MOLECULAR AND CELLULAR BIOLOGY, Apr. 1990, p. 1439-1451

Vol. 10, No. 4

0270-7306/90/041439-13$02.00/0 Copyright © 1990, American Society for Microbiology

HPRJ, a Novel Yeast Gene That Prevents Intrachromosomal Excision Recombination, Shows Carboxy-Terminal Homology to the Saccharomyces cerevisiae TOPJ Gene ANDRES AGUILERA AND HANNAH L. KLEIN* Department of Biochemistry, New York University Medical Center and Kaplan Cancer Center,

New

York,

New, York 10016

Received 5 October 1989/Accepted 11 December 1989

The HPR1 gene has been cloned by complementation of the hyperrecombination phenotype of hprl-l strains by using a color assay system. HPR1 is a gene that is in single copy on chromosome IV of Saccharomyces cerevisiae, closely linked to ARO1, and it codes for a putative protein of 752 amino acids (molecular mass, 88 kilodaltons). Computer searches revealed homology (48.8% conserved homology; 24.8% identity) with the S. cerevisiae TOP] gene in an et-helical stretch of 129 amino acids near the carboxy-terminal region of both proteins. The ethyl methanesulfonate-induced hprl-1 mutation is a single-base change that produces a stop codon at amino acid 559 coding for a protein that lacks the carboxy-terminal TOP] homologous region. Haploid strains carrying deletions of the HPRJ gene show a slightly reduced mitotic growth rate and extremely high rates of intrachromosomal excision recombination (frequency, 10 to 15%) but have a undetectable effect on rDNA recombination. Double-null mutants hprl top) grow very poorly. We conclude that Hprl is a novel eucaryotic protein, mutation of which causes an increase in mitotic intrachromosomal excision recombination, and that it may be functionally related to an activity of the topoisomerase I protein.

Repeated DNA sequences are ubiquitous in the eucaryotic They may be located in tandem on the same chromosome or on different chromosomes. They are extremely abundant in higher eucaryotes, accounting for more than 30% of the genome (13, 28), and they also exist in lower eucaryotes such as yeasts (39). The repetition of some DNA sequences has a clear biological meaning, and specific mechanisms of recombination are associated with them. Some examples are the specific recombination processes leading to immunoglobulin diversity in mammals (21), antigen variation in Trypanosoma spp. (11), and MAT interconversion in Saccharomyces cerevisiae (53). Other repetitive sequences encode known functions, yet have no specific recombination process associated with them. All these repeated sequences are also subject to general homologous recombination, which can be reciprocal (crossing-over) or nonreciprocal (gene conversion) (27, 32, 37, 52). Gene conversion between repeated DNA sequences probably plays an important role in maintaining sequence homogeneity of multigene families as well as in generating diversity (8, 19, 63). Recombination between repeated DNA sequences in S. cerevisiae has been extensively studied (reviewed in references 31 and 47). In the rDNA cluster, intrachromosomal recombination occurs less frequently than expected based on the physical length of the rDNA region (46). Recombination between 8 and between Ty DNA sequences also occurs below the expected frequency (35). These observations suggest that there may be a mechanism that maintains recombination between these DNA sequences at a low level, presumably to avoid DNA deletions that could be deleterious for the cell (6). It has been recently shown that mitotic recombination in the rDNA cluster occurs at a high frequency in strains mutated in the TOP] gene (topoisomerase I), the TOP2 gene (topoisomerase II) (16, 30), or the SIR2 gene (a gene required

for MAT switching) (23). SIR2 is also involved in rDNA meiotic recombination but not in meiotic recombination of other repeats (23). TOP] and TOP2 are not involved in recombination of repeated DNA sequences located outside the rDNA cluster (16) or in interchromosomal recombination (25). Recombination between 8 sequences near SUP4 is controlled by TOP3, a gene coding for a new eucaryotic DNA topoisomerase I (62). Mutations in these genes do not lead to an increase in recombination in sequences other than rDNA or 8 sequences. To examine the genic regulation of intrachromosomal recombination between repeated sequences in S. cerevisiae, we have isolated mutants with elevated rates of intrachromosomal gene conversion and/or pop-out recombination (pop-out recombination defines an excision of the DNA fragment located between nontandem direct DNA repeats) (3). Among these was a mutation, hprl-l, that shows a 50- to 100-fold increase in pop-out recombination, with little effect on intrachromosomal gene conversion, homologous recombination (3), meiotic recombination, and sister chromatid exchange (4). Using a DNA duplication, leu2-k::URA3-ADE2::1eu2-k, constructed to allow visual inspection of the hprl-l hyperpop-out phenotype (high levels of red sectoring in a white colony [4]), we have cloned, genetically mapped, and sequenced the HPRI wild-type gene. Computer searches have revealed a striking homology to the yeast Topl protein in the near carboxy-terminal region of 129 amino acids. Interestingly, these amino acids are missing in the protein encoded by the original hyperrecombination allele hprl-J. Null mutants of HPRI are viable and show extremely high rates of pop-out recombination (10 to 15% of the cells have lost the duplication). Double null mutants containing hprl topl grow slowly, suggesting that Hprl may have a Topl-related function that acts to prevent intrachromosomal recombination between repeats, recombination that leads to deletions of interstitial sequences.

genome.

*

Corresponding author. 1439

1440

AGUILERA AND KLEIN

MOL. CELL. BIOL. TABLE 1. Strains used in this study'

Strain

Source

Genotype

YNN217 X3271-1C

MATa lys2-801 ura3-52 his3A200 ade2-101 MATa pet14 arolD rna3 ade8 trp4 Ieu2 mal SUP2 gaI4

RS190 KM84

MATa ade2-1 trpl-J canl-100 ura3-J leu2-3 112 his3-11,15 topl-8::LEU2 MATa his4-260 ade2-1 ura3-52 leii2-3,112 trpl-h Ivs2ABXho canI ade2-1 iura3-52 rDNA:: URA3 rDNA::ADE2 MATot Ieu2-k ura3 ade2-1 hprl-l MATot leu2-k::URA3-ADE2::leu2-k ura3 ade2-1 hprl-l MATot trpl his3As200 ira3-52 MATa petl4 arolD rna3 ade8 trp4 leu2 itra3-52 MA To petl4 arolD rna3 ade8 trpl trp4 ura3-52 Ieut2 MATot arolD trp4 ade2-101 Ieu2 ura3-52 his3A200 MATot arolD trpl Ieu2 ura3-52 his3A200 MATa leu2 trpl-h his4-260 his3A200 ade2-1 ura3 rDNA::URA3 rDNA::ADE2 MATa Ieu2 trpl-h his4-260 his3A200 ade2-1 ura3 rDNA::URA3 rDNA::ADE2 MATa Ieu2 ade2-1 ura3 his3A200 rDNA::URA3 rDNA::ADE2 rpgIA2 hpr1A2::HIS3 MATa Ieu2 trpl-h ade2-1 ura3 his3zX200 rDNA::URA3 rDNA::ADE2 rpglA2 hpr1A2::HIS3 MATot leu2-k::URA3-ADE2::Ieu2-k ura3 ade2 his3A200 MATot Ieu2-k::URA3-ADE2::leu2-k ura3 ade2 his3A200 MATot leu2-k::URA3-ADE2::Ieu2-k ura3 ade2 his3A200 hprl-l MATa ura3-52 ade2 his3A200 Ivs2-801 rgplAJ::HIS3 MATct ura3 ade2 his3A200 rgplAl::HIS3 MATa ura3 his3A200 ade2 rgpIA2hprIA2::HIS3 MATct Ieu2-k::URA3-ADE2::Ieu2-k ura3 ade2 his3A200 rgplA2hprMA2::HIS3 MATot leu2-k:: URA3-ADE2::Ieu2-k ura3 ade2 his3A200 hprlA3::HIS3 MATot leu2-k:: URA3-ADE2:: Ieu2-k ura3 ade2 his3A200 hprl A3::HIS3 MATot ade2 ura3 his3A200 hprlIA3::HIS3 MATot ade2 ura3 Ieu2-k::URA3-ADE2::Ieu2-k his3A200 hprJA3::HIS3 Ivs2-801 MATTa/MATa ade2-lIade2-1 leu2-k:: URA3-ADE2::leii2-kIleu2i68 trplltrpl ura3-521ura3

A3C11 A3C11-pAA2 369-14A AX9D AX16C AX9-9C AX16-4D AKM-1B AKM-15A AKM-1D AKM-15C A3Y-1A A3Y-3A A3Y-9A AA12-1C AA12-2D AA911-4B AA914-6D A3Y1-T6 A3Y3-T1 A831-1D A831-3B AD3-9A AD2-12B

AD3-12D

HPRl/hprl-l MAToa/MATa ade2-lIade2-1 leiu2-k::URA3-ADE2::Ieui2-klleiu2A68 trplltrpl ura3-521ura3 hprl-llhprl-l MA Ta/MA Ta ade2-l/ade2-1 lea-k:: URA3-ADE2: :Iei2-klleiu2A68 trplltrpl ura3-5211ra3 hprl-llhprl-l

M. Fasullo Yeast Genetic Stock Center Wallis et al. (62) R. Keil Aguilera and Klein (4) Aguilera and Klein (4) This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

This study This study

"Strains W814-29A (MAToa) and W814-31D (MATa) (provided by R. Rothstein) carrying the same genotype as the isogenic strain RS190 were also used. The allele number is not specified when it is not known.

MATERIALS AND METHODS Strains. The yeast strains used in this study are listed in Table 1. Plasmids. Two partial Sau3A genomic DNA libraries were used, one constructed in the YEp13 vector (42) and the other in the vector pBS32 (F. Spencer and P. Hieter, unpublished data). Vector pBS32 is YCp50, where the URA3 gene has been substituted by the LEU2 gene (F. Spencer and P.

Hieter, personal communication). Vector YEp13 is a multicopy vector carrying pBR322, the 21im replication origin, and LEU2. Vector YIpl contains a 6.1-kilobase (kb) EcoRI-SalI HIS3 fragment inserted in pBR322 (54). Vector YRp7 carries pBR322 and the 1.45-kb EcoRI ARSJ-TRPJ fragment (55). Vector pBS32 was reisolated from the Spencer and Hieter gene bank as a plasmid containing no insert from a yeast transformant in the course of the screening for the HPRI gene. We named this isolate YCp70. It is identical to pBS32 and contains pBR322, CEN4, ARSI, and LEU2. YCpA13, YCpA32, and YCpA76 are the three original plasmids isolated from the Spencer and Hieter gene bank. YCpA32BS7 was constructed by subcloning the 9-kb BamHI fragment from the genomic insert of YCpA32 into the BamHI site of YCp70. YCpA32BD was constructed by excision of the 9-kb BamHI yeast DNA fragment from YCpA32. YCpA32H was constructed by excising the 0.9and 2.3-kb HindlIl fragments from plasmid YCpA32. YCpABG17 was constructed by inserting the 3.0-kb BglII

fragment of YCpA76, containing the yeast DNA insert, into the BamHI site of YCp7O. YCpABX3 was constructed by inserting the 0.45-kb XbaI fragment of the downstream region of the DED1 gene of plasmid YIpl (54) into the unique XbaI site of plasmid YCpA76. YCpABBG9 was constructed by ligating the internal 1.0-kb BglII-BamHI fragment from the insert of plasmid YCpA13 with the 10.3-kb BamHI fragment of YCpA32. YCpABGG13 was constructed by inserting the 3.4-kb BglII-BglII fragment of the insert of YCpA76 (BglII was used under conditions of partial digestion) into the BamHI site of YCp70. YRpAABGH carries the deletion allele hprJA2::HIS3. The 4.5-kb SalI-BglII fragment of plasmid YCpA32 was ligated with the 1.7-kb BamHI HIS3 fragment from YIpl and YRp7 cut with BamHI-SalI. The resulting plasmid, YRpAA414, was cut with BamHI-XbaI and ligated with the 1.2-kb BglII-XbaI fragment of plasmid YCpA76 to create plasmid YRpAABGH. The 7.3-kb SalI-XbaI fragment, which contains the internal 3.4-kb BglII insert from YCpA32 replaced by the 1.7-kb BamHI HIS3 fragment, was used to transform diploid wild-type yeast strain A3Y-3A x YNN217. pBR-ABX4 carries the deletion allele hprlA3::HIS3. The 5.8-kb BamHI-HindIII fragment (containing 0.38 kb of pBR322) from YCpA13 was inserted into the BamHIHindlIl site of pBR322 to form plasmid pBR-ABH3. The 1.2-kb BglII-XbaI internal HPRI fragment was excised from pBR-ABH3 (BglIl was used under conditions of partial

VOL. 10, 1990

THE YEAST HPRI GENE SUPPRESSES REPEAT RECOMBINATION

digestion). The 8.4-kb remaining fragment was ligated with the 2.1-kb BamHI-XbaI HIS3 fragment obtained from YIpl (BamHI was used under conditions of partial digestion) to form plasmid pBR-ABX4. The 5.0-kb EcoRI-BglII fragment containing the internal BglII-XbaI insert from YCpA13 substituted by the 2.1-kb BamnHI-XbaI HIS3 fragment was used to transform diploids A3Y-1A x YNN217 and A3Y-3A x YNN217 and the haploid strain A3Y-3A. YCpA3D1 carries the hiprl-J allele. It was obtained by gap repair (43) after transformation of the homozygous hpr 1-1 diploid strain A3D-12D with the 14.7-kb linearized BamHIXbal fragment of YCpA13. The YCpA3D1 plasmid was isolated from an unstable Leu+ transformant that showed the hyper-pop-out phenotype. YEpAB3D1 and YEpAB41 are multicopy plasmids that were constructed by insertion of the 5.8-kb BarnHI-HinidIII fragment from YCpA3D1 (carrying the hpprl-I allele) and YCpA13 (carrying the HPR1 allele), respectively, into the Ba,nHI-HindIII site of YEp13. Media and growth conditions. Standard media were prepared as described previously (51). L-Canavanine sulfate or 5-fluoroorotic acid (5-FOA) was added to synthetic complete medium (SC) at concentrations of 60 and 750 ,ug/ml, respectively, unless otherwise indicated. All yeast strains were grown at 30°C, with rotatory shaking for liquid cultures. Yeast strains were transformed by the lithium acetate method (26). Cloning of the HPRI gene. Strains carrying the duplication system leu2-k::URA3-ADE2::leu2-k were used to screen for plasmids carrying inserts able to complement the lipr1-1 mutation. The construction of this duplication has been reported (4). To use gene libraries made in vectors containing the LEU2 gene, both leu2-k alleles are identical, carrying a 7-base-pair (bp) deletion of the KpnzI site. Leu+ transformants were selected on SC-Leu supplemented with 75 jig of FOA per ml. On this medium hprl-I strains form redsectored colonies as a consequence of the high frequency of excision (2 to 4%) of the URA3-ADE2 sequences from the duplication system (Fig. 1). The sublethal concentration of FOA enhances the visualization of the red sectors, by slowing down the growth of the Ura+ Ade+ sectors to equal the growth rate of the Ura- Ade- sectors. Wild-type HPRI strains do not form sectors on this medium, because the pop-out rate is too low to give visible sectors (Fig. 1). More than 13,000 colonies were screened with the YEp13 library, and no transformant able to complement the redsectoring phenotype of hprl-J strains was found. A total of 13,581 colonies were screened after transformation with the pBS32 library. From these, 76 low-sectoring or nonsectoring transformant colonies were selected and tested for the frequency of papillation on SC-FOA. Three different transformants were finally selected (one each obtained with each of the three strains used) that consistently gave wild-type levels of papillation on SC-FOA for all Leu+ segregants tested and for which the wild-type recombination phenotype cosegregated with the Leu+ phenotype. Plasmid DNA was isolated from the three transformants and propagated through Escherichia coli. A large-scale plasmid DNA preparation was made, and the restriction map of the inserts was determined by restriction analysis with 6-bp-recognizing restriction endonucleases. Cloning of the hprl-I allele. Plasmid YCpA13 was cut with BaimHI, which cuts outside the HPRI gene, and XbaI, which cuts in the 3' end of the HPRI gene. The resulting 14.6-kb linearized plasmid with most of the HPRI coding region removed was used to transform the lipr1-1 homozygous

1441

chr. IlI it''o

-JA

ADE2

UR'A3 'oLJ2-A-

-I''Z2-4Tzz p B R 3 22

hp r 1 - 1

HPR 1

FIG. 1. At the top is a diagram of the duplication system used to visually screen the liprl-I and HPRI alleles. The leu2-k allelle is duplicated to allow the use of LEU2 vector-based libraries to clone the HPRI gene. Below this. growth of diploid strains AD3-12D (liprl-Illhprl-) and AD3-9A (HPRIIiprI-1) on SC supplemented with 75 ,ug of FOA per ml is shown. The hyper-pop-out phenotype conferred by hprl-l is observed as red sectoring, which is not seen in HPRI strains (4). hiprl-I strains were transformed with two genomic libraries and plated on the same medium lacking leucine. Nonsectored colonies were selected for further studies as transformants with

plasmids containing the wild-type HPRI gene.

diploid mutant AD3-12D. Leu + unstable transformants were selected, and the frequency of recombination leading to Ura- pop-outs was determined. In half the transformants

tested, the frequency of Ura- recombinants was at the wild-type level, and the other half were hyperrecombinant, suggesting that the mutant site was located between the XbaI site and the 3' end of the gene. Genetic analysis and determination of recombination rates. Genetic analysis was performed by published procedures (51). Genetic distances were calculated in centimorgans (cM) by Perkins' formula (45). Recombination rates were calculated by the median method of Lea and Coulson (36). Experiments to obtain recombination rates were carried out as described previously (3, 4). Yeast strains transformed with LEU2-containing plasmids were grown on SC-Leu, and after 3 days independent colonies were plated on SC-FOA to determine the rate of Ura- recombinants. UV viability

experiments were performed as reported previously (3). The UV exposure used was 0, 30, 50, 70, and 90 J/m2. Methyl methanesulfonate (MMS) sensitivity experiments were performed as described previously (48). Samples were taken from liquid 0.5% MMS incubations after 0, 10, 20 and 30 min and plated on YEPD. The mutator phenotype was determined as the frequency of Canr colonies forming on SCL-canavanine from overnight YEPD liquid cultures. DNA and RNA manipulation. DNA was isolated and used for Southern analysis as already described (4). Yeast chromosomal DNA was isolated and run on orthogonal field alternation gel electrophoresis gels by the method of Carle and Olson (14) and blotted and hybridized as described previously (4) with an incubation of the gel in 0.1 M HCI for 15 min prior to denaturation to fragment the DNA for efficient transfer. RNA was extracted from exponentially growing yeast cells as described previously (15) and fractionated on 1.2% agarose-37% formaldehyde gels as reported previously (58).

1442

MOL. CELL. BIOL.

AGUILERA AND KLEIN

A p

Bg H

YCpA1 3

Bg

R

B

)

K

I (R

X

Bg P R, ,

Ir_.

x

B

P S *iI

I

YCpA76

R H II

H

R HR. H , ,':I

IR Bg

~R

Bg P R a

.,

Xb Bg P R I

YCpA32

,I1

kb,

It

Complementation of hprl-1

B YCpA32BS7 YCpABG17 YCpABX3

YCpABGG13 YCpABBG9 YCpA32H

YCpA32BD

++ ++ ++ ++

FIG. 2. (A) Restriction maps of the three inserts isolated from plasmids that complement the hprl-l mutation. An asterisk indicates the side of the insert closest to the HindilI site of pBR322. Plasmid YCpA13 was recovered from a transformant of diploid strain AD3-12A, YCpA76 was recovered from diploid strain AD2-12B, and YCpA32 was recovered from haploid strain A3C11-pAA2. Abbreviations of restriction sites: B, BamHl; Bg, BglII; H, HindIll; K, KpnI; P, Pstl; R, EcoRI; S, Sall; Xb, XbaI; X, XhoI. (B) Deletion analysis of the 7-kb overlapping region of the three inserts shown. All subclones were made in the BamHl site of the centromere-based plasmid YCp7O. Complementation tests were done with strain A3C11-pAA2 (hprl-l).

Hybridization was performed in 5x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-5 x Denhardt solution50% formamide-25 mM KH2PO4 (pH 7.4) for 24 h. DNA sequencing. DNA fragments from the HPRI gene were subcloned into M13mplO, M13mpl8, and M13mpl9. DNA fragments were isolated from the three inserts of the original HPRI-containing plasmids YCpA13, YCpA32, and YCpA76. Both DNA strands of the HPRJ gene were sequenced by the dideoxy-chain termination method (50) with T7 DNA polymerase (Sequenase) (56) and 5'-([a-35S]thio)triphosphate (9). M13 universal primers were used for all sequencing reactions, with the exception of the sequencing of one strand of the junctions between pBR322 and the HPRI inserts in plasmids YCpA32 and YCpA76. In this case a pBR322 BamHI site primer (counterclockwise) 16-mer was used. RESULTS Isolation of the HPR1 gene. The hprl-l haploid strain A3C11-pAA2 and hprl-llhprl-J diploid strains AD3-12D and AD2-12B carrying the duplication system leu2-k:: URA3ADE2::leu2-k were transformed with the YEp13 library of Nasmyth and Tatchell (42) and with the pBS32 (YCpLEU2)library of Spencer and Hieter (unpublished). Leu+ transformants were selected on SC-Leu supplemented with 75 ,ug of FOA per ml and screened for the red-sectoring phenotype

(Fig. 1; see Materials and Methods). The map of the three DNA inserts isolated able to complement the hprl-J phenotype is shown in Fig. 2A. The three inserts overlap by 7.0 kb. The three plasmids were used to retransform the original hprl-l strains A3CpAA2 and AD3-12D. In all cases, the Leu+ transformants showed wild-type levels of Ura- recombinants, a property that was linked to the Leu+ phenotype and lost when the plasmid was lost from the cells. This result suggests that the 7.0-kb overlapping region complements the hprl-l mutation. Southern analysis showed that this region is unique in the genome (data not shown). We found, however, that plasmid YCpA32, which was isolated from haploid strain A3CpAA2, was unable to complement the hyperrecombination phenotype of homozygous hprl-llhprl1 diploid strains, in contrast to the observed complementation of a hprl-l haploid strain. This suggests that the insert in YCpA32 lacks DNA sequences required for full complementing activity when compared with the inserts in plasmids YCpA13 and YCpA76, which fully complement the hyperrecombination phenotypes of both haploid and diploid hprl1 strains. Deletion analysis of the three DNA inserts was performed to define the shortest DNA fragment able to complement the hprl-I mutation. All subcloning experiments were performed with plasmid YCp7O containing pBR322, CEN4, ARSI, and LEU2. The subcloned regions (see Materials and

VOL. 10, 1990

THE YEAST HPRI GENE SUPPRESSES REPEAT RECOMBINATION

1443

A A

B

C

A

B C

-X. i;HPW1 ;,i __ P? X.

X_

v!. v

FIG. 3. Northern blot analysis of the HPRI gene. RNA was prepared from exponentially growing cells from strains S288C (HPRI) (lane A), A364A (HPRI) (lane B), A3C11-pAA2 (hprl-I) (lane C), A3Y3-T6 (hprIA3::HIS3) (lane D), and A3Y-3A (HPRI) (lane E). The 1.2-kb BgIII-Xbal internal HPRI fragment was used as a DNA probe. All strains showed a weak 3-kb hybridizing band that is absent in the strains carrying hprMA3. The internal yeast rRNAs were used as size markers.

B PD

Methods) were used to transform the hprl-l haploid strain A3CpAA2. The results (Fig. 2B) indicate that the complementing activity is located to the right of the BamHI site and goes beyond the XbaI site. The 1.2-kb BglII-Xbal internal hprl-l complementing fragment was used as probe in Northern (RNA) experiments analysis. A unique 3-kb mRNA band was detected in wildtype strains and hprl-l mutants (Fig. 3) that was not present in hprl deletion mutants (see below). Genetic mapping of the HPRJ gene. To assign the HPRJ gene to a chromosome, whole chromosomal DNA from three different wild-type strains was fractionated on orthogonal field alternation gel electrophoresis gels and hybridized with the 2.7-kb BamHI-XbaI fragment containing most of the HPRI gene. Chromosome IV was the only one that hybridized in all three strains (Fig. 4A). The chromosomal 1.9-kb BglII-BamHI fragment located to the left of the hprl-l complementing region (Fig. 2B) was replaced by a 1.7-kb BamHI HIS3 fragment from plasmid YIpl by one-step gene replacement (49) with the 6.1-kb SalI-XbaI fragment from plasmid YRpABX. This construct creates a deletion in a novel gene, RGPI (for reduced growth phenotype), which is not essential, although it is important for mitotic growth (A. Aguilera, P. Moskowitz, and H. Klein, Nucleic Acids Res., in press). A transformant strain with the HIS3 gene inserted at the RGPI locus was used to show that the isolated DNA inserts are genetically linked to hprl-l and to accurately map the HPRI gene on chromosome IV. The inserts isolated are genetically linked in the genome to the hprl-l allele (47 of 47 tetrads were parental ditype). HPRJ is tightly linked to AROI (Fig. 4B). Since no recombinants could be found between AROJ and HPRJ (marked with HIS3), we confirmed that HPRI is not AROI by showing that both plasmids YCpA13 and YCpA32 are unable to complement the arolD deficiency. Also, the hprl-l mutant as well as the hprJA2 deletion mutant (see below) do not show an Aro- phenotype, since they are able to grow on SC-Tyr. We compared the restriction map of the HPR1 region with the reported map for the SAC6 region (1), a gene closely linked to AROI. We believe that the insert in clone YCpA32 overlaps the DNA fragment cloned by Adams et al. (1), which places the HPR1 and SAC6 genes 14 kb apart. HPR1 encodes a 752-amino-acid protein with homology near the carboxy terminus to TOP]. The HPRI gene was se-

At .. T ,

A

.

s :

>PD

TT

3



..

HIS3 iso A

'

IC2

_

33 .

-

2 4 tD

*

FIG. 7. (A) Construction of the deletion hprJA2::HIS3. The 3.4-kb Bglll-BamHI fragment containing the 5' 132 bp of HPRI was substituted by the 1.7-kb HIS3 fragment from plasmid YIpl. The resulting 7.3-kb SalI-XbaI fragment from newly constructed plasmid pBR-AABGH was used to transform diploid strain A3Y-3A x YNN217 (HPRIIHPRI). His' diploid transformants were sporulated and dissected. Southern analysis of a complete tetrad is shown. Genomic DNA was digested with BglII. Probes used were the 3.0-kb BglII HPRI 5'-end fragment and the 1.7-kb BamHI HIS3 fragment. All His' spores (lanes C and D in the gel) carried the expected replacement, as deduced from the hybridizing bands. The 9.5-kb HIS3 hybridizing band common to all spores represents the his3A200 allele in chromosome XV. (B) Construction of the deletion hprJA3::HIS3. The 1.2-kb BglII-XbaI internal HPRI fragment was replaced by the 2.1-kb BamHI-BamHI-XbaI HIS3 fragment isolated from YIpl. The resulting 5.0-kb EcoRl-BglIll fragment was isolated from plasmid pBR-XH and used to transform haploid strain A3Y1A and diploid strain A3Y-3A x YNN217. His' diploids were sporulated and dissected. All spores were viable, and cosegregation was found between the slow growth and His' phenotypes. Southern analysis of one tetrad is shown. Genomic DNA was digested with EcoRI. Probes used were the 1.2-kb BglII-XbaI internal HPRJ fragment and the 1.7-kb BamHI HIS3 fragment. Both His' spores (lanes A and B) carried the expected gene replacement as deduced from the pattern of hybridizing bands. The 10-kb HIS3 hybridizing band common to all spores represents the his3A200 allele in chromosome XV. are shown in Table 3. Increasing the copy number of the HPRI allele as well as placing it in combination with other alleles in haploid strains had no effect on either growth or pop-out recombination over the wild-type value. Therefore HPRI can exist in high copy number in the cell with no detectable phenotype. This is consistent with the fact that similar number of transformants are obtained when HPRI is in the single-copy vector YCp7O or in the multicopy vector YEp13. Allele hprl-32, which is the truncated HPRI copy carried by plasmid YCpA32, is partially functional. When this allele was the only copy of the gene in the cell (introduced in centromeric plasmid YCpA32 in a hprlA3::HIS3 null mutant strain), a 20-fold increase in pop-out recombination was observed. However, when a hprl-32 copy was present in a haploid strain together with a hprl-l copy, only a 3.8-fold increase in the recombination rate was observed. These

results suggest that the 43 amino acids at the 3' end, which missing in the Hprl-32 protein, are not essential for the ability of the Hprl protein to suppress intrachromosomal pop-out recombination. It is worth noting that the Hprl-32 protein contains the a-helical domain homologous to Topl. The hyperrecombination rate produced by the allele hprl1 (304- to 329-fold over the wild-type value) is about 4-fold lower than that produced by the deletion alleles hprJA2 and hprIA3 (833- to 1333-fold over the wild type). This suggests that the hprl-l function is leaky. This is reinforced by the observations that two copies of the hprl-l allele in a haploid strain give a Ura- recombination rate only 100-fold over the wild-type value and that multiple copies of the hprl-l allele result in a rate only 19.2-fold over the wild-type value (Table 3). These partial complementation results in YEp vectors are similar to those obtained with another leaky mutation in S. cerevisiae (2). The strong reduction in the recombination are

1448

MOL. CELL. BIOL.

AGUILERA AND KLEIN TABLE 2. Relevant properties of HPRI and hpfl.A strains

Strain

Generation time (h)"

% Surviving UV (50 JIm2) irradiation'

% Surviving 0.5% MMS treatment (20 min)"

Frequency of Can~mutants

HPRl

1.58, 1.67 2.67, 2.67

48.5. 57.7 41.7, 69.8

64.1 66.7

4.2 6.7

hprlA

Rate of

Ura-

% Germination rrecombinantsI (no. of tetrads in rDNA locus analyzed)" (o17)d (o04c

94 (22) 91 (37)

6.0. 8.9 10.3, 18.0

" Separate determinations were made in strains carrying the deletion mutation hpr1A3::HIS3 derived from A3Y3-T1 and A3Y1-T6 strains and the isogenic HPRI A3Y-1A and A3Y-3A strains. b Separate determinations were made in strains carrying the deletion mutation hprlA2::HIS3 derived from AA911-4B and AA914-6D strains and HPRI A3Y-1A and A3Y-3A strains. The percent viability was determined relative to the number of viable cells receiving no UV treatment. No difference in viability between wild-type HPRI and mutant hprIl 2::HIS3 strains was found for UV exposures of 30 J/m2 (70 to 93% viability), 70 J/m2 (22 to 43% viability), or 90 J/m2 (7 to 21%). Similar results were obtained in hprJA3::HIS3 strains. ' Determinations were made by using the same strains that were used for UV sensitivity curves. No difference in viability between wild-type HPRI and mutant hprJA2::HIS3 strains was found for 0.5% MMS incubations of 10 min (84 to 87% survival relative to untreated cells) and 30 min (38 to 50%). Similar results were obtained in hprlA3::HIS3 strains. "The frequency of Can' mutants is the median frequency of six different cultures of strains A3Y-3A (HPRI) and A3Y3-T1 (Uprl.13::HIS3). ' Each rate is the median rate obtained with AKM-1B (HPRI) and AKM-15A (HPRI) strains and AKM-1D (hprIA2::HJS3) and AKM-15C (hprIA2::HIS3) strains from the analysis of five independent cultures each. Similar results were obtained in hprIA3::HIS3 strains. J'Germination levels of spores are from isogenic heterozygous HPRIIhprl13::HIS3 and homozygous hprIA3::HJS31hprI1A3::HIS3 diploids derived from strains A3Y-3A and A3Y1-T6.

rate observed in hprl-l transformants with YEp13-hprl-I (plasmid YEpA3D1) confirms that YEp13 can be used to

increasing the gene the 250 amino acids at the carboxyl end of the Hprl protein, which are missing in the Hprl-1 protein, are essential for preventing crossovers between tandem duplications. The low activity detected in the single hprl-l strain could be explained by the low efficiency of readthrough stop codons.

overexpress the HPRI gene product by copy number. These results indicate that

DISCUSSION HPRI defines a novel yeast gene with homology to TOP] at the near-carboxy-terminal end. The wild-type HPRI gene of S. cerevisiae has been cloned by the ability to complement the hyperrecombination phenotype of a hprl-l mutant. HPRI is a novel gene which is in single copy in the haploid genome of S. cerevisiae and maps to chromosome IV closely linked to AROI. The DNA sequence shows that HPRI can potentially encode a novel 752-amino-acid protein. A significant homology has been found between amino acids 571 and 699 of the open reading frame with a region of S. cerei'isiae TOP] (48.8% conserved and 24.8% identical residues). This region from Topl is similarly related to a region of the S.

poinbe Topl protein (51.6% conserved and 28.0% identical residues). This is an ox-helical structure in all three proteins and may have some common function. Three different topoisomerases have already been reported to affect different types of intrachromosomal recombination (16, 62), and the Hprl protein has a striking carboxy-terminal homology to Topl. This suggests that Hprl may have some similarities at the functional level in common with DNA type I topoisomerases. This conclusion is reinforced by two observations: (i) hprl-I mutants, which show a very strong hyper-pop-out phenotype, code for a Hprl protein which lacks the carboxy-terminal 250 amino acids containing the xt-helical domain homologous to Topl; and (ii) hprl topl null double mutants grow more slowly than single mutants. The fact that the active tyrosine found in S. (ere'visiae Topl protein is not found in the 129-amino-acid stretch lowers the likelihood that Hprl is a DNA topoisomerase. The Hprl TABLE 3. Rates of excision of the URA3 gene in the leu2-k::URA3-ADE2::leu2-k duplication in haploid Yeast strains carrying different HPRI alleles

dosage One or more HPRI

Strain" HPRI HPRI

is

X, l.B. ..

a.

is.

i. ..

a

a

S

FIG. 8. Tetrad analysis of diploid A831-2A x W814-29A. Cosegregation between double-mutant spores hprJA3: :HIS3 topl-8:: LEU2 and a very small colony size is observed. The bottom part represents the segregation of the topl-8::LEU2 allele (-) and the hprlA3::HIS3 allele (0). Nongerminated spores are also indicated (=). The same results were obtained with crosses A831-3B x RS196 and A831-3B x W814-31D.

YCp7O YEp13

2.8 2.0 3.7

Fold increase over wild type"

1.2 0.8

1.5 YCpA3D1 (hprl-l) 0.5 1.2 YCpA32 (hprl-32) 3.7 1.5 Two HPRI YCpA13 (HPRI) 0.6 1.5 Multiple HPRI YEpA13 (HPRI) 48 20.0 One hpr/ -32 hprlA3 YCpA32 (liprl-32) One hpr/ -32 and hprl-l YCpA32 (lipl1-32) 9 3.8 one hpl/-J One hprl-/ 730 304.2 hpr 1-I YCp7O 790 329.2 Ilprl1-1 YEp13 Two liprl-J 240 100.0 hprl-/ YCpA3D1 (hprl-1) 46 19.2 Multiple hprl-J hprl-l YEpA3D1 (hprl-I) 833.3 2.000 hprl'A hprlA2 YCp7O 3,200 1,333.3 hprl&A3 YCp7O 10-6 x "The wild-type value of 2.4 is used and is the average of the two

HPRI HPRI HPRI HPRI

41

Ura_nation rate Plasmid (10-6)

Genotype

Allele gene

median rates obtained in A3Y-3A strains transformed with YCp7O and YEpl3. 6 Strains used were A3Y-3A (HPRI) and the isogenic strains A3Y3-T1 and A3Y1-T6 (hprJ113) and the closely related strains A911-4B (hprIA2) and

A3C11-pAA2 (hprl-l(.

VOL. 10, 1990

THE YEAST HPRI GENE SUPPRESSES REPEAT RECOMBINATION

protein may interact with the Topl protein through the a-helical domain. Alternatively, both proteins may bind to another factor through this domain. The 129-amino-acid a-helical stretch homologous to Hprl is not found in the human and vaccinia virus Topl proteins (40). HPRI is involved in the maintenance of low levels of recombination that leads to the excision of DNA fragments located between DNA repeats. hpriA deletion strains show extremely high rates of pop-out recombination in the leu2-k::URA3-ADE2::1eu2-k duplication system characterized in this study. Between 10 and 15% of the cells of any colony tested in these deletion mutants have excised the URA3-ADE2 genes, a value 4-fold higher than the value for hprl-l mutants and about 1,000-fold higher than the value for the wild-type HPRJ strains. The hprl-l mutation, although leaky, must have very little HPRI function, since the hyperpop-out phenotype is very strong. Another allele isolated in this study, hprl-32, which has the coding region truncated at amino acid 709, shows only a 20-fold increase over the wild type in pop-out recombination. This indicates that the carboxy-terminal 43 amino acids of the Hprl protein are important but not essential for preventing intrachromatid pop-outs. The observation that the hprl-32 allele is able to complement almost to wild type levels the hyperrecombination phenotype of haploid hprl-l mutants but not that of diploid hprl-llhprl-l mutants may reflect a required threshold of a ratio of HPRI protein to DNA content for suppression of intrachromosomal recombination. Below this threshold ratio, intrachromosomal popout recombination is enhanced. The Hprl-32 mutant protein could be 100% functional but more unstable than the wild type and therefore could lead to a leaky mutant phenotype as a consequence of the low concentration of the Hprl protein in the cell. This alternative can also account for the phenotype of the hprl-l mutation. The Hprl-1 protein could be very unstable in the cell as a consequence of a different carboxyl end. If this were the case, the mutant protein that confers the stronger hyperrecombination phenotype would be the more unstable in the cell. Recombination in hprl-l and hprl-32 mutants confirms that the region between amino acids 559 and 709, which contains the Topl homologous region, is very important for specifically preventing intrachromatid recombination between repeats. These carboxy-terminal 250 amino acids have little, if any, role in the general mechanism of homologous recombination, sister chromatid exchange, meiotic recombination, or mitotic homologous recombination as has been shown for hprl-l mutants (4, 5). HPRI is not essential for DNA repair and replication. Hyperrecombination S. cerevisiae and E. coli mutants are usually defective in either DNA repair or DNA replication (3, 22, 24, 64). Accumulation of recombinogenic lesions in the DNA that are subsequently repaired through a recombinational pathway has been suggested to be the major cause for the hyperrecombinant activity of these mutants (24, 64). The hyperrecombination phenotype of hprl mutants is unlikely to be produced by an accumulation of such lesions in the DNA. Our results indicate that HPRI is not involved in DNA repair. hprlA cells are as resistant to UV irradiation and MMS exposure as the wild-type HPRI strains are and do not have any mutator phenotype (Table 2). In contrast, hyperrecombination mutations in DNA repair genes of E. coli (64) and S. cerevisiae (10, 41) may show either mutator phenotype or sensitivity to DNA-damaging agents. Our results also suggest that the role of the Hprl protein in DNA replication is minimal. Hyperrecombination mutations in

1449

key proteins of DNA replication either are conditional (the gene is essential) or show sensitivity to MMS in S. cerevisiae (3, 24, 34). None of these phenotypes have been found in hprlA mutants. Also, plasmid stability is not greatly affected in hprl-l and hprlA mutants (data not shown). Biological role of the Hprl protein. The DNA topoisomerase TOP], TOP2, and TOP3 genes and the silencer-binding SIR2 gene product are required to maintain low levels of mitotic recombination in the rDNA region or between 8 sequences (16, 23, 30, 62). Lack of function in these genes leads to elevated levels of recombination. HPRI also acts to prevent recombination leading to DNA deletions, but may act differently from the genes mentioned above, since it is essentially involved in intrachromosomal recombination between repeated DNA sequences other than the rDNA. Hprl and Topl proteins may participate in similar but independent cellular processes. DNA topoisomerases have been proposed to enhance recombination by changes in local superhelical density (16, 23, 62). Superhelical density has been correlated with transcription (12, 38), which has also been shown to enhance recombination. HOTI, a DNA sequence from the rDNA identified as a hot spot for recombination (29), has been identified as the promotor region for RNA polymerase I and acts as a hot spot only when transcription by RNA polymerase I is active (61). Recently it has been shown that transcription by RNA polymerase I in S. cerevisiae also induces recombination (57). The effect of transcription on recombination has also been found in bacteriophages (18). It has been proposed that DNA topoisomerases may control recombination by controlling the DNA supercoiling produced during transcription (30). It has been reported that DNA topoisomerase I from Xenopus laevis is involved in nucleosome organization (7). The Hprl protein, even if it is not a DNA topoisomerase, may interact with either the chromatin or some component of the chromosome structure. The absence of Hprl protein in the cell may change the superhelicity of DNA or the chromatin structure in such a way that repeated DNA sequences can easily interact to generate deletions by recombination that otherwise are prohibited. Eucaryotes may have developed mechanisms to prevent deletions occurring between different repeated DNA families. This type of function should be more important in higher eucaryotes, in which repetitive DNA is far more abundant than in S. cerevisiae. The understanding of the mechanism by which intrachromosomal excision of DNA located between repeated sequences is prevented by the Hprl protein may provide new insights about DNA organization in S. cerevisiae and other eucaryotes. ACKNOWLEDGMENTS We thank P. Moskowitz for her excellent technical assistance in DNA sequencing, R. Keil for providing a yeast strain with the genetically marked rDNA locus, and R. Rothstein for providing the topl-8 null mutants. This work was supported by Public Health Service grant GM30439 from the National Institutes of Health. LITERATURE CITED 1. Adams, A. E. M., D. Botstein, and D. G. Drubin. 1989. A actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation. Science 243:231-233. 2. Aguilera, A. 1988. Mitotic gene conversion of large DNA heterologies in Saccharomyces cerei'isiae. Mol. Gen. Genet. 211:455-458.

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