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Effects of Yeast DNA Topoisomerase III on Telomere Structure (Genome Stability/Genetic Recombination/Subtelomeric Elements/EST1 Gene/TOP3 Gene) RAYMOND A

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Effects of Yeast DNA Topoisomerase III on Telomere Structure (Genome Stability/Genetic Recombination/Subtelomeric Elements/EST1 Gene/TOP3 Gene) RAYMOND A Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2667-2671, March 1995 Biochemistry Effects of yeast DNA topoisomerase III on telomere structure (genome stability/genetic recombination/subtelomeric elements/EST1 gene/TOP3 gene) RAYMOND A. KIM*, PAUL R. CARONt, AND JAMES C. WANG Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138 Contributed by James C. Wang, December 23, 1994 ABSTRACT The yeast TOP3 gene, encoding DNA topo- gene (TOP3) encoding yeast topoisomerase III has subse- isomerase III, and EST] gene, encoding a putative telomerase, quently confirmed this assignment: the purified protein par- are shown to be abutted head-to-head on chromosome XII, tially but specifically relaxes highly negative supercoiled with the two initiation codons separated by 258 bp. This DNAs, and biochemical characterization of the yeast enzyme arrangement suggests that the two genes might share common indicates that it is more closely related to E. coli topoisomerase upstream regulatory sequences and that their products might III than to E. coli topoisomerase I (17). In vivo, the enzyme also be functionally related. A comparison ofisogenic pairs ofyeast appears to have a weak activity in the removal of negative TOP3+ and Atop3 strains indicates that the G1_3T repetitive supercoils; in cells expressing either topoisomerase I or II, the sequence tracks in Atop3 cells are significantly shortened, by weak activity of topoisomerase III is masked (17). One might about 150 bp. Cells lacking topoisomerase III also show a therefore expect that inactivation of a weaker topoisomerase much higher sequence fluidity in the subtelomeric regions. In III relaxation activity in the presence of two stronger ones- Atop3 cells, clusters of two or more copies of tandemly namely, those of topoisomerases I and I-should have little arranged Y' elements have a high tendency of disappearing effect on cell physiology. Surprisingly, null mutations in TOP3 due to the loss or dispersion ofthe elements; similarly, a URA3 in a TOP1J TOP2+ genetic background result in slower growth, marker embedded in a Y' element close to the chromosomal hyperrecombination between repetitive sequence elements, tip shows a much higher rate of being lost relative to that in and defective sporulation (16). TOP3+ cells. These results suggest thatyeast DNA topoisomer- The lack of a coherent picture relating the known enzymatic ase III might affect telomere stability, and plausible mecha- activity of yeast topoisomerase III to the phenotypes of cells nisms are discussed. lacking it has led to the speculation that the in vivo role of the protein is probably not directly related to its relaxation of The budding yeast Saccharomyces cerevisiae is known to pos- negative supercoils; instead, the enzyme might be involved in sess three distinct DNA topoisomerases, each ofwhich belongs the separation of intertwined DNA single strands, perhaps in to a separate topoisomerase subfamily (for compilation of conjunction with a helicase (6, 18). Interestingly, an extragenic members of the three subfamilies and their amino acid se- suppressor mutation that suppresses the slow-growth and quence alignments, see ref. 1). Yeast DNA topoisomerases I hyperrecombination phenotypes of top3 mutants has recently and II have been studied extensively. The former closely been mapped to a gene (SGS1) encoding a protein homolo- resembles DNA topoisomerase I of all other eukaryotes and is gous to the E. coli RecQ protein, the primary sequence of a member of a subfamily of type I topoisomerases that also which suggests that it might be a helicase (19). includes poxvirus topoisomerase and DNA topoisomerase V In a search for sequence homologies between yeast topo- of the thermophilic prokaryote Methanopyrus kandleri (2). The isomerase III and other proteins, we found that the yeast TOP3 enzyme relaxes both positive and negative supercoils, and and EST1 genes, the latter encoding a putative telomerase genetic and biochemical evidence suggests that the enzyme is (20), might be positioned head-to-head at the same chromo- involved in transcription and replication (reviewed in refs. 3 somal location, with 258 bp between the two initiation codons and 4). Yeast DNA topoisomerase II is a type II DNA (see below). This finding was unexpected, as TOP3 and EST1 topoisomerase. Like yeast topoisomerase I, it also relaxes both were mapped to two different chromosomes, XII and VIII, positive and negative supercoils (reviewed in refs. 5 and 6). respectively (16, 20). A divergent pair of genes separated by a Thus, yeast Atopl mutants, lacking DNA topoisomerase I, are short distance of 258 bp would suggest that the two genes might viable, as its normal cellular roles can presumably be filled by be coregulated, raising the possibility that DNA topoisomerase topoisomerase II (reviewed in refs. 3, 5, and 6). The type II III might be involved in the maintenance of telomere structure. enzyme has the unique ability of unlinking intertwined pairs of Here we report evidence in support of these possibilities. newly replicated DNA rings of chromosomes, and in its absence cells cannot survive segregation of the chromosomes MATERIALS AND METHODS during mitosis. The type II enzyme is also involved in chro- Yeast Strains and Plasmids. S. cerevisiae strains CH1105 mosome condensation and decondensation (7-10), and the (MA Ta ade2-101 leu2-Al lys2-801 trpl-Al ura3-52) and possibility of its involvement in the organization of chromo- CH1585 (MATa leu2-AJ trpl-A63 ura3-52 his3-200) were somes has also been suggested (refs. 11-14; see refs. 10 and 15 obtained from C. Holm (University of California, San Diego); for a contrary view). strain JCW15 was derived from CH1105 by switching its In contrast to its more extensively studied congeners, yeast mating type. The Atop3 derivatives of these strains were DNA topoisomerase III remains largely an enigma. It was obtained by the one-step gene transplacement method (21), initially identified as a member of the DNA topoisomerase using a segment from the plasmid described below. A 3.3-kb subfamily represented by Escherichia coli DNA topoisomer- EcoRI-Xba I fragment of TOP3 in pUC18 was moved into ases I and III, from a comparison of primary sequences (16). pBluescript (Stratagene) as an EcoRI-Sal I fragment. The Purification of the protein from yeast cells overexpressing the *Present address: Bristol-Myers Squibb, 5 Research Parkway, Depart- The publication costs of this article were defrayed in part by page charge ment 106, Wallingford, CT 06492. payment. This article must therefore be hereby marked "advertisement" in tPresent address: Vertex Pharmaceuticals, 40 Allston Street, Cam- accordance with 18 U.S.C. §1734 solely to indicate this fact. bridge, MA 02139. 2667 Downloaded by guest on September 24, 2021 2668 Biochemistry: Kim et al. Proc. Natl Acad Sci USA 92 (1995) resulting plasmid was digested with Hpa I and Nae I, which cut which TOP3 and its upstream sequences reside. The sequenc- respectively at positions 1038 and 1067 of the TOP3 sequence ing results as well as additional sequencing data on DNA (16), and an 850-bp TRP1 marker was inserted between these samples amplified from the Atop3 strains JCW53 and JCW153 sites. An EcoRI-Sal I segment from the TRPJ-bearing plasmid confirmed unequivocally that TOP3 and EST] are abutted was then used to transform the various strains to Trp+ (17). head-to-head (data not shown). Where our sequencing data A plasmid bearing yeast ARG4 was obtained from N. overlapped with the published results, no difference was found Kleckner (Harvard University), and plasmids bearing TOP3 between our data and the published sequence of EST1 (20), and rDNA were from laboratory stock (17, 22). Probes for the and only one discrepancy was found between our data and the telomere-associated Y' element and EST1 gene were derived published sequence of TOP3 (16), with C51 in the published respectively from YCp50-131Y (23) and from a 5.8-kb EcoRI sequence being a G in ours. The sequencing results with DNA fragment of yeast genomic DNA bearing TOP3 and part of samples derived from the Atop3 strains used in the present ESTI in M13mpl8 (17). Introduction of a URA3 marker into work also show that during the construction ofthese strains, no a telomere-associated Y' element was carried out as described inadvertent changes were introduced into the upstream re- by Louis and Haber (24), by using a Pvu I-EcoRI fragment of gion; if such changes had occurred, they could alter' the plasmid pEL-2. regulation of the ESTI gene and thus complicate the inter- Cloning- and Sequencing of the TOP3-ESTI Intergenic pretation of the results to be described below. Region. A pair of primers, 5'-CAGGAAGCATCTGAAC- Effect of top3 Null Mutation on Telomere Length. The GTGA-3' and 5 '-GAAATCCCTTGAAGTTGATCTG- presence of TOP3 and EST1 as a pair of very closely linked C-3', whose sequences were based on those of the noncoding genes sharing untranscribed upstream sequences suggested strands near the beginning of the ESTI and TOP3 open that TOP3, like EST1 (20), might be involved in telomere reading frames (16, 20), respectively, were used to sequence function. Genomic DNA samples from an isogenic pair of the intergenic region on a plasmid clone and to amplify by PCR TOP3+ and Atop3. strains were therefore prepared and di- the intergenic segment in DNA samples isolated from the gested with Xho I restriction endonuclease, which cuts at a Atop3::TRP1+ strains used in the present work, JCW53 and highly conser'ved site in the subtelomeric Y' element -1.3 kb JCW1S3. Chromosomal DNA samples prepared from the two frotn the chromosomal tips carrying the Y' elements (23, 24, strains were used: in the PCRs. The amplified DNAs were 26, 28-31). Xho I fragments were resolved by agarose gel individually purified by agarose gel electrophoresis as a 0.5-kb electrophoresis and blot hybridized with radiolabeled fragment and cloned into the Sma I site of pUC18.
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