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Copyright 0 199 1 by the Genetics Society of America

Genetic Analysis of the Gyrase A-Like Domain of DNA Topoisomerase I1 of Saccharomyces cerm’siae

Winston Thomas, Rachelle M. Spell, Michael E. Ming’ and Connie Holm Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138

Manuscript received January 18, 199 1 Accepted for publication April 12, 199 1

ABSTRACT We have undertaken a genetic analysis of heat-sensitive and cold-sensitive mutations in TOP2, the

gene encoding yeast DNA topoisomerase 11. Deletion mapping was used to localize 14 heat-sensitive and four cold-sensitive top2 mutations created by a method biased toward mutations in the 3’ two- thirds of the gene. The mutations all appear to be located in the region of DNA topoisomerase I1 that shows homology to the “A” subunit of bacterial DNA gyrase. The heat-sensitive mutations and one cold-sensitive mutation lie in the center of the gene near the sequence that encodes the active site tyrosine. The three other cold-sensitive mutations map farther toward the 3’ end of the gene. The cold-sensitive mutations exhibit intragenic complementation, and the complementation groups correspond to the physical map. We sequenced nine top2 mutations and found that the mutations are usually single missense mutations, frequently involve proline, and affect conserved regions of the protein. Suppressor analysis yielded two intragenic suppressors and seven independent isolates of an allele-specific extragenic suppressor we named tosl; tosl is not allelic to any genes predicted to encode type I topoisomerase-related proteins. The two intragenic suppressors were tested for allele-specificity; the results revealed a complex pattern of suppression between heat-sensitive and cold-sensitive top2 alleles. These top2 mutations may have compensatory effects on the general stability of the protein.

D by NA topoisomerase I1 alters the topology of DNA

making a double-stranded cut in a DNA molecule, passing another double-stranded segment through this break and then resealing the break (BROWN and COZZARELLI 1979; LIU, LIU and ALBERTS 1980). Because of its strand-passing ability, topoisomerase 11 plays an essential role in chromosome segregation (DINARDO, VOELKEL and STERNGLANZ 1984; HOLM et al. 1985; UEMURA et al. 1987; HOLM, STEARNS and BOTSTEIN 1989), chromosome condensation (UEMURA et al. 1987), and the resolution of recombined chromosomes in meiosis (ROSE, THOMAS and HOLM 1990). Topoisomerase I1 has also been implicated in other cellular processes where the gross movement of DNA is involved, such as mitotic recombination (CHRISTMAN, DIETRICH and FINK 1988; KIM and WANC 1989), transcription (LIU and WANC 1987; BRILL and STERNCLANZ 1988; WU et al. 1988; GIAEVER and WANG 1988; TSAO, Wu and LIU 1989), and the organization of chromatin in mitotic chromosomes (EARNSHAW et al. 1985; BERRIOS, OSHEROFF and FISHER 1985; GASSER et al. 1985; AMATI and GASSER 1988). T o better understand how DNA topoisomerase I1 functions in the cell, we have pursued a genetic analysis of TOP2, the structural gene for topoisomerase I1 in the yeast Saccharomyces cerevisiae.

Yeast topoisomerase I1 is related functionally and

’ Present address: Harvard Medical School, Boston, Massachusetts 021 15.

(:enctics 128: 703-716 (August, 1991)

structurally to bacterial DNA gyrase, which is also a type I1 topoisomerase. However, while eucaryotic DNA topoisomerase 11 can only relax supercoiled DNA (GOTO and WANG 1982; GOTO, LAIPIS and WANG 1984), bacterial gyrase can actively introduce negative supercoils into double-stranded DNA (GEL- LERT et al. 1976). There are structural differences as well between the two enzymes. Yeast topoisomerase I1 is a homodimer (GOTO, LAIPIS and WANG 1984), while Escherichia coli gyrase contains two subunits in an A2B2 structure (MIZUUCHI, O’DEA and GELLERT 1978). In the predicted amino acid sequences, significant colinear similarity is seen between the amino- terminal half of yeast topoisomerase I1 and the bacterial gyrase B subunit, and between the carboxy- terminal half of yeast topoisomerase I1 and the bacterial gyrase A subunit (LYNN et al. 1986). This pattern of sequence similarity suggests that the yeast TOP2 gene could be the result of a fusion between ancestral “gyrB” and “gyrA” genes (LYNN et al. 1986). Thus it seems likely that gyrase B and gyrase A homologous domains in eucaryotic topoisomerase I1 have functions corresponding to their bacterial coun- terparts. Consistent with this hypothesis, tyrosine-783 of yeast topoisomerase I1 was correctly predicted to be the amino acid that is covalently bound to the DNA by a phosphotyrosine bond during strand cleavage and passage (LYNN et al. 1986; WORLAND and WANC 1989). In E. coli gyrase, the gyrase B subunit contains

704 W. Thomas et al.

the site of ATP hydrolysis (MIZUUCHI, O'DEA and GELLERT 1978; SUCINO and COZZARELLI 1980), and the gyrase A subunit is the catalytic domain (HOROW- ITZ and WANG 1987).

In this report we present the results of a genetic analysis of 18 temperature-sensitive (heat-sensitive and cold-sensitive) top2 mutations. We found that (1) the heat-sensitive and cold-sensitive top2 mutations are probably all located in the gyrase A-like domain, (2) the cold-sensitive mutations form two complementation groups that correspond to the positions of the mutations on the physical map, and (3) the mutations generally affect conserved regions of the enzyme. To gain insight into the relationship between the structure and function of topoisomerase 11, we also con- ducted a suppressor analysis. We isolated extragenic suppressors of top2-14 that lie predominantly within o n e linkage group and are allele-specific. Another allele, tope-13, gave rise only to intragenic suppressors. Analysis of these suppressors revealed a complex pattern of intragenic suppression, which suggests that intragenic suppression is due to an effect on a general property of DNA topoisomerase I1 rather than affecting direct interactions between amino acids.

MATERIALS AND METHODS

Strains, plasmids and media: The genotypes of the yeast strains used in this study are listed in Table 1. These yeast strains are all closely related to strain S288C. E. coli strains HBlOl (BOYER and ROULLAND-DUSSOIX 1969), JM110 (YANISCH-PERRON, VIERA and MESSING 1985), and DH5a (HANAHAN 1983) were used to amplify plasmids.

Plasmid pCH510 (Figure 1B) (HOLM et al. 1985) is an integrating yeast plasmid that contains the TOP2 gene with a deletion of the 5' end. It was constructed by inserting the BglII fragment of TOP2 (GOTO and WANG 1984) into the BamHI site of plasmid YIp5 (SCHERER and DAVIS 1979). Plasmid pCH505 (Figure 1B) (also known as pIIHZ-1 and generously provided by TADAATSU GOTO and JAMES C. WANG) is an integrating yeast plasmid that contains the TOP2 gene with a deletion of the 3' end. This plasmid was constructed by inserting a BamHI-Hind111 (partial digestion) fragment of the TOP2 gene into plasmid YIp5 that had been digested with BamHI and HindIII. Plasmids pCH505 and pCH510 were transformed (AUSUBEL et al. 1987) into E. coli strains HBlOl , JM 1 10, or DH5a and isolated either by a mini-prep procedure (AUSUBEL et al. 1987) or by large- scale plasmid preparations using an ethidium bromide/ce- sium chloride gradient essentially as described by DAVIS, BOTSTEIN and ROTH (1980).

Yeast rich medium (YPD), minimal medium (SD) and sporulation medium were made as described by SHERMAN, FINK and HICKS (1 986). 5-Fluoro-orotic acid (5-FOA) plates for selection of yeast ura3 mutants (BOEKE, LACROUTE and FINK 1984) and LB plates containing ampicillin for selection of E. coli transformants (AUSUBEL et al. 1987) were made as described.

Genetic techniques, yeast DNA isolation and transformation: Standard genetic techniques were used in yeast mating, sporulation, tetrad analysis and complementation tests (MORTIMER and HAWTHORNE 1969; SHERMAN, FINK and HICKS 1986). Yeast genomic DNA was isolated from

spheroplasted cells as described (AUSUBEL et al. 1987). DNA restriction enzyme (New England Biolabs) digestions and T4 DNA ligase (New England Biolabs) reactions were performed as described (AUSUBEL et al. 1987). Yeast transformation by the lithium acetate method was performed essentially as described by ITO et al. (1983) and modified by SHERMAN, FINK and HICKS (1 986).

Isolation of temperature-sensitive top2 mutants: Heat- sensitive and cold-sensitive top2 mutants were isolated using the integrative replacement/disruption method (SHORTLE, NOVICK and BOTSTEIN 1984). Plasmid pCH510 was mutagenized in vitro (HOLM et al. 1985) by a random misincor- poration method as described by SHORTLE et al. (1 982) and transformed into yeast strain CH561. Integration of the plasmid was directed to TOP2 by digestion with the restriction enzyme KpnI, which linearizes the plasmid by making a single cut in the TOP2 sequence. Integration into the chromosome produces one complete copy of TOP2 and one partial copy of TOP2 with a 5' deletion. The complete copy contains mutagenized DNA in a large fraction of the integrants. Integrants expressing a heat-sensitive or cold-sensitive phenotype were identified by replica-plating to complete plates lacking uracil incubated at 14", 25" or 35". Selection for excision of the plasmid on 5-FOA plates produced stable congenic wild-type and mutant strains. The congenic pairs were used for cell cycle experiments (see below). We confirmed that each new temperature-sensitive mutation was at TOP2 by tetrad analysis.

Fine-structure mapping of the TOP2 gene: The positions of top2 mutations conferring heat sensitivity and cold sensitivity were determined by a deletion mapping protocol (ORR-WEAVER, SZOSTAK and ROTHSTEIN 198 1; SHORTLE, NOVICK and BOTSTEIN 1984; ROTHSTEIN 199 1). Plasmids pCH505 and pCH510 were digested with various combinations of restriction enzymes to produce single cuts or deletions within their TOP2 sequences (see Table 2 for the restriction enzymes used and Figure 1 for the location of their recognition sites). In each case the plasmid carrying the deletion was purified away from the restriction fragment by gel electrophoresis and then electroeluted from the agarose. Each cold-sensitive and heat-sensitive top2 strain was then transformed with each pCH505 and pCH5 10 deletion plasmid. Integration of the plasmid is directed to the TOP2 locus by the homologous top2 ends of the linearized plasmid (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981). Upon integration, the missing segment is replaced using chromosomal information (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981). The transformants were first selected at the permissive temperature (25") on plates lacking uracil and then tested by spotting the transformants onto YPD plates with a pronged inoculator and then incubating the plates at 14 O ,

25 O or 35 O . The viability of the transformants at the restrictive temperature (1 4" for cold-sensitive mutants, 35" for heat-sensitive mutants) indicates whether the mutations lie in the same area of the gene as the restriction fragment that was deleted from the plasmid.

Since integration results in one active and one inactive copy of the gene, it was possible that the interpretation of the deletion mapping could have been difficult. Using the simplest assumptions, one would expect that the position of the recombination event leading to integration would determine the precise phenotype of the transformant. For example, in the case of plasmid pCH510, if recombination occurred to the 5' side of the chromosomal mutation, then the mutation would end up in the inactive copy of t o p 2 the transformant would have wild-type information in the active copy, and its phenotype would be wild type (Ts+). However, if the recombination event occurred to the 3' side of the

Genetic Analysis of Yeast TOP2

TABLE 1

Yeast strains

705

Strain Genotype Origin

CH302" CH305" CH322" CH323" CH325" CH326" CH327" CH352 CH354 CH561 CH686" CH688" CH695" CH697" CH699" CH703" CH707" CH732" CH734" CH923" CH926" CH929 CH932" CH933" CH936 CH937 CH938 CH943" CH944 CH95 1" CH952" CH954" CH959" CH960" CH961 CH962" CH963 CH986" CH987" CH 1020 CH 1047 CH 1048 CH 1072 CH 1073 CH1136 CHI165 CH 1233 CH 1298" CHI401 A831-2A

MATa top2-2::URA3 ura3-52 lys2-801am his4-539am MATa top2-4::URA3 ura3-52 lys2-801am his4-539am MATa top2-2 ura3-52 lys2-801am his4-539am MATa top2-2 ura3-52 lys2-801am his4-539am MATa top2-4 ura3-52 lys2-801am his4-539am MATa top2-5 ura3-52 lys2-801am his4-539am MATa top2-6 ura3-52 lys2-801am his4-539am MATa top2-1 ura3-52 MATa top.?-1 ura3-52 his4-619 MATa TOP2 ura3-52 lys2-801am his4-539am MATa top2-13::URA3 ura3-52 lys2-801am his4-539am MATa top2-7 ura3-52 lys2-801am his4-539am MATa top2-9 ura3-52 lys2-80Iam his4-539am MATa top2-IO ura3-52 lys2-80Iam his4-539am MATa top2-I 1 ura3-52 lys2-80Iam his4-539am MATa top2-12 ura3-52 lys2-801am his4-539am MATa top2-8 ura3-52 lys2-801am his4-539am MATa top2-13 ura3-52 lys2-801am his4-539am MATa TOP2 ura3-52 lys2-801am his4-539am MATa top2-14 ura3-52 lys2-801am his4-539am MATa TOP2 ura3-52 lys2-80lam his4-539am MATa to@-14 ura3-52 leu2-3,112 MATa top2-15::URA3 ura3-52 lys2-801am his4-539am MATa top2-15 ura3-52 lys2-801am his4-539am MATa top2-15 ura3-52 leu2-3,112 lys2-801am MATa top2- 15 leu2-3,112 MATa top2-15 leu2-3,112 lys2-80lam MATa top2-16 ura3-52 lys2-80lam his4-539am MATa top2-16 leu2-3,112 lys2-801am MATa top2-16 ura3-52 lys2-801am his4-539am MATa TOP2 ura3-52 lys2-801am his4-539am MATa TOP2 ura3-52 lys2-801am his4-539am MATa top2-17 ura3-52 lys2-801am his4-539am MATa TOP2 ura3-52 lys2-801am his4-539am MATa top2-17 leu2-3,I I 2 lys2-801am MATa top2-17 ura3-52 leu2-3,112 lys2-801am MATa top2-17 leu2-3,112 MATa top2-16::URA3 ura3-52 lys2-801am his4-539am MATa top2-I 7::URA3 ura3-52 lys2-801am his4-539am MATa top2-13 leu2-3, I12 his4-539am MATa top2-13,18::URA3 ura3-52 lys2-801am his4-539am MATa top2-16,19::URA3 ura3-52 lys2-801am his4-539am MATa top2-14 tosl-1 leu2-3,112 ura3-52 MATa top2-14 tosl-2 leu2-3,112 ura3-52 MATa top2-14 tosl-1 ura3-52 his4-539am MATa TOP2 tos l -1 ura3-52 lys2-801am his4-539am MATa top2-19 ura3-52 lys2-801am his4-539am MATa top2-14::URA3 ura3-52 lys2-801am his4-539am MATa TOP3::HIS3 his3-11,15 leu2-3,112 ade2-1 MATa hprl-A3::HIS3 his3-A200 ura3-52 leu2-k ade2 ura3

HOLM et al. (1 985) HOLM et al. (1 985) HOLM et al. (1 985) HOLM et al. (1985) HOLM et al. (1 985) HOLM et al. (1 985) HOLM et al. (1985) This study This study HOLM et al. (1 985) 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 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 R. ROTHSTEIN H. KLEIN

" These strains were derived directly from strain CH561 (also known as DBY1034) by transformation.

chromosomal mutation, the mutation would end up in the active copy, and the transformant would have the mutant phenotype. These latter transformants, although initially Ts-, when spotted to YPD plates at the restrictive temperature should frequently give rise to Ts+ papillae via gene conversion and mitotic recombination. Thus, top2 strains transformed with a plasmid bearing a deletion not overlap- ping the position of the mutation or a single cut should give rise to two different phenotypes: complete wild type growth

or papillating growth at the restrictive temperature. Before beginning the mapping experiments it was important to confirm that these phenotypes would not cause misinterpre- tation of the data.

We showed that we could recognize both wild type transformants and papillating transformants by first determining that various control transformants exhibited the expected properties. We transformed heat-sensitive (Hs-) strain CH322 (top2-2) with plasmid pCH510 that had been di-

W. Thomas et al. 706

A

“gyrase E” “gyrase A“

I y r T 8 3

4 4 2 4 6 6 9 4 7 1 4 6 1 1 5 6 8 2 4 6 2 2 6 4 9 3 1 1 6 3 5 6 6 3 7 0 5 ‘ “ 4 6 3 6 .

II I H 6 K BC Ec Hp M Hp H S Ec

B

URA 3 URA3

FIGURE 1 .-The TOP2 restriction map and the plasmids used in this study. (A) The TOP2 restriction map showing the restriction sites used in this study. The numbers above the line indicate the position in base pairs from the translation start site. The most 3’ RclI site lies outside the TOP2 coding sequence. For reference, the DNA topoisomerase I 1 active site tyrosine (WORLAND and WANC 1989) and areas of amino acid sequence similarity with bacterial gyrase B and gyrase A (LYNN et al. 1986) are indicated above the TOP2 restriction map. (B) Plasmids pCH510 and pCH505 were constructed by adding TOP2 sequence to the yeast vector YIPS (SCHERER and DAVIS 1979) which contains URA3 as a selectable rnarker (for details see MATERIALS AND METHODS). Plasmid pCH5 10 contains the top2 gene with a 5’ deletion from theBglII site. Plasmid pCH505 contains the top2 gene with a 3’ deletion from the most 9’ HindIII site. The TOP2 sequence is indicated by the filled box. Restriction enzyme abbreviations are as follows: H (HindIII), B (HglII), K (KpnI), Bc ( B c l l ) , Hp (HpaI) , M (MstII), S (SpeI).

gested with either the restriction enzype KpnI or SpeI, which cut at the 5’ or 3‘ end of TOP2, respectively. As expected, the KpnI-cut plasmid gave rise to transformants that had a nonpapillating wild-type phenotype (20/20 transformants), and the SpeI-cut plasmid gave rise predominantly to transformants tested that produced wild type papillae at the restrictive temperature (1 3/16 transformants). We re- peated this experiment, and similar ones using plasmid pCH505, with every Ts- strain. The data from these experiments suggest that the recombination event occurs at or near the restriction enzyme cut site. We further confirmed that Ts+ papillae are the result of mitotic recombination and gene conversion by testing them for uracil auxotrophy. Since Ts+ papillae are produced by mitotic recombination and gene conversion, which occur at roughly equal frequen- cies, approximately half of the Ts+ papillae should be Ura- (due to mitotic recombination), and approximately half should be Ura+ (due to gene conversion). Consistent with this prediction, 52% (30/58) of the Ts+ papillae were Ura-. In contrast, in control colonies grown at the permissive temperature less than 2% (1/58) were Ura-. These results show that for the purposes of mapping the Ts- mutations, it was appropriate to score Ts+ papillae as “wild-type” growth, indicating that the top2 mutation was not located within the deletion.

Molecular cloning and DNA sequencing of the temperature-sensitive mutations: The temperature-sensitive top2 mutations were cloned by the method of ROEDER and FINK (1 980). We took advantage of the fact that the mutations

were created in such a way that the original temperature- sensitive top2 strains contained partially duplicated copies of TOP2, separated by plasmid sequences (see Isolation of temperature-sensitive top2 mutants). Thus, we could clone the top2 mutations by excising the plasmid and top2 sequences, and ligating the ends of the linear fragment to form a circular DNA molecule. Genomic DNA from the Ts- top2 partial duplication strain was digested to completion with KpnI, diluted, and treated with T 4 DNA ligase (New Eng- land Biolabs) at a DNA concentration of approximately 1 pg/ml. E. coli strain DH5a was transformed with the ligation mix, and transformants were selected on LB plates containing ampicillin. T o confirm that the cloned plasmids contained the mutant top2 sequences, plasmid DNA was isolated from the E. coli transformants and tested for its ability to confer temperature sensitivity to a TOP2 yeast strain upon integration at the TOP2 gene. Yeast transformants were selected at the permissive temperature and then scored for temperature sensitivity.

Cloning the top2-14 mutation from the partial duplication strain required an extra step. The slow growth of top2-14 strains at the permissive temperature selects for gene conversion of the to@-14 mutation to wild-type sequence. To prevent the loss of the top2-14 mutation, a linearized plasmid deleted for the region to which top2-14 mapped was inte- grated into the chromosome of a to@-14 strain (CH923). The deleted plasmid sequence is repaired with the mutant chromosomal sequence upon integration (ORR-WEAVER, SZOSTAK and ROTHSTEIN 1981). This procedure created a partial duplication of top2-14; both top2 copies contain the mutation. Thus, the mutant sequence could not be lost by gene conversion. The top2-14 mutation was then cloned as above.

The final step in preparation for sequencing each mutation was to subclone the appropriate restriction fragment into a plasmid designed for direct DNA sequencing. The restriction fragment to which each mutation had been mapped by deletion mapping was purified by agarose gel electrophoresis and then subcloned into the plasmid pBSKS (Stratagene). The corresponding restriction fragments from unmutagenized plasmid pCH5lO were also subcloned for use as controls in the sequence analysis. Double-stranded dideoxy sequencing (SANGER, NICKLEN and COULSON 1977) was done with sequencing primers (Stratagene) and the Sequenase Sequencing Kit (U.S. Biochemical).

For the mutations in fragments too large for complete sequencing from either end, primers were made for direct sequencing of the mutation in the original plasmid (pCH5 10). The primers for the coding strand were: 14S1, 5’ TTCGAACTTGGACAGAC 3‘ (corresponds to TOP2 base pair (bp) 1854 to 1870); 14S2, 5’ CAACTTGCAC-

CATCGCCAAGA 3’ (bp 1509-1528) and 18S1, 5’ AGTGGAGCCAGAGTGGT 3‘ (bp 2439-2455); 18S2 5’ AGGATTGAACAAATTGG 3’ (bp 2668-2684). The primers for the anti-coding strand were: 14A1, 5’ ATCAT-

GACTGCTCACCATGG 3’ (bp 2224-2208); 18A1, 5’ GATGTCCAAGTTCTGGC 3‘ (bp 2732-2716); 18A2, 5’ TTAAATCTTTCATAAAA 3’ (bp 2897-2881). ”S sequencing reactions were run on 6% acrylamide salt gradient gels or in a buffer gradient of 0.5 to 0.6 X TBE and 0.0 M to 1 .O M sodium acetate (SHEEN and SEED 1988) ( 1 X TBE is 0.089 M Tris-borate, 0.089 M boric acid and 0.02 M EDTA). The wild-type sequence was run side by side with the mutant sequences. Base pair changes were confirmed by reading both mutant strands.

Cell cycle experiments: T o determine whether the new

CATACGT 3’ (bp 2 170-2 186); 14S3,5’ GGGGTTACAA-

TACCCTGCAAAG 3‘ (bp 1905-1889); 14A2 5‘ AT-


cold-sensitive top2 mutants exhibited a loss of viability specifically when progressing through the cell cycle at the restrictive temperature, the viability of cultures arrested with a-factor was compared to the viability of identical cultures not arrested with a-factor. These experiments were performed essentially as described (HOLM et al. 1985). For each cold-sensitive top2 allele, we used congenic pairs of mutant and wild-type strains (see above). Exponentially growing cultures of strains CH732 (top2-13), CH734 (TOP2),CH923(top2-14),CH926(TOP2),CH933(top2-15), CH954 (TOP2), CH951 (t@2-16), CH952 (TOP2), CH959 (top2-17), and CH960 (TOP2) were split into two portions. a-factor ( 5 pg/ml; Sigma) was added to one portion to arrest the cells at the beginning of the cell cycle (BUCKING-THROM et al. 1973). After 1.5 doubling times (2.3-3.0 hr) at the permissive temperature (30"), the cultures were again split. One portion was incubated at the restrictive temperature (14"), and the other at the permissive temperature, each for one doubling time. The four portions were then sonicated briefly and plated for viability on YPD plates at the permissive temperature. The number of colonies appearing on each plate after three days was used to calculate the number of viable cells in the original liquid culture.

Isolation of suppressors of cold-sensitive top2 mutations: The following strains were used to isolate suppressors of Cs- top2 mutations: CH732 (MATa top2-13), CH1020 (MATa top2-13), CH923 (MATa top2-14), CH929 (MATa top2-14), CH938 (MATa top2-15), CH937 (MATa top2-15), CH943 (MATa top2-16), CH944 (MATa top2-16), CH962 (MATa top2-17), and CH961 (MATa top2-17). The temperatures at which each of these strains were viable were first determined by spotting each strain on YPD plates over a range of temperatures. Revertants were then isolated at the highest restrictive temperature (ie., least stringent temperature), 13" to 17", for each strain. To be sure that we isolated independent revertants, we employed the following procedures. Starting from an individual colony at the permissive temperature, each Cs- top2 strain was grown to saturation in liquid YPD and plated on solid YPD at 30" to give well separated single colonies. One hundred individual colonies were picked for each strain and used to inoculate separate liquid YPD cultures, which were grown to saturation. These saturated cultures were plated to give a density of 5 X lo7 cells per plate and incubated at the appropriate temperature for the individual strain. Cold-resistant (Cs') colonies were picked after 8 days of growth and purified by streaking for single colonies on YPD plates. Individual colonies were retested for cold-sensitivity and also tested for heat-sensitivity. Linkage of the Cs+ phenotype to TOP2 was tested by mating each suppressor candidate to strain CH734 (TOP2), sporulating the diploid, and analyzing the resulting tetrads.

Experiments to test the allele-specificity of intragenic suppressors: To test the allele-specificity of intragenic suppressors, plasmids bearing top2 genes that contained both a heat-sensitive (Hs-) mutation and a Cs- mutation were constructed using standard recombinant DNA procedures. The individual Hs- and Cs- top2 mutations used in these con- structions had been previously cloned in plasmid pCH5lO. The Hs- top2 mutations used for this experiment map to the KpnI-MstII restriction fragment (see RESULTS). This fragment was excised by cutting the plasmid with the restriction enzymes KpnI and MstII, and it was purified by agarose gel electrophoresis. This top2 fragment containing the Hs- mutation was then used to replace the corresponding fragment in a gel-purified plasmid pCH510 backbone that contained the Cs- top2 mutation. This procedure produced a

integration X I I

TOP2

707

1002

active copy disrupted

FIGURE 2,"Integration of plasmid pCH510 containing the Hs- Cs- top2 double mutation. A haploid TOP2 strain was transformed with plasmid pCH5 10 that contained a Hs- and a Cs- mutation and had been digested with KpnI. Integration of the plasmid was directed to TOP2 by the single KpnI cut within TOP2 sequence. This procedure produces a transformant that contains both mutations in the active copy of the partial duplication. Suppression was determined by examining the phenotype of the transformant at the permissive (25") and restrictive temperatures (14' and 35"). Filled box represents TOP2 sequence derived from the plasmid; open box indicates TOP2 sequence derived from the chromosome. The "X" symbolizes the recombination event leading to integration; the boxed "Hs" or "Cs" represents the top2 Hs- or Cs- mutations. The thin line indicates plasmid YIp5 sequence.

plasmid bearing a hybrid top2 gene containing both a Hs- and a Cs- mutation.

The hybrid plasmids were used to test the intragenic suppression properties of the top2 mutations. The hybrid top2 plasmids were amplified in E. coli, cut with KpnI to direct integration to the TOP2 locus, and then used to transform haploid yeast strain CH56 1 to uracil prototrophy. Integration of this linearized plasmid into the chromosome produces one complete copy of the TOP2 gene and one copy with a 5' deletion (Figure 2). The two top2 copies are separated by plasmid sequences that include the yeast URA3 gene. Yeast transformants were selected on plates lacking uracil at the permissive temperature and then scored for viability at 14", 25 " and 35 ' to identify intragenic suppression. Since in each case the transformant carried both a Hs- and a Cs- mutation, to judge suppression we used the following criteria. If the transformant was heat sensitive but not cold sensitive we concluded that the Hs- mutation suppressed the Cs- mutation. Conversely, if the transformant was cold sensitive but not heat sensitive we concluded that the Cs- mutation suppressed the Hs- mutation. Finally, if the transformant was both cold sensitive and heat sensitive we concluded that there was no suppression. There were no cases in which the transformant was neither heat sensitive nor cold sensitive.

To eliminate possible problems in interpreting the data, we confirmed that the transformants contained both the Hs- and Cs- top2 mutations by performing the following control experiments. The presence of the Hs-top2 mutation in a phenotypically Hs+ Cs- transformant was verified in two ways. First, the transformant was plated, on 5-FOA (BOEKE, LACROUTE and FINK 1984) to select for the loss of the URA3 gene from the chromosome via mitoti recombi-

COPY


P d B L mitot ic recombination

gene conversion

FIGURE 3,"Confirmation of the presence of the Hs- top2 muta- t i o n i n the transformants intended to contain both a Hs- and a Cs- top2 mutation. An accurate interpretation of the phenotypes of the double mutants required confirmation of the presence of both the Hs- and the Cs- top2 mutations in the transformants that were intended to contain them. For example, i n the case of a phenotypic;dly Hs+ Cs- transformant it was necessary to verify the presence o f the Hs- top2 mutation. T w o methods were used. (A) In the first lnetllod the primary transformants were grown on 5-FOA plates to select for the loss of the URA? gene, and they were then tested for cold and heat sensitivity. If the recombination event leading to 5- k'OA resistance occurred between the Hs- and Cs-top2 mutations, the resulting papila would contain a Hs-top2 mutation and express the Hs- phenotype. The Hs- phenotype confirms the presence of the Hs- mutation in the original transformant. (B) In the second method, the primary transformants were grown at 14" on YPD plates to select for Cs+ revertants. Cs+ revertants can arise via gene conversion or mitotic recombination. Depending on the position of the mitotic recombination event or gene conversion tract, these revertants may contain the Hs- mutation. The Cs+ revertants were tested for uracil auxotrophy and heat sensitivity. These diagrams are intended for illustration; the events leading to Hs- papillae will not necessarily always occur as shown. Symbols are as in Figure 2.

nation (Figure 3). When the recombination event leading to excision of the plasmid sequence lay between the Hs- and Cs- top2 mutations, then only the Hs- top2 mutation re- mained in the chromosomal top2 gene after mitotic recombination. Thus, some papillae on 5-FOA plates had a heat- sensitive phenotype. Second, the transformant was plated at 14" to select for Cs+ papillae (Figure 3). Cs+ papillae arise from two approximately equally likely events: gene conversion and mitotic recombination. An intrachromosomal gene conversion event could give rise to a Cs+ Ura+ papilla. However, if the conversion tract did not extend to the Hs- top2 mutation, then the Hs- top2 mutation would not be co-converted. In this case the Cs+ papilla would have a Hs- phenotype, confirming the presence of the Hs- top2 mutation. Mitotic recombination is the other mechanism by which Cs' papillae arose from phenotypically Hs+ Cs- transformants. If the mitotic recombination event was between the Hs- and Cs- mutation, the resulting Cs+ papilla would be Hs-. Thus, the Hs- phenotype once again confirmed the presence of the Hs- top2 mutation. We performed these determinations for every phenotypically Hs+ Cs- transformant, and in each case we confirmed the presence of the Hs- mutation in the Hs+ Cs- transformant.

The presence of the Cs- top2 mutation in a phenotypically Hs- Cs+ transformant was verified using a similar strategy. T h e Hs- Cs+ transformant was plated at 35" to select for Hs+ papillae. Similar to the case above, Hs+ papillae can arise by mitotic recombination or gene conversion. In each case tested the Cs- top2 mutation is 3' t o t he Hs- top2 mutation, therefore the products of mitotic recombination are not enlightening in this instance. However, gene con-

vertants are informative. The Hs+ papillae that resulted from gene conversion events were identified by their Ura+ phenotype and then scored for cold sensitivity a t 14". A gene convertant exhibiting a Cs- phenotype indicated that the Cs- top2 mutation was present in the primary transformant. As with the Hs- top2 mutations described above, we verified the presence of the Cs- top2 mutation in each phenotypically Hs- Cs+ transformant in a similar manner.

RESULTS

Fine-structure map of TOP2: T o explore the possibility of multiple functional domains within DNA topoisomerase 11, we constructed a fine-structure map of 18 temperature-sensitive (Ts-) top2 alleles. Prior to determining the DNA sequence of the mutations, we used a deletion-mapping method (ORR-WEAVER, SZOSTAK and ROTHSTEIN 198 1 ; SHORTLE, NOVICK and BOTSTEIN 1984) to localize each mutation to a specific restriction fragment of the TOP2 gene. This approach has the advantage of insuring that the mutation identified by sequencing is the one causing the phenotype; it also reduces the amount of DNA sequencing required to find each mutation. A series of single cuts and deletions were made within the TOP2 sequence of plasmids pCH510 and pCH505 (for details see MATERIALS AND METHODS). These plasmids, with a deletion or a single cut, were then transformed into Ts- top2 strains, and the transformants were retested for growth at the permissive and restrictive temperatures.

We mapped 18 Ts- top2 mutations using various deletion plasmids. For illustration, detailed mapping data for the cold-sensitive (Cs-) top2 mutations are shown in Table 2; similar data were obtained for the Hs- top2 mutations (data not shown). The number of transformants that grew at the restrictive temperature was compared to the number that grew at the permissive temperature for every transformation. Using this type of analysis, each mutation could be mapped to a single restriction fragment (Figure 4). The fourteen Hs- mutations map to restriction fragments in the center of the gene. In contrast, three of four Cs- mutations map farther toward the 3' end of the gene. Although we do not know precisely how close to the end of a restriction fragment we can map, we success- fully mapped top2-4, top2-15 and top2-17, which sequence analysis later showed to be 22, 26 and 25 base pairs away from an end of a fragment.

Sequence Analysis of Ts- top2 Mutations: After localizing each top2 mutation to a single restriction fragment, we determined which base pair was changed by analyzing the DNA sequence of the appropriate restriction fragment. We determined the DNA sequence of nine mutations: five Cs- and two Hs- mutations, all made by in vitro mutagenesis; and two Hs- mutations, isolated as spontaneous intragenic suppressors of the Cs- phenotype of top2-13. The restriction


TABLE 2

Deletion-mapping data for cold-sensitive top2 mutations

709

pCH5 10" pCH.505"

Allele KpnI Spel HpaI HpaI Hpal BclI @el BgllI Mstl l Bcll Bcll HpaI BglIl KpnI + SpeI + Mst l l + Mstl l + Kpnl + KpnI +

top2-13 26 7 4 0 7 0 0 30 18 26 - - - - 30 30 17 30 30 24 30 30 30 30

- - - - - -

top2-14 2 1 0 3 - 3 0 - 1 - - l 9 - 9 0 30 30 42 58 30 42 30 30 30 30

top2-15 2 10 12 0 6 0 0 30 20 20 30 30 30 30 30 30 30 30 30 30

top2-16 26 5 4 0 8 0 0 30 14 15 30 30 22 30 30 30 30 30 30 30

- - - -

- - - - - - - - -

- - - - - - - - - -

t0p2-17 53 21 0 0 0 0 0 - - 56 21 38 60 60 60 60 60 60 60 60 60 60

- - - - - - - -

- 24 15 26 30 30 30

- -

10 9 6 30 30 30

30 14 29 30 30 30

- - -

- - -

- 29 11 26 30 30 30

- -

- 55 0 56 60 60 60

- -

top2 mutant strains were transformed with plasmids from which various restriction fragments had been deleted. For each entry, the upper tiumber is the number of transformants scored as wild-type growth at the restrictive temperature (14"). The lower number is the total number of transformants examined in each case. The restriction enzyme(s) used to create each deletion is indicated at the top of each column. I he location of the restriction sites in each plasmid is indicated in Figure 1. _.

I' The plasmid in which the deletion of TOP2 was made by digestion with restriction enzymes.

TOP2

"gyrase E" "gyrase A"

tyIllsl H B K Bc Bc Hp M Hp H S BC

tOp2-4 10p2- 1 l o p 2 . 2 l o p Z - 1 3

lOp2-7 lOp2-17 lOp2-15 I O p 2 - b l O p 2 - 3

t o p 2 - 5 top2-1 l l IOP2-8 t o p 2 - 1 1 l O P 2 - 9

t o p z - 1 8 t o p 2 - 1 8 t o p z - 1 9

t o p 2 - 1 4 l o p 2 - 1 2

FIGURE 4.-The map position of the Ts- top2 mutations on the TOP2 restriction map. Each top2 mutation was mapped to a restric- t i o n fragment within the TOP2 gene using information like that shown in Table 2. The Cs- top2 alleles are indicated by the super- script cs. top2-I8 is listed twice because it is a double mutation that ;tf'fects two adjacent restriction fragments. Symbols and restriction enzyme abbreviations are as in Figure 1.

fragments bearing each of the nine Ts- mutations were subcloned to a plasmid designed for DNA sequencing (see MATERIALS AND METHODS), and their DNA sequence was determined (Table 3). The sequence analysis showed that the mutations in top2-I3 (tryptophan 1123 to arginine, serine 1124 to threo- nine) and top2-I6 were identical. Because top2-13 and top2-16 were derived from the same mutagenized pool of plasmids, they are probably two independent isolates of the same mutational event. T o avoid confusion we will hereafter refer only to top2-13. The other Cs- mutations are top2-14 (leucine 720 to proline), top2- 15 (glycine 11 19 to alanine, methionine 1120 to isoleucine), and top2-17 (isoleucine 1031 to phenylala-

TABLE 3

top2 mutations affect conserved residues

lop2 allele Nucleotide change" Amino acid change" Conservationh

top2-2

top2-I? top2-4

top2-14 I5

top2-I7 t0p2- I8

top2-I 9

T 2900 + C C 2462 + A T 3367 + C T 3370 + A T 2159 + C G 3356 -+ C G 3360 + T A 3091 + T GTT 2482-84 + A A2512-T T2123-C

Leu 967 -+ Pro Pro 821 + Gln T r p 1123 + Arg Ser 1 124 + T h r Leu 720 + Pro Gly 11 19 +Ala Met 1 120 + fle Ile 1030 + Phe Val 828 + A Arg 838 -+ T r p Leu 708 + Pro

' Indicates the nucleotide(s) or amino acid(s) affected by the top2 mutation.

The number of identical or conservatively different amino acids at the corresponding amino acid in the wild-type type 11 topoisomerase of the following seven organisms: S. cereuisiae, H. sapiens, D. melanogaster, S . pombe, E. coli, E . subtilis, and phage T 4 (WYCKOFF et al. 1987; TSAI-PFLUGFELDER et al. 1988). For reference, yeast topoisomerase 11 and bacterial gyrase are 22% identical overall (LYNN et al. 1986.

' A indicates a deletion of one amino acid. * Phage T4 sequence does not extend to the region affected by

these mutations.

nine). The two Hs- mutations created in vitro both involve a proline residue: top2-2 (leucine 967 to proline), and t o p 2 4 (proline 824 to glutamine). The spontaneous Hs- intragenic suppressors are top2-18 and top2-19. top2-18 is a double mutation (deletion of leucine 828, arginine 838 to tryptophan); top2-19 is a single base pair change (leucine 708 to proline) and is one of four mutations involving proline.

Phenotypic analysis of Cs- top2 mutants: Because three of the Cs- mutations are located in a different region of the T O P 2 gene than are the Hs- mutations,


TABLE 4

a-Factor viability experiments

top2 allele a-Factor Viability"

top.2-13 + 0.73 - 0.14

top2-14 + 0.88 0.32 -

top.2-15 + 0.78 - 0.08

t0p2-17 + 0.92 - 0.23

Number of viable cells at the restrictive temperature, divided by the number at the permissive temperature, normalized to the congenic TOP2' strain.

it was possible that they would confer a novel phenotype. Heat-sensitive top2 strains become inviable when incubated at the restrictive temperature, but only if the cells are actively traversing the cell cycle (HOLM et al. 1985); if the cultures are prevented from traversing the cell cycle by treating them with the mating pheromone a-factor, they remain completely viable. T o determine whether these phenotypes are also char- acteristic of the Cs- top2 strains, we examined the viability of Cs-top2 mutants CH732 ( top2-13) , CH923 ( top2-14) , CH933 ( top2-15) , and CH959 ( top2-17) . As with Hs- top2 strains, exponentially growing Cs- top2 strains exhibit a striking loss of viability after being incubated at the restrictive temperature for one doubling time (Table 4). However, this phenotype is less severe in top2-14 or top2-17 strains, which is consistent with their slightly leaky Cs- phenotype (data not shown). (In addition to their somewhat leaky phenotype at 14 O , top2-14 strains also grow less well at 30 O and 35" than the other Cs- top2 strains.) When the Cs-top2 strains are prevented from traversing the cell cycle by treatment with a-factor, they all show en- hanced levels of viability (Table 4). These results are similar to those of experiments with Hs- top2 strains (HOLM et al. 1985). Thus, it appears that the Cs- mutations, as with the Hs- mutations, are lethal only if the cells are traversing the cell cycle at the restrictive temperature.

Complementation analysis: Although it appears that the Cs- mutations confer similar phenotypes, their disparate locations suggested that they might nonetheless affect different functional domains of the protein. In diploids heterozygous for mutations that affect two different functional domains of a protein, intragenic complementation is occasionally observed (e.g., DUNTZE and MANNEY 1968; D'ANDREA et al. 1987). To test the possibility that different alleles of T O P 2 might exhibit intragenic complementation, we performed complementation tests of the top2 Ts- alleles.

All possible pairwise crosses of the recessive Ts-

top2 mutants and wild-type strains were performed, and the diploids were scored for viability at the restrictive temperature. All of the Hs- top2 mutations fail to complement one another. The Cs- top2 mutations, however, exhibit intragenic complementation: top2-14 defines one complementation group, while top2-13, top2-15 and top2-17 define the other. As expected, a topPCs-ltop2Hs- diploid is neither Cs- nor Hs-. The intragenic complementation seen with the Cs- mutations suggests the possibility that the Cs- mutations may identify two functional domains of yeast DNA topoisomerase 11. However, this result must be interpreted with caution because top2-14 is the only member of its complementation group.

Isolation of intragenic and extragenic suppressors: T o explore structural and functional interactions within DNA topoisomerase I1 and possible interactions with other proteins in the cell, we selected intragenic and extragenic suppressors of cold-sensitive mutations in the T O P 2 gene. Suppressors were isolated by selecting for reversion of the Cs- phenotype; to facilitate further genetic analysis, we screened the cold-resistant revertants for heat sensitivity. At least two selections, one for each mating type, were performed for each Cs- top2 allele. Linkage of the Cs+ Hs- phenotype to T O P 2 was determined by tetrad analysis. Cosegregation of the Cs+ and Hs- phenotypes indicated that suppression was conferred by a mutation in top2. Conversely, independent segregation revealed that suppression was due to a mutation in a gene other than TOP2.

Both intragenic and extragenic suppressors of Cs- top2 mutations were isolated. Although all of the Cs- top2 mutants gave rise to Cs+ revertants at the rate of approximately 3-6 X lo-* Cs+ revertants per cell division, only a small fraction of the suppressors were Hs-. In fact, strains bearing top2-15 or top2-17 mutations gave rise only to Hs+ revertants. Of the 231 independent top2-13 Cs+ revertants, only two (0.9%) intragenic suppressors also expressed a secondary Hs- phenotype. These results show that top2-13 can be suppressed by two independently isolated Hs- top2 mutations.

Only one allele ( top2-14) yielded extragenic mutations that suppress its Cs- phenotype. Tetrad analysis showed that the suppressor mutations were unlinked to top2-14 in 24 of the 36 Cs+ revertants that were subjected to tetrad analysis. However, in eight of these unlinked suppressors cold sensitivity did not segregate 2:2 in sup top2-14 X top2-14 crosses, as would be expected for a single Mendelian gene. We refer to the remaining 16 unlinked suppressors of to@-14 as tos mutations (for topoisomerase suppressor). Because many of the tos mutants were not clearly recessive for suppression, it was not possible to place them into groups using complementation analysis. Therefore we

Genetic Analysis of Yeast TOP2 71 1

used tetrad analysis to group the extragenic suppressor mutations. MATa tosX top2-14 Cs+ segregants were mated to the original MATa tosY top2-14 Cs+ revertants, the diploids were sporulated, and the tetrads were dissected. Tetrad analysis showed that seven of the 16 tos mutations fell into one linkage group, which we refer to as tosl . Since we recovered multiple isolates of tosl, we focused our efforts on this extragenic suppressor gene.

Our low yield of extragenic suppressors prompted us to investigate the possibility that we were missing potential extragenic suppressors by requiring them to confer a Hs- phenotype. Thus, we performed tetrad analysis on 25 Cs+ Hs+ revertants of top2-13, top2-15, and top2-17, by crossing them to appropriate TOP2 strains. In every case the Cs+ phenotype segregated as would be expected if it was due to a compensatory mutation at tope, indicating that the suppressor mutation was intragenic. Therefore, we believe it unlikely that we missed a significant reservoir of extragenic suppressors of top2 by requiring that they be Hs-.

Analysis of the extragenic suppressor tosl: T o understand better the role of the TOSl gene product in the cell, it would be useful if tosl mutations conferred a phenotype in addition to suppression of top2- 14. Although initial tests showed that tosl top2-14 strains were Hs-, subsequent analysis showed that the Hs- phenotype was an enhancement of the weak Hs- phenotype normally conferred by top2-14. In the ab- sence of the top2-14 mutation, tosl itself does not confer heat sensitivity; tosl TOP2 strains have normal growth rates at 14", 25" and 35". The tosl TOP2 strains also appear to be no more sensitive than TOSl TOP2 strains to UV exposure, hypertonic media, or heat shock, and they do not appear to have defects in mating or sporulation (data not shown). Thus, the effects of tosl can only be recognized and analyzed in a top2-14 background.

Work in E. coli suggested that a possible suppressor of top2 mutations would be a mutation in a structural gene encoding a type I topoisomerase function; mutations in E. coli topA, the structural gene for DNA topoisomerase I, are suppressed by mutations in the genes encoding the subunits of the type 11 topoisomerase, DNA gyrase (PRUSS, MANES and DRLICA 1982; DINARDO et al. 1982). There are at least three genes in S. cerevisiae that appear to be related to, or share a functional similarity with, type I topoisomerases: TOP1, TOP3 and H P R l . Thus, we tested the possibility that TOSl was identical to one of these genes. Linkage studies showed that TOSl is not allelic to TOPI, the structural gene for yeast DNA topoisomerase I (THRASH et al. 1985; GOTO and WANG 1985); although TOPl is centromere-linked (absolutely linked to cenl5 in 23 tetrads; THRASH et al. 1985), TOSl is not strongly centromere-linked relative to the

centromere-linked marker leu2 (2.9 cM from cen3; MORTIMER et al. 1989). Tetrad analysis showed that tosl is not an allele of TOP3, which has been suggested to encode a type I topoisomerase on the basis of sequence similarity between the predicted gene products of TOP3 and E. coli topA (WALLIS et al. 1989); when strain CH1136 (top2-14 tosl-1 HZS3+) was mated to strain CH1401 (TOP2+ TOSl+ his3 TOP3::HZS3+, kindly provided by R. ROTHSTEIN), segregation of the HZS3+ gene immediately adjacent to TOP3 was incon- sistent with allelism to TOSl. Finally, linkage studies showed that TOSl was not allelic with H P R l , which encodes a protein with predicted amino acid sequence similarity with yeast DNA topoisomerase I (AGUILERA and KLEIN 1990; for review see WANG, CARON and KIM 1990); when strain CHI072 (top2-14 tosl-1 HPRl+ HZS3+) was crossed with strain A831-2A (TOP2+ TOSl+ his3 hprl-3::HZS3+, kindly provided by H. KLEIN), the segregation of the inserted HZS3' was once again incompatible with allelism.

While the linkage analysis did not provide the iden- tity of TOSl, we employed other genetic methods to obtain information regarding the mechanism of suppression by tosl; specifically, we tested the allele specificity of tosl suppression. A suppressor that en- tirely bypasses the need for topoisomerase I1 would be expected to suppress all top2 alleles. Conversely, suppressors that compensate through a physical interaction would be expected to be allele specific (BOT- STEIN and MAURER 1982; ADAMS, BOTSTEIN and DRU- BIN 1989). T o determine if tosl suppresses other Cs- top2 alleles, strain CH1165 (TOP2 tosl) was mated to strains CHI020 (top2-137, CH936 (top2-15"), CH963 (top2-17"), and, as a control, CH929 (top2-14"). Ad- ditionally, to determine if tosl suppresses Hs- top2 alleles, strains CH1072 (top2-14 t o s l - 1 ) and CH1073 (top2-14 tosl-2) were mated to strains CH322 (top2- 2hs), CH323 (t0p2-3~'), CH325 (t0p2-4~'), CH1233 (t0p2-19~'), and as a control CH923 (top2-14). (These Hs- top2 alleles were chosen because they represent the three restriction fragments to which Hs- top2 alleles map.) In tetrads from all the experimental crosses, cold sensitivity or heat sensitivity segregated 2:2, indicating that tosl does not suppress the Cs- or Hs- phenotypes of these top2 alleles. Thus, tosl mutations are allele-specific suppressors of top2-14.

Intragenic suppressors of t op2 Analysis of the nucleotide changes in the intragenic suppressors of top2-13 raised questions about the interactions between the amino acids altered by these mutations. Specifically, the suppressor mutations top2-18 and top2-19 are located many hundreds of base pairs to the 5' side of top2-13 and top2-17, near the sequence encoding the active site tyrosine (WORLAND and WANG 1989). One hypothesis was that folding of the protein results in direct physical contact between the


TABLE 5

Phenotypes of top2 double mutants

top2-13" top2-17" TOP2+

top% 18 hr Cs+ Hs- Cs' Hs- Cs+ Hs-

top2-4 hs Cs- Hs+ Cs- Hs- Cs+ Hs- TOP2+ Cs- Hs+ Cs- Hs+ Csf Hs+

top2- I Y hr Cs+ Hs- Cs+ Hs- Cs+ Hs-

Double mutations were constructed in all pairwise combinations, i n cis, and transformed into haploid strains. The transformants were then tested for temperature sensitivity. Hs- (heat-sensitive) and Cs- (cold-sensitive) indicate the phenotypes of the double mutants.

amino acids affected by top2-18, top2-19, and top2-13, and that suppression is due to compensatory changes at these contacts. Alternatively, the interaction could be indirect. A distant mutation could act as a suppressor by propagating an effect through the polypeptide chain or between domains of the protein. As with the extragenic suppressors discussed above, if there is a physical interaction, then the suppressors might be expected to be allele-specific.

T o determine whether the intragenic suppression is allele-specific, we created novel combinations of Cs- and Hs- top2 mutations in cis using the cloned mutant top2 genes. Appropriate restriction fragments were exchanged between plasmids containing the cloned genes to create all possible Cs- , Hs- top2 double- mutant combinations with the following top2 alleles: t0p2-4~", t0p2-18~' , t0p2-19~' , top2-13"" and top2-17"". The double-mutant plasmids were transformed into a wild-type yeast strain, and integration was directed to the TOP2 locus. The presence of both the Cs- and Hs- top2 mutations in the transformants was verified in each case by control experiments described in MA- TERIALS AND METHODS. Suppression was determined by examining the phenotype of the transformants at 14", 25" and 35".

The pattern of intragenic top2 suppression (Table 5) from these experiments indicates that there is specificity in the interaction between the top2 mutations, but that the interaction is probably indirect rather than a direct physical interaction. The mutations top% 1 8 and top2-19, isolated as Hs- suppressors of the top2- 13 Cs - phenotype, also suppress the Cs- phenotype of tope-17, which is 276 base pairs away from top2-13. This result raised the possibility that any Hs- mutation in the region of top2-18 and top2-19 might exhibit suppression of top2-13 and top2-17. T o test this hypothesis we examined the effects of top2-4 which is a well characterized Hs- allele that was isolated by a random in vitro mutagenesis procedure with no selection for suppression of any top2 allele (Holm et al. 1985). Unlike top2-18 and top2-19, top2-4 does not suppress the Cs- phenotype of top2-1 7 ; a toP2-4,I 7 double mutant is both Hs- and Cs- . Surprisingly, however, the Cs- allele top2-13 suppresses the Hs-

phenotype of top2-4; a top2-4,13 double mutant is Cs- but Hs+. Thus, the intragenic suppression is not a simple one-to-one correspondence between alleles, but rather a more complex pattern of interactions.

DISCUSSION

We have constructed a fine-structure map of the yeast T O P 2 gene identifying regions of DNA topoisomerase I1 that are required for the enzyme to per- form its essential function in the cell. Fourteen Hs- top2 mutations cluster in the center of the gene, near the region that encodes the active site tyrosine. Four Cs-top2 mutations fall into two groups on the physical map, and these groups correspond to groups defined by complementation tests. We sequenced four Cs- and four Hs- mutations and found that the mutations usually change a single DNA base pair, frequently involve proline, and affect evolutionarily conserved regions of DNA topoisomerase 11. Both intragenic and extragenic suppressors of Cs-top2 mutations were found. One allele, top2-13, yielded two intragenic suppressors that simultaneously confer a Hs- phenotype. We explored the allele specificity of the intragenic suppressors and uncovered a complex pattern of suppression. Only one Cs- top2 allele, top2-14, gave rise to suppressor mutations in genes unlinked to T O P 2 Seven of the suppressors were alleles of one gene, T O S l ; mutations in TOSl exhibit allele-specific suppression of top2-14.

All 18 of the Ts- top2 mutations appear to be located in the region of the gene that encodes an amino acid sequence similar to the middle domain of the three domains proposed by UEMURA, MORIKAWA and YANAGIDA ( 1 986). On the basis of sequence com- parisons of the Schirosaccharomyces pombe T O P 2 gene with bacterial gyrase, UEMURA, MORIKAWA and YAN- AGIDA have suggested that there is an amino-terminal gyrase B-like ATP-binding domain, a central gyrase A-like catalytic domain, and an extreme carboxy-terminal region of alternating acidic and basic amino acids that interacts with chromatin. It is not surprising that we found no mutations in the most 5' of these proposed domains, because the plasmid (pCH5 10) used in the screen for Ts- top2 mutants lacks the sequence encoding the 162 amino-terminal amino acids. Furthermore, we did not expect to find mutations 5' to the KpnI site (corresponding to amino acid 3 16) because integration of the plasmid was directed to the T O P 2 locus by cutting at this site. In other studies mutations have been found in the amino terminal region of type I1 topoisomerases. In S . pombe DNA topoisomerase I1 one Cs- mutation has been located in the gyrase B-like domain, at amino acid 134 (UEMURA et a l . 1987), and has been postulated to affect the ATP binding site. In E. coli, nonconditional nalidixic acid-resistant mutations in the gyrase B sub-

Genetic Analysis of Yeast TOP2 713

unit have been found at amino acids 426 and 447 (YAMACISHI et al. 1986) and are thought to affect the region of the protein responsible for binding the gyrase B subunit to the gyrase A subunit.

It is interesting that we did not isolate any mutations in the 3' region of TOP2 proposed by UEMURA, Mo- RIKAWA and YANAGIDA to encode a region of topoisomerase I1 that interacts with chromatin. While it is difficult to interpret negative results, the lack of top2 mutations in this region of the gene may be because mutations in this region lack a Ts- phenotype or because we isolated an insufficient number of mutations. Alternatively, the carboxy-terminal region of the protein may not be essential for topoisomerase I1 to function in the cell. This latter hypothesis is consistent with the finding that a s. pombe TOP2 allele with a large carboxy-terminal deletion complements S. pombe top2 null, Hs- and Cs- mutations (K. SHIOZAKI and M. YANACIDA, cited in YANACIDA and STERNC- LANZ 1990). Additionally, we know of no mutations having been isolated in this region of E. coli, Bacillus subtilis or S. pombe type I1 topoisomerase genes.

The eight Ts- top2 mutations that we sequenced affect amino acids that have been conserved during evolution and are themselves within conserved regions of DNA topoisomerase I1 (Table 3). For example, the isoleucine that is changed by the top2-17 mutation is conserved in seven other type I1 topoisomerases (WYCKOFF et al. 1989; TSAI-PFLUGFELDER et al. 1988). All of the sequenced Ts- top2 mutations change at least one amino acid to one that is not found at the corresponding position in any of these other type I1 topoisomerases.

Although all of the Hs- top2 mutations fail to complement one another, one Cs-top2 mutation, top2-14, complements all of the other Cs- mutations. This result suggests several possibilities. First, topoisomerase I1 may have multiple in vivo roles that can be performed by a hybrid dimer containing subunits with mutations in different functional domains. This type of subunit complementation has been observed in multimeric proteins containing more than one functional domain (e.g., DUNTZE and MANNEY 1968; D'ANDREA et al. 1987). A second possibility is that topoisomerase I1 is part of a nuclear structure, and the intragenic complementation is due to compensatory interactions within the structure. Finally, an attractive possibility is that top2-14 might affect the catalytic domain of topoisomerase 11, and top2-13, top2-15 and top2-I 7 might perturb the region of contact between the subunits in the topoisomerase 11 homodimer (P. CARON, personal communication). This hypothesis is consistent with the disparate map positions of the two groups of Cs- mutations. Under this last hypothesis, wild-type sequence in the region of contact of a hybrid enzyme might compensate for

the top2-13, top2-15 or top2-17 lesion and restore the proper interaction between subunits. Although all these hypotheses are certainly reasonable, it is important to note that top2-14 is the only member of its complementation group; therefore we may be observ- ing only a special effect of this allele.

The extragenic suppressor tosl: Although allele- specificity of extragenic suppressors is often informative (e.g., JARVIK and BOTSTEIN 1975; MOIR et al . 1982), the unusual properties of top2-I4 make interpretation of the tosl allele-specificity difficult. One possible interpretation of the allele-specific suppression is that topoisomerase I1 and the TOSI gene product physically interact. In this case suppression would be due to a compensatory change in the TOSl gene product. Another possibility is that a mutant TOSl gene product can bypass the requirement for the topoisomerase I1 function affected by top2-14. If tosl were such a bypass suppressor it might also suppress the nearby Hs- top2 mutations. However, we found that tosl does not suppress any of four Hs-top2 alleles that are located near to@-14. For example, tosl does not suppress top2-19 which is only 36 base pairs from top2-14. Another interpretation of the observed allele specificity is simply that it derives from the unusually leaky Cs- phenotype conferred by top2-14; we cannot discount the trivial possibility that the allele specific suppression may derive simply from top2-14 being the easiest allele to suppress.

Although we do not know the function of the TOSl gene product, we have eliminated the possibility that TOSl is allelic to any of three yeast genes that appear to encode type I topoisomerase-related proteins: TOPI, TOP3 or HPRI . The hypothesis that suppressors of top2 mutations could be in genes encoding type I topoisomerases is suggested by research on E. coli topoisomerases (PRuSS, MANES and DRLICA 1982; DINARDO et al . 1982). While suppression of topoisomerase I1 mutations by an alteration in a protein similar to topoisomerase I is an attractive hypothesis, our linkage analyses clearly indicate that TOSl is not any of the three genes predicted to encode proteins with sequence similar to type I topoisomerases: TOPI, TOP3 or HPRI. However, we cannot eliminate the possibility that TOSl encodes yet another topoisomerase-related protein.

While we do not know the function of the TOSl gene product, TOSl might encode a protein that regulates, modifies, degrades, or physically interacts with topoisomerase 11. The hypothesis that the TOSl gene product could be a regulator or modifier of topoisomerase I1 is consistent with evidence that topoisomerase I1 is phosphorylated in vitro (ACKERMAN, GLOVER and OSHEROFF 1985; SAHYOUN et al. 1986; S. GASSER, personal communication) and that the amount and stability of chicken topoisomerase I1 is


cell cycle-dependent (HECK, HITTELMAN and EARN- SHAW 1988). Alternatively, TOSl may encode a structural protein with which topoisomerase 11 interacts, such as a mitotic scaffold protein. There is evidence from a variety of systems, including chicken (EARN- SHAW et al. 1985; GASSER et al. 1985), Drosophila (BERRIOS, OSHEROFF and FISHER 1985), and yeast (AMATI and GASSER 1988), that topoisomerase I1 may be a component of a mitotic scaffold. The possibility that the top2-14 lesion affects the binding of topoisomerase I1 to the scaffold is consistent with the result that top2-14 complements the other Cs- top2 alleles. However, as discussed earlier, although the top2-14 lesion could affect an interaction with another protein, the to@-14 lesion maps near the active site tyrosine and near many Hs- top2 alleles that did not give rise to extragenic suppressors (our unpublished observa- tion). Further study is required to understand the cellular role of the TOSl gene product.

Intragenic to#2 suppression: The observed pattern of intragenic suppression indicates that many possible combinations of top2 mutations are mutually suppress- ing. We isolated top2-18 and top2-19 as suppressors of top2-13, yet they both also suppress top2-17, a mutation 276 base pairs 5' to top2-13. Furthermore, gra- tuitous suppression was also exhibited by two alleles, top2-4 and top2-13, that were isolated in a random mutagenesis scheme that imposed no selection for suppression of any top2 allele.

Intragenic suppressors that suppress more than one allele of a single gene have been seen in other systems. For example, missense mutations in the E. coli trpR gene, which encodes the trp repressor, were used to isolate intragenic suppressors (KLIG, OXENDER and YANOFSKY 1988). Many of these intragenic suppressor mutations act globally in that they suppress more than one of the original missense mutations. In the trp repressor system it appears that the intragenic suppressors suppress multiple mutations because they in- crease the activity of the trp repressor protein (KLIG, OXENDER and YANOFSKY 1988). Some of the intragenic suppressors in TOP2 may have a similar effect.

An alternative possibility is that the intragenic top2 suppression pattern results from the effect of the mutations on the stability of the encoded protein. Global intragenic suppressor mutations that appear to affect protein stability have been isolated in the gene encoding staphylococcal nuclease (nuc) (SHORTLE and LIN 1985). Missense mutations yielded three different intragenic suppressors that were able to suppress the nuclease-minus phenotype of multiple alleles (SHOR- TLE and LIN 1985). Guanidine hydrochloride denaturation studies (SHORTLE 1986) indicated that the original missense mutations caused unfolding of the enzyme at low concentrations of guanidine hydrochloride, while the suppressor mutations resulted in un-

folding of the enzyme only at high concentrations. Thus, it appears that the suppressors are able to suppress multiple distant nuc alleles because they affect the stability of the protein in an opposite manner. The TOP2 intragenic suppressors may cause suppression by a similar mechanism.

Consistent with this hypothesis, it is interesting to note that the top2 mutations that have been sequenced affect conserved, nonpolar amino acids. Conserved amino acids tend to be buried within the folded protein structure in a nonpolar, hydrophobic environ- ment (reviewed in ALBER 1989; BOWIE et al. 1990). In addition, many of the mutations involve amino acid changes to or from a proline residue. The amino acid proline is often associated with reverse turns in protein secondary structure; thus the substitution of a proline residue is likely to disrupt protein structures such as a-helices and @-pleated sheets. These changes may sufficiently affect the stability of the protein such that the entire protein becomes thermosensitive. This hypothesis is consistent with the recent finding that of three mutations affecting the stability of the fushi tarazu protein of D. melanogaster two are a proline to leucine change and one is a proline to serine change (KELLERMAN, MATTSON and DUNCAN 1990).

The hypothesis that the top2 mutations characterized in this study affect protein stability is also consistent with the lack of allele-specificity displayed by the intragenic suppressors. The complex suppression pattern of intragenic top2 suppression suggests that the suppression is unlikely to be due to contacts between specific amino acids. Therefore, the most economical hypothesis for the intragenic suppression mechanism is that the phenotypes of Hs- top2 and Cs- top2 mutations are due to effects on the stability of the protein, and the appropriate combination of two mutations restores wild-type stability at either the high or low restrictive temperature.

We would like to thank JANICE KRANZ for the a-factor viability experiments, RUTH SCHMIDT and ROBERT RAY for isolation of top2 mutations, and MEI ANN LIU for mapping top2-18 and top2-19. We thank R. ROTHSTEIN, H. KLEIN and A. AGUILERA for generously providing yeast strains. We also thank DEANNE THOMAS, DAVID EISENMANN and the members of the Holm laboratory for critical reading of the manuscript, and JUDY EMERSON for manuscript preparation. This work was supported by grants to C.H. from the National Institutes of Health (GM 36510) and the PEW Charitable Trust. W.T. was supported in part by a National Institutes of Health training grant (GM 07590).

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