Yeast DNA Topoisomerase II Is Encoded by a Single-Copy, … · 2017. 1. 26. · The enzyme DNA gyrase is encoded by two genes, gyrA and gyrB, and the availability of mutants and specific

Cell, Vol. 36, 1073-1080, April 1984, Copyright0 1984 by MIT

Yeast DNA Topoisomerase II Is Encoded by a Single-Copy, Essential Gene

0092.8674/84/04107308$02.00/0

Tadaatsu Goto and James C. Wang Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138

Summary

The gene TOP2 encoding yeast topoisomerase II has been cloned by immunological screening of a yeast genomic library constructed in the phage X expression vector, hgtl 1. The ends of the message encoded by the cloned DNA fragment were delimited by the Berk and Sharp procedure (Sl nuclease mapping) for the 5’ end and mapping of the polyA tail portion of a cDNA fragment for the 3’ end. The predicted size of the message agrees with the length of the message as determined by Northern blot hybridization analysis. The identity of the gene was confirmed by expressing the gene in E. coli from the E. coli promoter lac UV5 to give catalytically active yeast DNA topoisomerase II. Disruption of one copy of the gene in a diploid yeast creates a reces- sive lethal mutation, indicating that the single DNA topoisomerase II gene of yeast has an essential function.

Introduction

It is well established that DNA topoisomerases are ubiqui- tous in living organisms (for recent reviews, see Cozzarelli, 1980; Gellert, 1981 a, 1981 b; Wang, 1981). Genetic analysis and functional studies of the enzymes have been, however, largely limited to those of the procaryotes. In E. coli, the most extensively studied system to date, three topoisomerases are known. The enzyme DNA gyrase is encoded by two genes, gyrA and gyrB, and the availability of mutants and specific inhibitors of this enzyme has led to a rapid accumulation of knowledge of its biological roles. One important function of the enzyme appears to be the maintenance of the negatively supercoiled state of DNA in vivo (Gellert et al., 1976b; Drlica and Synder, 1978). The supercoiling of DNA may in turn affect a number of biological processes including the initiation of DNA replication and the transcription of certain genes (reviewed by Coz- zarelli, 1980; Gellert, 1981a, 1981 b; see also Fairweather et al., 1980; Wang, 1983; Menzel and Gellert, 1983). Another function of gyrase appears to be the resolution of two intertwined chromosomes after a round of replication (T. Steck and K. Drlica, personal communication). The gene TopA encoding E. coli topoisomerase I has been identified (Trucksis and Depew, 1981; Sternglanz et al., 1981) cloned (Wang and Becherer, 1983) and sequenced (Y.-C. Tse and J. C. Wang, unpublished data). A role for the enzyme in regulating the degree of supercoiling has been suggested (Sternglanz et al., 1981; Trucksis et al., 1981; DiNardo et al., 1982; Pruss et al., 1982; Gellert et

al., 1983) and genetic and biochemical studies of other possible roles of the enzyme are underway. Recently, a new topoisomerase in E. coli, DNA topoisomerase Ill, has been isolated (Dean et al., 1983). The gene encoding this enzyme is unknown.

In eucaryotes, two DNA topoisomerases, a type I and a type II enzyme, have been well characterized biochemically (see previously cited reviews and Sander and Hsieh, 1983; Shelton et al., 1983; Goto and Wang, 1982). Although eucaryotic topoisomerases and their procaryotic counter- parts share many characteristics, there are notable differences. The eucaryotic type I enzyme relaxes both positively and negatively supercoiled DNA (Champoux and Delbecco, 1972) whereas the procaryotic type I enzymes efficiently relax only negatively supercoiled DNA (Wang, 1971; D. Lockshon and D. R. Morris, personal communication). The eucaryotic type II enzyme, unlike bacterial gyrase, does not catalyze the supercoiling of DNA; it relaxes both positively and negatively supercoiled DNA in the presence of ATP. These and other biochemical differences between the eucaryotic and procaryotic topoisomerases may reflect differences in their physiological roles. Although the procaryotic topoisomerases appear to be involved in the maintenance of negative superhelicity in DNA, it is unclear that the eucaryotic topoisomerases serve a similar function. The bulk of the eucaryotic genome appears to be free of torsional stress (Sinden et al., 1980); however, several recent studies suggest that DNA supercoiling may have profound effects on gene expression in eucaryotes (reviewed in Weisbrod, 1982; see also Luchnik et al., 1982; Harland et al., 1983; Abraham et al., 1983).

Studies on the physiological roles of eucaryotic topoisomerases have been hindered by the lack of mutants and specific inhibitors of the enzymes. In order to learn more about the functions of eucaryotic topoisomerases, we have been studying the enzymes from the lower eucaryote yeast. The ease of genetic analysis and the recent development of recombinant DNA methodologies with this organism make yeast an ideal system for these studies. Moreover, previous studies on the yeast type I (Durnford and Champoux, 1978; Badaracco et al., 1983; Goto et al., 1984) and type II (Goto and Wang, 1982; Goto et al., 1984) DNA topoisomerases have demonstrated that the yeast enzymes are very similar to other eucaryotic topoisomerases.

To clone the genes encoding the yeast topoisomerases, we have raised specific antibodies against these enzymes, and used these antibodies to detect the antigenic determinants expressed in E. coli (see Helfman et al., 1983; Young and Davis, 1983a, 1983b). In this paper, we report the cloning of the structural gene TOP2 encoding yeast DNA topoisomerase II. Expression of the active yeast enzyme in E. coli has been achieved, confirming that the gene identified by immunological screening is indeed the structural gene encoding yeast DNA topoisomerase II. Furthermore, inactivation of one copy of the gene in a diploid strain and tetrad analysis of the spores derived

Cell 1074

from the resulting heterozygote show clearly that TOP2 is an essential gene, and that one copy is present in each haploid genome. From screening a collection of temperature-sensitive yeast mutants, DiNardo et al. (1984) have recently identified a mutation that is most likely in TOP2 they have also concluded that the single-copy gene is essential.

Results

Identification of Sequences Encoding Yeast DNA Topoisomerase II in Xgtl 1 Yeast Genomic Library We have screened a yeast genomic library constructed with the phage X expression vector, Xgtll (Young and Davis, 1983a, 198313). Each of the Xgtl 1 recombinants contains a yeast DNA fragment inserted within the /acZ gene of the phage genome; therefore the recombinants are expected to produce hybrid proteins of @galactosidase and yeast proteins encoded by the inserts. The proteins from phage plaques were transferred to a nitrocellulose sheet and screened with antibodies directed against yeast topoisomerase II, as described by Young and Davis (1983b). Of 3 X lo5 phage plaques screened, five repro- ducibly gave positive signals (see Figure 1). These were designated XF13 to XF17. Restriction mapping confirms that all five share sequences in their yeast inserts. The restriction maps of hF14 and XF15 appear to be identical, and the two are most likely siblings. Unexpectedly, not all positives are in the same orientation in the Xgtl 1 genome: XF13 and XF17 are oriented in one way, and the other three in the opposite way (see Discussion).

Frgure 1. Plaque Screening with Anti-toporsomerase II Antibody

Autoradiograms of three frlters are shown: (A) 2 X 103 phage Xgtll recombinants with no positives; (6) phage XF13 (a positive clone) after plaque purification; and (C) mixture of phages shown in (A) and (B). The three strong spots seen near the edge of filter (C) are position markers. The three filters were simultaneously treated with the same antibody solution and then with ProteinA solution.

The expected length of the coding sequence for the 150,000 dalton yeast topoisomerase II (Goto and Wang, 1982; Goto et al., 1984) is about 4.5 kb. However, most of the DNA fragments obtained are less than 4 kb in length. Therefore, in order to isolate the complete topoisomerase II or TOP2 gene, we have screened a YEp24 plasmid library that contains yeast DNA inserts lo-20 kb in length (Carlson and Botstein, 1982) using the yeast DNA insert from XF13 as a hybridization probe. One of the positives from this screening was purified and its insert mapped with a number of restriction enzymes. The top line in Figure 2 depicts a portion of this restriction map. Southern blot hybridization analysis of yeast nuclear DNA gives an identical restriction map, ruling out the possibility of cloning artifacts.

Alignment of the restriction sites in the yeast DNA inserts in kF13 to XFI 7 shows that all the inserts are derived from the mapped region of the yeast genome. The locations of these inserts and their orientations in the Xgtl 1 vector are shown in Figure 2; the letter Z denotes the junction with the N-terminal portion of the P-glactosidase gene in the phage vector.

The Transcript of the TOP2 Gene In order to map the 3’ end of the TOP2 transcript, we screened a yeast cDNA library (McKnight and Mc- Conaughy, 1983) with the yeast insert from XF13 as a probe. This library was constructed by annealing vector DNA molecules tailed with oligo(dT) at a unique end to the polyA tails of the messenger RNAs (McKnight and Mc- Conaughy, 1983; Okayama and Berg, 1982). This procedure fixes the orientation of the cDNAs in the clones, allowing the identification of the polyA end of inserts by restriction mapping. A clone containing a 0.8 kb fragment that hybridizes with the probe was found, and the location of the fragment within TOP2 was identified by restriction

6 R HR RK R H A I II I

Fl3 ZI I

Fl4.15 I IZ

F16 ‘Z

F17 Zl

CCNA MAA

Figure 2. Restriction Map of the TOP2 Clones

The restriction map shown at the top was determined with a genomic clone. The symbols used for restriction enzymes are (B) Bam HI, (R) Eco RI, (H) Hind Ill, (K) Kpn I, and (A) Ava I. The vertical arrow indicates the 5’ end of mRNA as determined by Sl nuclease mapping. The horizontal bars under the map indicate the positrons and sizes of Inserts in Xgtll and cDNA clones. The hF17 insert extends 1.3 kb beyond the Ava I site. Z denotes the amino-terminal portion of the P-galactosidase gene (see text). AA identifies the polyA end of the cDNA insert; the position and size of the Insert was determined by locating unique Pvu I and Pvu II sites (not shown) wrthin the fragment.

Structural Gene of Yeast DNA Topoisomerase II 1075

mapping. This is shown at the bottom of Figure 2. Since the length of the polyA tail in the cDNA clone is not known, the location of the polyadenylation site of TOP2 has an uncertainty of one to two hundred bases.

From Sl nuclease mapping with a 5’-end-labeled DNA probe (Berk and Sharp, 1977; Weaver and Weissman, 1979) it was found that the transcriptional start lies within the 700 bp Eco RI fragment as indicated by the vertical arrow in the upper map shown in Figure 2. The transcriptional start and the polyadenylation site shown in Figure 2 are separated by about 4.5 kb. This length is in excellent agreement with the length of the TOP2 message determined by Northern blot hybridization (Figure 3).

Confirmation of Gene Identity: Expression of Active Yeast DNA Topoisomerase II in E. coli The results described above do not preclude the possibility that we have inadvertently cloned a gene other than the structural gene of yeast DNA topoisomerase II, since we cannot rule out the possibility that the antibodies in our preparation recognize more than one yeast protein. To demonstrate that the cloned gene is in fact TO/Q, we have expressed the cloned yeast gene in E. coli to give catalytically active yeast topoisomerase II.

We first investigated whether TOP2 is expressed in E. coii without further manipulations of the gene. The 6.4 kb Barn HI-Ava I fragment containing the entire TOP2 gene (Figure 2) was cloned into a multicopy plasmid derived from pBR322. This recombinant plasmid did not direct the synthesis of a detectable amount of antigenic determinants of yeast topoisomerase II in E. coli. We have therefore constructed a plasmid that allows the expression of TOP2

e 4362

e 3612

e 2938

e 1424

Figure 3. Northern Blot Hybridization of the TOP2 Transcript

Oligo(dT)-selected yeast RNA was run In a 1% agarose gel containing formaldehyde, blot-transferred to a nitrocellulose sheet, and hybridized to radioactively labeled XF13 DNA. Length (in bases) and mobility of single- stranded DNA markers are indicated.

from an E. coli promoter, lac UV5. A plasmid containing the entire TOP2 gene was cut at the unique Barn HI site; as indicated in Figure 2, the Barn HI (B) site is about 1.8 kb from the start of the message. Bal31 nuclease digestion of the Barn HI-cut plasmid was then carried out to remove an average of 1.8 kb of DNA from each end. After addition of Barn HI linkers, the DNA was cut with Barn HI and Ava I. Fragments containing the yeast topoisomerase II gene were inserted in an expression vector downstream from an E. coli lac UV5 promoter. The expression vector (Wang et al., 1983) also contains a 1 .l kb lac repressor gene with an iq up-promoter mutation. Efficient expression from the promoter should occur only in the presence of the inducer IPTG. The clones were screened by the colony-screening procedures of Young and Davis (1983a) with and without IPTG for the presence of antigenic determinants that are recognized by antibodies against the yeast enzyme. Sev- eral clones that showed strong signals in response to IPTG induction were purified. Plasmid DNA was prepared from each clone and the junction between TOP2 and the lac UV5 promoter was located. The clone that gave the strong- est signal contained a plasmid, designated pllElO1, which has the junction about 30 bp upstream of the transcriptional start determined by Sl nuclease mapping. Partial purification of the yeast topoisomerase II activity from this clone was then carried out as described under Experimen- tal Procedures. This purification removes contaminating nucleases as well as E. coli topoisomerases.

Figure 4 shows the results of the activity assays done with partially purified protein from the clone containing pllElO1. Lanes a-g in Figure 4 depict results of the unknotting assay of type II DNA topoisomerase activity (Liu et al., 1981; Goto and Wang, 1982). Lane a shows the agarose gel electrophoresis pattern of the knotted phage P4 DNA used in the assay (Liu et al., 1981). The bulk of the DNA is in the form of nicked rings containing knots of various degrees of complexity, and runs as a broad smear. The sharp band above the smear consists of nicked rings without knots and the sharp band below that band is linear P4 DNA. Lane g shows the pattern after treatment, in the presence of ATP, of the knotted DNA with DNA topoisomerase II purified from yeast; knotted rings are converted to the form without knots. The partially purified activity from E. coli catalyzes this conversion if ATP is present (lane c), but not if ATP is absent (lane b), as expected for a type II activity. Lanes d-f show the activity in the presence of ATP plus rabbit antibodies against yeast DNA topoisomerase II (lane d), novobiocin (lane e), or oxolinic acid (lane f). Clearly, the activity is inhibited by antibodies against the yeast enzyme but not by drugs that specifically inhibit E. coli DNA gyrase, or its derivative, DNA topoisomerase II’ (Gellert et al., 1976a, 1977, 1979; Sugino et al., 1977, 1978; Brown et al., 1979). We have previously shown that yeast topoisomerase II is not inhibited by these drugs at the concentrations (50 rg/ml) used in these assays (Goto and Wang, 1982; Goto et al., 1984). Lanes h-j depict the results of assays of the activity with negatively supercoiled

Cell 1076

Figure 4. Detection of the Yeast Topoisomerase II Activity Produced in E. coli Containing pilElO

The topoisomerase activity from IPTG-induced E. coli harboring pIlElO was purified through phosphocellulose and aminopentyf-agarose columns as described under Experimental Procedures. Four units (see Goto et al., 1964) of this activity were added to the samples shown in lanes b-f, i, and j. The reactions were done under standard conditions (Goto et al., 1984) unless otherwise stated. Lanes a-g show the results of unknotting assays with phage P4 knotted DNA. (a) control (DNA only): (b) without ATP; (c) with ATP; (d) with 0.7rg of anti-topoisomerase II antibody; (e) with 50 pg/ml novobiocin; (f) with 50 rg/ml oxolinic acid; (g) with 4 U of topoisomerase II purified from yeast. Lanes h-i show the results of relaxation assays with pBR322. (h) control (DNA only); (i) without ATP; (j) with ATP. Under standard conditions, the amount (4 U) of enzyme used in each lane is approximately twice as much as required for the completion of the unknotting reaction. The percentage of the inhibition of E. coli DNA gyrase has been reported to be 100% with 3 @g/ml of novobiocin (Gellert et al., 1976b), and 96% with 50 rg/ml of oxolinic acid (Gellert et al., 1977).

plasmid DNA. The ATP-dependent relaxation of the negatively supercoiled DNA is observed, further confirming that the activity is yeast DNA topoisomerase II. Under these assay conditions, E. coli DNA gyrase catalyzes the negative supercoiling of DNA; all the other known E. coli DNA topoisomerases (I, II’, and Ill) catalyze relaxation of negatively supercoiled DNA, but the reaction is ATP-independ- ent (Wang, 1971; Brown et al., 1979; Gellert et al., 1979; Dean et al., 1983).

Demonstration that TOP2 is a Single-Copy, Essential Gene To test whether topoisomerase II performs an essential function, we have carried out a one-step gene-disruption experiment using the fragment-mediated transformation procedure described in Rothstein (1983). A plasmid containing the yeast TOP2 gene fragment spanning the right- most Eco RI site in Figure 5A to the first Bgl II site to its right (solid bar in Figure 5A) was first constructed. A 1.2 kb Hind III fragment containing the selectable marker Ufa3 was then cloned into the Hind Ill site in the Eco RI-Bgl II segment (Figure 5A). The Eco RI-Bgl II piece, now containing the URA3 insert, was excised from the vector and

URA3 4 ,’ ‘. ,’ P 6

.\ \. ,’ H? K H

A AA A

1 >

A 3.1 kb 6.3 kb t t-h

C TopoU probe Ura 3 probe I I

‘C D MHz C D HlH2 1

9.5 ---, 6.3 + + 6.3

+ 5.2

4.1 4 e 4.1 + 3.7

Figure 5. Demonstration That TOP2 is a Single-Copy, Essential Gene

(A) Relevant restriction sites of TOP2 and URA3 loci are shown. Symbols used for restriction enzymes are (P) Pst I, (B) Barn HI, (H) Hind III, (Bg) Bgl II, (K) Kpn I, and (A) Eco RI. The hollow arrow symbolizes the TOP2 transcript (from 5’ end to 3’ end). A 1.2 kb Hind Ill fragment containing the UPA3 gene was inserted in the Hind Ill site of the subcloned Eco RI-Bgl II fragment (solid bar). The lengths of two Hind Ill-Pst I fragments relevant to the analysis in (C) (see below) are indicated. The unique Pst I site in the 1.2 kb MA3 fragment is located at about 0.2 kb from one end as indicated in the figure. (B) After sporulation of a transformed diploid, ten asci were dissected. Eight gave viable spores as shown. Spores from a single ascus are aligned vertically. After this photograph was taken, viable spores were examined for their phenotypes wrth regard to uracil. All of them were found to be uracil auxotrophs. (C) Results of the Southern blot experiments are shown. Samples are Pst l-digested total DNA from various strains: (C) the parental diploid before transformation, (D) the parental diploid afler transformation, (Hi) and (H2) a pair of viable spores from a single ascus. Parallel sets of samples were run in a 1% agarose gel and blot-transferred to a nitrocellulose sheet. One set was hybridized with the Eco RI-Bgl II TOP2 fragment (solid bar in Figure 5A). and the other, with the 1.2 kb MA3 fragment, as indicated at the top of the gel.

used to transform a diploid yeast strain AB320 ura3-1 (Rothstein et al., 1977). Because DNA ends are recombi- nogenic in yeast (Orr-Weaver et al., 1981) integration of the fragment into the yeast chromosomes occurs prefer- entially at the TOP2 locus. The net effect of this integration would be the insertion of the lJRA3 fragment into TOP2 in the chromosomes. A dozen individual Ura’ transformants


were selected, and one was allowed to sporulate for tetrad analysis. Figure 58 depicts eight tetrads. Clearly, only two of the four spores are viable. All the viable spores are Ura-, suggesting that URA3 segregates with the lesion that causes lethality. This segregation pattern in turn suggests that the inactivation of the TOP2 gene is lethal in haploid.

In order to ascertain that the genetic events described above were correlated with physical changes in the genome, total DNA was prepared from Ura+ transformants and viable spores for Southern blot hybridization analysis. Figure 5C shows the results of this experiment. For the four lanes on the left side, the probe used was a cloned TOP2 fragment (solid bar in Figure 5A), and the restriction enzyme used was Pst I. In the control lane (C), using DNA from the untransformed diploid, a single 9.5 kb Pst I fragment is seen, as expected for a single-copy gene. DNA from Ura’ transformants of the diploid strain all give the pattern shown in lane D; in addition to the 9.5 kb fragment, two fragments 6.3 kb and 4.1 kb in length are seen. This pattern is expected if the linear fragment containing URA3 has integrated into the TOP2 gene of one of the two haploid genomes in the diploid (Figure 5A). Lanes HI and H2 depict the patterns obtained with DNA prepared from each of a pair of viable spores from a single ascus. Only the single 9.5 kb band is seen, showing that the viable spores are the ones that have inherited the intact TOP2 gene. The same conclusion is obtained when the 1.2 kb URA3 fragment is used as the probe in the blot hybridization (Figure 5C right four lanes). Two bands, of 5.2 kb and 3.7 kb, corresponding to the endogenous URA3 gene, are seen throughout. Lane D shows hybridization to the 6.3 kb and 4.1 kb bands from the inactivated TOP2 gene; as expected, these do not appear in lanes HI and H2, implying that the viable spores contain intact TOP2 genes, The gene-inactivation experiment and the hybridization patterns described above show that the inactivation of the TOP2 gene is lethal in a haploid but not in a diploid, and that there is one copy of the gene per haploid genome.

Discussion

Immunological screening of a yeast genomic DNA library in phage hgtll (Young and Davis, 1983a, 1983b) has enabled us to identify DNA segments containing coding sequences of yeast topoisomerase II. From the positions of the 5’ terminus of the transcript of the gene determined by Sl nuclease mapping (Berk and Sharp, 1977) and the polyadenylation site mapped in a cDNA clone, it is concluded that the structural gene of yeast topoisomerase II, TO/?, spans a region 4.5 kb in length. This length is in agreement with that of the messenger RNA of this gene determined by blot hybridization. The molecular weight, 150,000, of the purified enzyme (Goto and Wang, 1982) is consistent with these results. The expression of enzy- matically active yeast DNA topoisomerase II from a cloned fragment in E. coli unequivocally shows that the gene cloned is indeed the structural gene encoding the enzyme.

This experiment further shows that the enzymatic activity of this enzyme is contained within a single polypeptide chain. These results, taken together, also suggest that TOP2 has no introns.

Inactivation of the yeast TOP2 gene by insertion of the URA3 gene clearly demonstrates that the gene is essential, and that each haploid genome of yeast contains a single copy of the gene. DiNardo et al. (1984) have recently identified a temperature-sensitive lethal mutation that is most likely in TOi??, and have independently concluded that the gene bearing the mutation is an essential, single- copy gene. Their results further suggest that one of the main functional defects of their mutant is in the resolution of intertwined pairs of double-stranded DNA molecules at the end of a round of replication (DiNardo et al., 1984; see also Sundin and Varshavsky, 1980,198l). It has also been reported recently that the tsAlS9 locus in mouse L cells, a gene required for nuclear DNA replication, may encode a novobiocin-binding polypeptide that is required for DNA topoisomerase II activity (Colwill and Sheinin, 1983). In- volvement of type II topoisomerase in DNA replication has been most clearly established with E. coli DNA gyrase. A large body of evidence suggests that gyrase is involved in the initiation, and perhaps the elongation phase as well, of DNA replication in E. coli (for review see Cozzarelli, 1980; Gellen, 1981 a, 1981 b; see also Fairweather et al., 1980). Recently, Steck and Drlica (personal communication) noted that 90% of the nucleoids isolated from temperature- sensitive gyrase mutants held at a nonpermissive temperature exhibit a doublet morphology. Moreover, treatment of this material in vitro with purified gyrase decreases the percentage of the doublet nucleoids to 50%. These ob- servations suggest that gyrase is also involved in the resolution of daughter molecules at the end of DNA replication in E. coli. Phage T4 topoisomerase II, which is similar to eucaryotic type II enzymes in its inability to supercoil DNA, has also been implicated in the initiation of T4 DNA replication (Stetler et al., 1979; Liu et al., 1979). If eucaryotic topoisomerase II performs analogous functions, yeast would provide an excellent opportunity to define the roles of type II topoisomerase in DNA replication at the molecular level because of the recent development of in vitro replication systems for this organism (Jazwinski and Edelman, 1979, 1982; Kojo et al., 1981; Celniker and Campbell, 1982; Jazwinski et al., 1983).

In our screening of the yeast genomic library in Xgtll , we have scored five positives that contain inserts from the yeast TOP2 gene. Since the Xgtll vector is designed to express inserts fused to the P-galactosidase gene on the vector, it is unexpected that, in three of the positives (XFl4, XF15, XFI 6) the yeast DNA is oriented so that its direction of transcription is opposite to that of 8-galactosidase. It is unlikely that the yeast DNA fragments in these hybrids contain sequences that can be used as promoters by E. coli RNA polymerase. The same yeast DNA segments, or a longer yeast DNA fragment (the 6.4 kb Barn HI-Ava I fragment; see Results) containing these regions, do not

Cell 1078

give positives when cloned into multicopy plasmids unless they are oriented correctly downstream from an adequate E. coli promoter, such as lac UV5. Thus it is plausible that the expression of the yeast protein in XF14-16 is from a late phage X promoter in the vector. Furthermore, restriction mapping of XFI 7 shows that most of the @galactosidase gene including its promoter has been deleted; this deletion might account for the observation that XF17 con- sistently gives the weakest signal among the five positives (results not shown).

Finally, cloning of the structural gene of yeast DNA topoisomerase II has made it possible to apply a number of recently developed methods for mapping the chromosomal position of the gene and for constructing mutants for genetic and biological studies. Results of these studies will be reported elsewhere (C. Holm, D. Botstein, T. Goto, and J. C. Wang, unpublished data).

Experimental Procedures

Strains and Plasmids E. coli strain E7074 F’/aclProA+,B+A(lac pro)supE thi was kindly provided by Dr. J. Beckwith of the Harvard Medical School. The strain has been found by one of us (T. G.) to have high transformation efficiency with plasmids, and was used as the host in most of the E. coli transformations in this work. The Saccharomyces cerevisiae strain a/a ade2-I /ys2-1 trpd 2 leul-12 canl-100 me&l ufa31 (Rothstein et al., 1977) used in the gene- inactivation experiment was obtained from Dr. P. Silver of Harvard. The yeast genomic library in phage Xgtl 1 and the host strain Y1090 (Young and Davis, 1983b) were kindly provided by Drs. Ft. Young and R. Davis of Stanford University. Several yeast genomic (Carlson and Botstein, 1982) and cDNA (McKnight and McConaughy, 1983) libraries in plasmid vectors were kindly provided by Drs. C. Holm and D. Botstein of M.I.T. Cloning of the various fragments of yeast TOP2 gene was carried out in multicopy plasmids derived from pBR322.

Antibody Preparation and Plaque Screening The purification procedures for yeast DNA topoisomerase II have been described (Goto et al., 1984). Rabbit antibodies against the purified enzyme were prepared according to the published procedures of J. Vaitukaitis (1981). Avidity of the serum was assayed by the inhibition of the enzyme. One microliter of a 2000.fold dilution of the serum preparation used completely inhibited 1 U yeast DNA topoisomerase II in our standard assay (Goto et al., 1984). The dot-blot test, which is similar to the actual screening procedure, was also used to measure the effectiveness of the serum. Staining of purified topoisomerase II spotted on a nitrocellulose sheet with immunoglobulin purified from the anti-topoisomerase II serum, followed by indirect labeling with ‘2SI-labeled Staphylococcus aureus proteinA, detected a lower limit of about 100 pg of the enzyme.

The immunoglobulin fraction was purified from the serum by ammonium sulfate precipitation and DEAE-cellulose chromatography (Hurn and Chan- tier, 1980). lmmunoglobulin (10 mg/ml) was diluted 100.fold with 20% calf serum in TBS (50 mM Tris-HCI, pH 8. 150 mM NaCI). Antibodies that recognize E. coli proteins were removed by the treatment of the solution with filter-immobilized, total proteins from E. coli; lawns of host cells (Y1090) were grown on 82 mm nitrocellulose filters, lysed with a chloroform vapor, and treated as described for Xgtl 1 colony screening (Young and Davis, 1983a). Incubation of the antibody solution with the filters resulted in removal of most anti-coliform antibodies as evidenced in a marked decrease in the background in subsequent screening of plaques and colonies. Screening of a Xgtl 1 genomic library was carrred out as described (Young and Davis, 1983b). Approximately IO4 plaques per 82 mm fitler were screened. Positive plaques were punfied by one to two additional cycles of screening. Other manipulations of phages and preparation of phage DNA were done according to standard procedures (Davis et al., 1980).

Hybridization Procedures Probes were gel-purified from phage h clones digested with the appropriate restriction enzymes and labeled by nick translation to give a specific activity of about l@ cpm/&g. The yeast genomic and cDNA libraries were screened by high-density colony screening procedures (Hanahan and Meselson, 1980). The conditions for colony and Southern blot hybridization (Southern, 1975) were as described by Wu (1980). Northern blot hybridization of pofyA+ RNA was carried out according to the procedures described in Maniatis et al. (1982).

Partial Purification of Yeast DNA Topoisomerase II from E. coli E. coli E7074 harboring pllElO1, which contains the yeast TOP2 gene downstream from a lac UV5 promoter and a lac i4 gene (see Results), was grown in LB broth and induced for topoisomerase II production with IPTG (0.4 mM). After incubation at 37°C for 1.5 hr, cells were harvested by centrifugation, resuspended in TEG (50 mM Tris-HCI, pH 7.4, 1 mM N&EDTA, 10% glycerol, 1 mM P-mercaptoethanol, and 0.5 mM phenyl- methylsulfonyl fluoride) plus 0.6 M KCI, and lysed by sonication. The lysate was cleared by centrifugation. and nucleic acids removed by precipitation with polyethyleneimine (Miles Laboratories) (Burgess and Jendrisak, 1975). Solid ammonium sulfate was added to 35% saturation (0°C) and the precipitate was removed. To the supernatant, ammonium sulfate was added to 60% saturation, and the precipitate was collected, resuspended, and dialyzed against TEG + 0.2 M KCI. The solution was applied to a phosphocellulose column, and the column was washed with TEG + 0.35 M KCI. Proterns were eluted by a linear gradient from 0.35 M to1 M KCI in TEG. Active fractions, located by activity assay (and also by dot-blot tests with antibody), were pooled, dialyzed against TEG + 0.2 M KCI, and applied to an aminopentyl agarose column. The column was washed with the dialysis buffer, and proteins eluted with TEG + 0.5 M KCI.

Other Techniques ProteinA (Calbiochem-Behring) was rodinated with the Bolton-Hunter reagent (Amersham and New England Nuclear). Digestions with restriction enzymes and Bal 31 nuclease (New England Biolabs) were carried out according to the protocols of the supplier. Unknotting and relaxation assays for yeast topoisomerase II activity have been described (Goto and Wang, 1982; Goto et al., 1984). Yeast transformation was carried out with spher- oplasts (Hinnen et al., 1978). Yeast nuclear DNA and polyA+ RNA were prepared as described in Cryer et al. (1975) and in Sripati and Warner (1978) respectrvely.

Acknowledgments

We are most grateful to Drs. Richard Young and Ronald Davis for providing us the Xgtl 1 system and detailed instructions, to Dr. Judith Vaitukaitis for her instructrons on the preparation of rabbit antisera, to Dr. Pamela Silver for many helpful discussions and her assistance in yeast transformation, to Ms. Monrca Penn for her help in the dissection of yeast asci, to Mrs. Kathleen Becherer for her superb assistance in the construction of a number of plasmids, and David Horowitz and Robin Wharton for their thoughtful advice and encouragement throughout the work. We also thank Drs. Connie Holm and David Botstein for many stimulating discussions. This work has been supported by grants from the U. S. Public Health Service (GM24,544) and the American Cancer Society (NP-424).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

Received December 22, 1983

References

Abraham, J., Feldman, J., Nasmyth, K. A., Strathem, J. N., Klar, A. J. S., Broach, J. R., and Hicks, J. B. (1983). Sites required for position-effect regulation of mating-type information in yeast. Cold Spring Harbor Symp. Quant. Biol. 47, 989-998.

Badaracco, G., Plevani, P., Ruyechan, W. T., and Chang, L. M. S. (1983).


Purification and characterization of yeast topoisomerase I. J. Biol. Chem. 258,2022-2026.

Berk, A. J., and Sharp, P. A. (1977). Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of Sl endonuclease-digested hybrids, Cell 12,721~732.

Brown, P. O., Peebles, C. L., and Cozzarelli, N. R. (1979). A topoisomerase from Escherichia coli related to DNA gyrase. Proc. Nat. Acad. Sci. USA 76, 6110-6114.

Burgess, R. R., and Jendrisak, J. J. (1975). A procedure for the rapid, large- scale purification of Escherichia coli DNA-dependent RNA polymerase involving polyminP precipitation and DNA-cellulose chromatography. Bio- chemistry 14.4634-4638.

Carlson, M., and Botstein, D. (1982). Two differentially regulated mRNAs with different 5’ ends encode secreted and intracellular forms of yeast invertase. Cell 28, 145-154.

Celniker, S. E., and Campbell, J. L. (1982). Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes. Cell 31. 201-213.

Champoux, J. J., and Dubecco, R. (1972). An activity from mammalian cells that untwists superhelical DNA-a possible swivel for DNA replication. Proc. Nat. Acad. Sci. USA 69, 143-146.

Colwill, R. W., and Sheinin, R. (1983). fs AlS9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity. Proc. Nat. Acad. Sci. USA 80, 4644-4648.

Cozzarellr, N. R. (1980). DNA gyrase and the supercoiling of DNA. Science 207,953-960.

Cryer, D., Eccleshall, R., and Marmur, J. (1975). Isolation of yeast DNA. Meth. Cell Biol. 12, 39-44.

Davis, R. W., and Botstein, D., and Roth, J. R. (1980). Advanced bacterial genetics. (Cold Spring Harbor, New York Cold Spring Harbor Laboratory) pp. 70-82. 106-I 15.

Dean, F., Krasnow, M. A., Otter, R., Matzuk, M. M., Spengler, S. J., and Cozzarelli, N. R. (1983). Escherichia coli type-l topoisomerases: identification, mechanism, and role rn recombination. Cold Spring Harbor Symp. Quant. Biol. 47, 769-777.

DiNardo, S., Voelkel, K. A., Sternglanz, R., Reynolds, A. E., and Wright, A. (1982). Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell 31, 43-51.

DiNardo, S., Voelkel, K., and Sternglanz, R. (1984). DNA topoisomerase mutant of S. cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc. Nat. Acad. Sci. USA, in press.

Dnica, K.. and Synder, M. (1978). Superhelical Escherichia coli DNA: relaxation by coumermycin. J. Mol. Biol. 120, 145-154.

Durnford, J. M., and Champoux, J. J. (1978). The DNA untwisting enzyme from Saccharomyces cerevisiae; partial purification and characterization. J. Blot Chem. 253, 1086-1089.

Fairweather, N. F.. On, E., and Holland, I, B. (1980). Inhibition of deoxyn- bonucleic acrd gyrase: effects on nucleic acid synthesis and cell division in Escherichia coli K-12. J. Bacterial. 742. 153-161,

Gellert, M. (1981a). DNA topoisomerases. Ann Rev. Eiochem. 56, 879- 910.

Geliert, M. (1981b). DNA gyrase and other type II topoisomerases. In The Enzymes, 14, P. Boyer, ed. (New York Academic Press) pp. 345-366.

Gellert. M., Mizuuchr, K.. O’Dea. M. H., and Nash, H.A. (1976a). DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc. Nat. Acad. Sci. USA 73, 3872-3876.

Gellert. M., C’Dea, M. H., Itoh, T., and Tomizawa, J. (1976b). Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Nat. Acad. Sci. USA 73, 4474-4478.

Gellert M., Mizuuchi, K., O’Dea, N. H., Itoh, T., and Tomizawa, J. (1977). Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity Proc. Nat. Acad. Ser. USA 74, 4772-4776.

Gelled, M., Fisher, L. M., and O’Dea, M. H. (1979). DNA gyrase: purification and catalytrc propertres of a fragment of gyrasa B protein. Proc. Nat, Acad. Sci. USA 76, 6289-6293.

Gelled, M., Menzel, R., Mizuuchi, K., O’Dea. M. H., and Friedman, D. I. (1983). Regulation of DNA supercoiling in Escherichia colt Cold Spring Harbor Symp. &ant. Biol. 47, 763767.

Goto, T.. and Wang, J. C. (1982). Yeast topoisomerase II: an ATP- dependent type II topoisomerase that catalyzes the catenation, decatena- tion, unknotting, and relaxation of double-stranded DNA rings, J. Biol. Chem. 257, 58665872.

Goto, T., Laipis, P., and Wang, J. C. (1984). The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevrsiae. J. Biol. Chem., in press.

Hanahan, D.. and Meselson, M. (1980). Plasmid screening at high colony density Gene 10, 63-67.

Harland, R. M., Weintraub, H., and McKnight, S. L. (1983). Transcription of DNA injected into Xenopus oocytes IS influenced by template topology. Nature 302, 38-43.

Helfman, D. M., Feramrsco, J. l?., Fiddes, J. C., Thomas, G. P., and Hughes, S.H. (1983). Identification of clones that encode chicken tropomyosin by direct immunological screenrng of a cDNA expression library. Proc. Nat. Acad. Sci. USA 80, 31-35.

Hrnnen, A., Hicks, J., and Fink, G. (1978). Transformation in yeast. Proc. Nat. Acad. Sci. USA 75, 1929-1933.

Hurn, 8. A. L., and Chantler, S. M. (1980). Production of reagent antibodies, Meth. Enzymol. 70, 104142.

Jazwinski. S. M., and Edelman, G. M. (1979). Replication in vitro of the 2 rrn DNA plasmid of yeast. Proc. Nat. Acad. Sci. USA 76, 1223-1227.

Jazwinski, S. M., and Edelman. G. M. (1982). Protein complexes from active replicative fractions associate in vitro with the replication origin of yeast Z-pm DNA plasmid. Proc. Nat. Acad. SCI. USA 79, 3428-3432.

Jazwinski. S. M., Niedzwiecka, A., and Edelman, G. M. (1983). In vitro association of a replication complex with yeast chromosomal replicator, J. Biol. Chem. 258, 2754-2757.

Kojo, H., Greenberg, B. D., and Sugino, A. (1981). Yeast 2 p plasmid DNA replication in vitro: origin and direction. Proc. Nat. Acad. Sci. USA 78, 7261- 7265.

LIU, L. F., Liu, C.-C., and Able&, B. M. (1979). T4 DNA topoisomerase: a new ATP-dependent enzyme essential for initiation of T4 bacteriophage DNA replication. Nature 281, 456-461,

LIU, L. F., Davis, J. L., and Calendar, R. (1981). Novel topologically knotted DNA from bacteriophage P4 capsids: studies with DNA topoisomerases. Nucl. Acids Res. 9, 3979-3989.

Luchnik. A. N., Bakayev, V. V., Zbarsky, I. B., and Georgiev, G. P. (1982). Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin. EMBO J. I, 1353-1358.

Manratrs, T., Fritsch, E., and Sambrook, J. (1982). Molecular cloning: a laboratory manual. (Cold Spring Harbor, New York Cold Spring Harbor Laboratory) pp. 202-203.

McKnight, G. L., and McConaughy, B.L. (1983). Selection of functional cDNAs by complementatron in yeast, Proc. Nat. Acad. Sci. USA 80, 4412- 4416.

Menzel, R., and Gellert, M. (1983). Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell 34, 105-I 13.

Okayama, H., and Berg, P. (1982). High-efficiency cloning of full-length cDNA. Mol. Cell. Biol. 2, 161-170.

@r-Weaver, T. L., Szostak, J. W.. and Rothstein, R. J. (1981). Yeast transformations: a model system for the study of recombination. Proc. Nat, Acad. Sci. USA 78, 6354-6358.

Pruss. G. J., Manes, S. H., and Drlica, K. (1982). Escherfchia coli DNA topoisomerase I mutants: increased supercoiling IS corrected by mutations near gyrase genes. Cell 31, 35-42.

Rothstein, R. J. (1983). One-step gene disruption in yeast. Meth. Enzymol. 101,202-211.

Rothstein. R. J.. Esposito, R. E., and Esposito, M. S. (1977). The effect of ochre suppression on meiosis and ascospore formation in Saccharomyces. Genetics 85, 35-54.

Sander, M.. and Hsieh. T.-s. (1983). Double strand DNA cleavage by type

Cell 1080

II DNA topoisomerase from Drosophila melanogaster. J. Biol. Chem. 258, 8421-8428.

Shelton, E. Ft., Osheroff, N., and Brutlag, D. L. (1983). DNA topoisomerase II from Drosophila melanogaster: purification and physical characterization. J. Biol. Chem. 258, 9530-9535.

Young, R. A., and Davis, Ft. W. (1983b). Yeast RNA polymerase II genes, isolation with antibody probes. Science 222, 778-782.

Sinden, R. R., Carlson, J. O., and Pettijohn, D. E. (1980). Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogus measurements in insect and human cells. Cell 27, 773- 783.

Southern, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517.

Sripati, C. E., and Warner, J. R. (1978). Isolation, characterization and translation of mRNA from yeast. Meth. Cell Biol. 20, 61-81.

Sternglanz, R., DiNardo, S., Voelkel, K. A., Nishimura, Y.. Hirota, Y., Becherer, K., Zumstein, L., and Wang, J. C. (1981). Mutations in the gene coding for Escherichia coli DNA topoisomerase I affect transcription and transposition. Proc. Nat. Acad. Sci. USA 78, 2747-2751.

Stetler, G. L., King, G. J., and Huang, W. M. (1979). T4 DNA-delay proteins, required for specific DNA replication, form a complex that has ATP- dependent DNA topoisomerase activity. Proc. Nat. Acad. SCI. USA 76, 3737-3741.

Sugino, A., Peebles, C. L.. Kreuzer, K. N., and Cozzarelli, N. R. (1977). Mechanism of action of nafidixic acid: purification of Escherichia coli na/A gene product and its relationship to DNA gyrase and a novel nicking- closing enzyme. Proc. Nat. Acad. Sci. USA 74, 4767-4771.

Sugino, A., Higgins, N. P., Brown, P. O., Peebles, C. L., and Couarelli, N. R. (1978). Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc. Nat. Acad. Sci. USA 75. 4838-4842.

Sundin, O., and Varshavsky, A. (1980). Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell 27, 103- 114.

Sundin, O., and Varshavsky, A. (1981). Arrest of segregation leads to accumulation of highly intertwined catenated dimers: dissection of the final stages of SV40 DNA replication. Cell 25, 659-669.

Trucksis, M., and Depew, R. E. (1981). Identification and localization of a gene which specifies production of Escherlchia coli DNA topoisomerase I. Proc. Nat. Acad. Sci. USA 78, 2164-2168.

Trucksis, M., Golub, E. I., Zabel, D. J., and Depew, R. E. (1981). Escherichia coli and Salmonella typhimurium supX genes specify deoxyribonucleic acid topoisomerase I. J. Bacterial. 747, 679-681.

Vaitukaitrs. J. L. (1981). Production of antisera with small doses of immu- nogen: multiple intradermal injections. Meth. Enzymol. 73, 46-52.

Wang, J. C. (1971). Interaction between DNA and an Escherichia coli protein W. J. Mol. Biol. 55, 523-533.

Wang, J. C. (1981). Type I topoisomerases. In The Enzymes, 74. P. Boyer, ed. (New York: Academic Press) pp. 331-344.

Wang. J. C. (1983). DNA supercoiling: structural effects and biological consequences. In Genetic Rearrangement, K. F. Chater, C. A. Cullis, D. A. Hopwood, A. A. W. B. Johnston, and H. W. Woolhouse, eds. (London: Croom Helm) pp. l-26.

Wang, J. C., and Becherer, K. (1983). Cloning of the gene fopA encoding for DNA topoisomerase I and the physical mapping of the cysB-fopA-trp region of Escherichia colt Nucl. Acids Res. 7 7, 1773-l 790.

Wang, J. C.. Peck, L. J., and Becherer. K. (1983). DNA supercoiling and its effects on DNA structure and function. Cold Spring Harbor Symp. Quant. Bid. 47, 85-91.

Weaver, R. F., and Weissman, C. (1979). Mapping of RNA by a modification of the Berk-Sharp procedure: the 5’ termrni of 15s i3-globin mRNA precurser and mature 10s @globin mRNA have identical map coordinates. Nucl. Acids Res. 7, 1175-l 193.

Weisbrod, S. (1982). Actrve chromatin. Nature 297, 289-295.

Wu. C. (1980). The 5’ end of Drosophila heat shock genes in chromatin are sensitive to DNase I. Nature 286, 854-860.

Young, R. A., and Davis, R. W. (1983a). Efficient isolatron of genes by using antibody probes. Proc. Nat. Acad. Sci. USA SO, 1194-l 198.

Yeast DNA Topoisomerase II Is Encoded by a Single-Copy, … · 2017. 1. 26. · The enzyme DNA gyrase is encoded by two genes, gyrA and gyrB, and the availability of mutants and specific

Documents