Gene Disruption of a G4-DNA-Dependent Nuclease In
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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6002-6006, June 1995 Genetics Gene disruption of a G4-DNA-dependent nuclease in yeast leads to cellular senescence and telomere shortening (guanine-quartet/KEMI/SEPI/checkpoint/meiosis) ZHIPING Liu, ARNOLD LEE, AND WALTER GILBERT Department of Molecular and Cellular Biology, The Biological Laboratories, 16 Divinity Avenue, Harvard University, Cambridge, MA 02138-2092 Contributed by Walter Gilbert, April 3, 1995 ABSTRACT The yeast gene KEMI (also named (11). A highly conserved feature is that one of the strands is SEPI/DST2/XRNI/RAR5) produces a G4-DNA-dependent very G-rich and always exists as a 3' overhang at the end of the nuclease that binds to G4 tetraplex DNA structure and cuts in chromosome. Telomeres carry out two essential functions. a single-stranded region 5' to the G4 structure. G4-DNA They protect, or stabilize, the ends of linear chromosomes, generated from yeast telomeric oligonucleotides competitively since artificially generated ends (by means of mechanical inhibits the cleavage reaction, suggesting that this enzyme sheering, x-ray irradiation, or enzymatic cleavage) are very may interact with yeast telomeres in vivo. Homozygous dele- unstable (12-14). Furthermore, they serve to circumvent the tions ofthe KEMI gene in yeast block meiosis at the pachytene incomplete DNA replication at the ends of linear chromo- stage, which is consistent with the hypothesis that G4 tetra- somes (15) by extending the G-rich strand through a de novo plex DNA may be involved in homologous chromosome pairing synthesis catalyzed by telomerase to counterbalance the se- during meiosis. We conjectured that the mitotic defects of quence loss at an end in lagging-strand replication (16, 17). In kemr Isepl mutant cells, such as a higher chromosome loss the presence of alkali ions and at neutral pH, oligonucleotides rate, are also due to failure in processing G4-DNA, especially corresponding to telomeric G-rich sequences can spontane- at telomeres. Here we report two phenotypes associated with ously form a class of stable DNA structures, called G4-DNA, a keml-null allele, cellular senescence and telomere shorten- in which four strands are held together by the formation of ing, that provide genetic evidence that G4 tetraplex DNA may Hoogsteen bonding between guanines in a guanine-quartet play a role in telomere functioning. In addition, our results (G-quartet) with the sugar backbones running in either par- reveal that chromosome ends in the same cells behave differ- allel or antiparallel orientation (10, 18, 19). The presence of ently in a fashion dependent on the KEMI gene product. such stacks of G-quartets holding four DNA strands together has been confirmed by x-ray crystallography and two- The yeast KEMJ/SEPI gene encodes a G4-DNA-dependent dimensional NMR studies (20-22). Telomeric G-rich strand nuclease, an enzyme that binds to a G4 tetraplex (G4-DNA) DNA folded into a G-quartet form is no longer a substrate for structure and cuts in a single-stranded region 5' to the G4 telomerase addition, although it may still serve the function of structure (1, 2). This gene has been variously identified by capping the ends of linear chromosome and negatively regulating mutations that block nuclear fusion during yeast conjugation telomere synthesis by telomerase (23). Therefore, a failure to (3) or enhance the mitotic stability of a minichromosome process G-quartet structures, if they formed at chromosome bearing a weak ARS (autonomously replicating sequence) (4) termini, should lead to telomere-related defects. or by the biochemical properties of the gene product as a strand-exchange protein (5, 6) or an RNase (7). We showed MATERIALS AND METHODS that various G4-DNAs, including one generated from a yeast telomeric G-rich oligonucleotide, competitively inhibited the Strains and Plasmids. Plasmid pJI112 was a gift from G4-DNA-dependent cleavage reaction, while single-stranded Gerald Fink (3), and yeast strain DBY1829 [MATa ura3-52 DNA did not (1), suggesting that this nuclease is a bona fide his3-A200 leu2-3,112 trpl-l(am) lys2-801] was obtained from G4-DNA-dependent enzyme. Homozygous deletions of the Tim Huffaker. KEM1 gene disruption was carried out by KEMl/SEP1 gene block meiotic cells at the pachytene stage cutting pJI112 with Pvu II and HindIII enzymes, transforming (8, 9), supporting the hypothesis that the G4-DNA plays a role DBY1829 cells by the lithium acetate method, and selecting for in meiosis (10). In mitotic cells, however, only minor defects, URA+ transformants. keml-null cells were later confirmed by such as chromosome loss, slow growth, and a hypersensitivity their characteristically larger cell size, slow growth, and to benomyl (an antimicrotubule drug), have been reported for genomic Southern blot analysis. The medium and plates for mutations in this gene (3, 5, 7). The fact that the G4-DNA yeast culture were prepared as described (24). nuclease activity was identified in a partially purified yeast Serial Culturing and Plating. A single yeast colony was used protein fraction eluted from a telomeric DNA affinity column to inoculate 10 ml of YPD medium and grown at 30°C on a (1) prompted us to look for telomere defects in keml/sepl-null rollerdrum until the cell density reached 1.5 x 107 cells per ml, cells. We show here that a keml deletion leads to cellular based on hemacytometer counting. An aliquot of the culture senescent behavior and to telomere shortening. This finding was used to inoculate 10 ml of YPD at a density of 5.0 x 103 suggests that the removal of G4 tetraplex DNA plays a role in cells per ml, and the rest of the cells were harvested by telomere functioning and that the genomic instability observed centrifugation, resuspended in -1 ml of 15% (vol/vol) glyc- in keml mutant cells is probably due to a failure in processing erol, and stored at -70°C. The fresh culture was grown in the telomeres. same way for -2 days, and when the cell density reached 1.5 Telomeres are the ends of linear chromosomes. In most x 107 cells per ml, the inoculation and harvesting were repeated organisms examined so far, they are simple repetitive se- as described above. There were 15 doublings, or generations, quences conforming to a general consensus (G1_8)(A/T)1_4 between each serial passage (an estimation based on the hemacytometer counting). At the end of the serial culturing, The publication costs of this article were defrayed in part by page charge the frozen cells were thawed, and several 1:10 diluted (in 15% payment. This article must therefore be hereby marked "advertisement" in glyceral) stocks were made for each culture and stored at accordance with 18 U.S.C. §1734 solely to indicate this fact. -70°C; the rest of the cells were used to extract genomic DNA 6002 Downloaded by guest on October 2, 2021 Genetics: Liu et al. Proc. Natl. Acad. Sci. USA 92 (1995) 6003 based on a standard method (24). For plating, the diluted stocks were thawed, and the cell density was determined by hemacytometer counting. Cells were then diluted serially to 2.5 x 104 cells per ml, and 50 ,ul of the cell suspension was spread out on a quadrant of a YPD plate. Genomic Southern Blot Analysis. Genomic Southern blot analysis was carried out essentially as described (25). About 2 Ag of yeast genomic DNA was digested with 15 units of Xho I enzyme for 6-8 h and then separated on a 0.7% agarose gel. The electrophoresis was at 2 V/cm in TBE buffer and was stopped when bromophenol blue dye reached to 20 cm from the wells. The gel was then denatured and blotted onto a GeneScreen membrane. The probe was (GT)20, labeled at the 5' end with [y-32P]ATP and T7 polynucleotide kinase. The hybridization was carried out in an Autoblot hybridization oven (Bellco) at 55°C overnight. The blot was washed for one 2-min period at room temperature, for two 30-min periods at 55°C, and finally for one 30-min period at 60°C and then either scanned with a PhosphorImager or autoradiographed. For BAL-31 digestion experiment, yeast genomic DNA was first digested with 2 units of BAL-31 enzyme (BRL) at 37°C for the time specified and stopped by phenol extraction once. The DNA was recovered by ethanol precipitation, redissolved in H20, digested with Xho I to completion, and analyzed as described above. B 700 RESULTS 600 J Senescence We examined several a) Phenotype. keml-deletion .0 . yeast strains and found that null mutations in this gene gave E 500: rise to cellular senescence and to telomere For shortening. 0 400 wild type example, we constructed a keml-deletion strain (named a, keml-D allele hereafter), by one-step gene disruption in a 300 strain in which a 3.2-kb sequence in KEM1 Ca haploid (26), coding 200 ! kernl-null m gene was replaced by the yeast URA3 gene. After purifying > URA+ transformants once on a selective plate, we picked a 100 and cultured it in single colony serially rich medium. After each 0: 15 of we inoculated a fresh culture generations growth, (at 5 0 50 100 150 200 X 103 cells per ml) with an aliquot of the preceding culture and then harvested and stored the rest of the cells in 15% glycerol Generation number at -70°C. Fig. 1 shows the growth on a rich plate of equal FIG. 1. (A) Senescence phenotype of a keml-null strain. KEMI numbers of thawed cells from cultures at different ages. For the deletion was constructed by one-step gene disruption in a haploid mutant cells, the colony number fell at 45, 75, and 105 strain DBY1829 [MATa ura3-52 his3-A200 leu2-3,112 trpl-l(am) generations, and drastic cell death occurred at about genera- lys2-801].