploids compared with the wild-type control strain (Table 7). The increased recovery of double recombinants could be due to increased numbers of independent recombination events in the HIS4LEU2 and LEU2-MAT intervals or to increased gene conversion at LEU. Our ability to address this point was limited by our ability to recover asci from sepilsepi strains with four viable spores. We observed that in 42 four-spored asci recovered from a sepilsepi diploid, there were 15 double crossover events, 1 event with a conversion at both LEU2 and MAT, and 3 events with a crossover in the LEU2-MAT interval combined with a conversion at HIS4. In 94 fourspored asci recovered from a SEPJ/SEPJ diploid, there were 7 double crossover events, 1 event with a conversion at LEU2, and 2 events with a crossover in the HIS4-LEU2 interval combined with a conversion at MAT. Because the frequency of gene conversion events among sepl double recombinants (2.7%) was not increased over that seen in SEP] double recombinants (4.3%), we suggest that the increased frequency of double crossover events on chromosome III in sepl/sepi diploids is not due to increased gene

2604

TISHKOFF ET AL.

MOL. CELL. BIOL. TABLE 7. Analysis of crossing over in sepi mutantsa % Recombinant classes observed

Strain

RKY1348

HIS' LEU- or HIS- LEUW

Relevant genotype

HIS4 LEU2

MATa

SEP]

leu27:hisG MATo (III) SEPI (VII) i (VII) HIS4 LEU2 MATa (

his4x

RKY1349

his4x

sep) leu2: :hisGUMAo sepl7

LEU+ MATot or

HIS+ LEUMATa or HIS LEU MATa (C)

Nonmaters

(A)

LEU- MATa (B)

14.3

20.6

1.4

19.5

31.4

5.9

b

NC

Cd