Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation.

The termini of Saccharomyces cerevisiae chromosomes consist of tracts of C1-3A (one to three cytosine and one adenine residue) sequences of approximately 450 base pairs in length. To gain insights into trans-acting factors at telomeres, high-copy-number linear and circular plasmids containing tracts of C1-3A sequences were introduced into S. cerevisiae. We devised a novel system to distinguish by color colonies that maintained the vector at 1 to 5, 20 to 50, and 100 to 400 copies per cell and used it to change the amount of telomeric DNA sequences per cell. An increase in the number of C1-3A sequences caused an increase in the length of telomeric C1-3A repeats that was proportional to plasmid copy number. Our data suggest that telomere growth is inhibited by a limiting factor(s) that specifically recognizes C1-3A sequences and that this factor can be effectively competed for by long tracts of C1-3A sequences at telomeres or on circular plasmids. Telomeres without this factor are exposed to processes that serve to lengthen chromosome ends.     

Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae.

We have investigated two reactions that occur on telomeric sequences introduced into Saccharomyces cerevisiae cells by transformation. The elongation reaction added repeats of the yeast telomeric sequence C1-3A to telomeric sequences at the end of linear DNA molecules. The reaction worked on the Tetrahymena telomeric sequence C4A2 and also on the simple repeat CA. The reaction was orientation specific: it occurred only when the GT-rich strand ran 5' to 3' towards the end of the molecule. Telomere elongation occurred by non-template-directed DNA synthesis rather than any type of recombination with chromosomal telomeres, because C1-3A repeats could be added to unrelated DNA sequences between the CA-rich repeats and the terminus of the transforming DNA. The elongation reaction was very efficient, and we believe that it was responsible for maintaining an average telomere length despite incomplete replication by template-directed DNA polymerase. The resolution reaction processed a head-to-head inverted repeat of telomeric sequences into two new telomeres at a frequency of 10(-2) per cell division.     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

A mechanism for the control of patch size in mammalian cell DNA excision repair

The hypothesis is suggested that size of the region excised in repair of UV-induced damage in mammalian cell is determined by the occurrence at random of a recognition sequence which terminates this excision process. The statistics of first occurrence times for a specific nucleotide sequence in a random chain are derived and shown to lead to an approximately random distribution of sizes around the average. The heterogeneity in sizes arising from a model are shown not to conflict with existing measurements. A sequence of length three or four is sufficient to account for the measured average size.     

Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length.     

Nickel enhances telomeric silencing in Saccharomyces cerevisiae

Certain nickel compounds including crystalline nickel sulfide (NiS) and subsulfide (Ni3S2) are potent human and animal carcinogens. In Chinese hamster embryo cells, an X-linked senescence gene was inactivated following nickel-induced DNA methylation. Nickel also induced the inactivation of the gpt reporter gene by chromatin condensation and a DNA methylation process in a transgenic gpt+ Chinese hamster cell line (G12), which is located near a heterochromatic region. To determine if nickel can cause gene silencing independently of DNA methylation, based only on the induction of changes in chromatin structure, we measured its effect on gene silencing in Saccharomyces cerevisiae. Growth of yeast in the presence of nickel chloride repressed a telomeric marker gene (URA3) and resulted in a stable epigenetic switch. This phenomenon was dependent on the number of cell doubling prior to selection and also on the distance of the marker gene from the end of the chromosome. The level of TPE (telomeric position effect) increased linearly with elevations of nickel concentration. Addition of magnesium inhibited this effect, but magnesium did not silence the reporter gene by itself. The level of silencing was also assessed following treatment with other transition metals: cobalt, copper and cadmium. In the sublethal range, cobalt induced similar effects as nickel, while copper and cadmium did not change the basal level of gene expression. Silencing by copper and cadmium were evident only at concentrations of those metals where the viability was very low.     

Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae.

The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae undergoing transition from the anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-type strain. We confirm that mitochondria of aerobic cells of a RAD strain lack the excision repair capacity of UV-induced pyrimidine dimers at all doses tested (1-15 J/m2). In contrast, in promitochondria of anaerobic cells of the wild-type strain excision repair appears to take place. This process is very efficient at low doses (at 0.5-5 J/m2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50%. The promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the rad2 mutant. Finally photoreactivation is found to be operating in nuclei, mitochondria and promitochondria.     

Specific binding of single-stranded telomeric DNA by Cdc13p of Saccharomyces cerevisiae

Cdc13p is a single strand telomere-binding protein of Saccharomyces cerevisiae; its telomere-binding region is within amino acids 451-693, Cdc13(451-693)p. In this study, we used purified Cdc13p and Cdc13(451-693)p to characterize their telomere binding activity. We found that the binding specificity of single-stranded TG(1-3) DNA by these two proteins is similar. However, the affinity of Cdc13(451-693)p to DNA was slightly lower than that of Cdc13p. The binding of telomeric DNA by these two proteins was disrupted at NaCl concentrations higher than 0.3 m, indicating that electrostatic interaction contributed significantly to the binding process. Because both proteins bound to strand TG(1-3) DNA positioned at the 3' end, the 5' end, or in the middle of the oligonucleotide substrates, our results indicated that the location of TG(1-3) in single-stranded DNA does not appear to be important for Cdc13p binding. Moreover, using DNase I footprint analysis, the structure of the telomeric DNA complexes of Cdc13p and Cdc13(451-693)p was analyzed. The DNase I footprints of these two proteins to three different telomeric DNA substrates were virtually identical, indicating that the telomere contact region of Cdc13p is within Cdc13(451-693)p. Together, the binding properties of Cdc13p and its binding domain support the theory that the specific binding of Cdc13p to telomeres is an important feature of telomeres that regulate telomerase access and/or differentiate natural telomeres from broken ends.     

NMR observation of T-tetrads in a parallel stranded DNA quadruplex formed by Saccharomyces cerevisiae telomere repeats.

We report here the NMR structure of the DNA sequence d-TGGTGGC containing two repeats of Saccharomyces cerevisiae telomere DNA which is unique in that it has a single thymine in the repeat sequence and the number of Gs can vary from one to three. The structure is a novel quadruplex incor-porating T-tetrads formed by symmetrical pairing of four Ts via O4-H3 H-bonds in a plane. This is in contrast to the previous results on other telomeric sequences which contained more than one T in the repeat sequences and they were seen mostly in the flexible regions of the structures. We observed that the T4-tetrad was nicely accommodated in the center of the G-quadruplex, but it caused a small underwinding of the right handed helix. The T tetrad stacked well on the adjacent G3-tetrad, but poorly on the G5 tetrad. Likewise, T1 also formed a stable T-tetrad at the 5' end of the quadruplex. To our knowledge, this is the first report of T-tetrad formation in DNA structures. These observations are of significance from the points of view of both structural diversity and specific recognitions.     

The size of mitochondrial DNA from a cytoplasmic petite mutant of Saccharomyces cerevisiae

Summary Mitochondrial DNA has been isolated from a cytoplasmic petite mutant of Saccharomyces cerevisiae which has retained only about 2% of the mitochondrial wild type genome. The denatured DNA was analyzed by agarose gel electrophoresis and a homogeneous, single band of DNA was found. Petite and wild type mitochondrial DNAs exhibited similar gel electrophoretic mobilities. Using denatured DNA from the E. coli phages T4 and T3 for comparison a molecular weight of 55×10⁶ daltons has been calculated for the double-stranded petite mitochondrial DNA. On the basis of this observation most of the mitochondrial DNA of this petite mutant appeared to consist of a polymer of about 50 repeats to account for a size similar to that of the wild type molecule. Thus a regulatory mechanism might exist which keeps constant the physical size of the mitochondrial DNA molecule in spite of the elimination of large fractions of the wild type genome.Dedicated to Dr. Dr. h. c. Peter Michaelis on the occasion of his 75th birthday     

Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.     

Involvement of Rho-type GTPase in control of cell size in Saccharomyces cerevisiae

Maintaining specific cell size, which is important for many organisms, is achieved by coordinating cell growth and cell division. In the budding yeast Saccharomyces cerevisiae, the existence of two cell-size checkpoints is proposed: at the first checkpoint, cell size is monitored before budding at the G1/S transition, and at the second checkpoint, actin depolymerization occurring in the small bud is monitored before the G2/M transition. Morphological analyses have revealed that the small GTPase Rho1p participates in cell-size control at both the G1/S and the G2/M boundaries. One group of rho1 mutants (rho1A) underwent premature entry into mitosis, leading to the birth of abnormally small cells. In another group of rho1 mutants (rho1B), the mother cells failed to reach an appropriate size before budding, and expression of the G1 cyclin Cln2p began at an earlier phase of the cell cycle. Analyses of mutants defective in Rho1p effector proteins indicate that Skn7p, Fks1p and Mpk1p are involved in cell-size control. Thus, Rho1p and its downstream regulatory pathways are involved in controlling cell size in S. cerevisiae.     

Exposure of single-stranded telomeric DNA causes G2/M cell cycle arrest in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Cdc13p is a single-stranded TG(1-3) DNA binding protein that protects telomeres and maintains telomere length. A mutant allele of CDC13, cdc13-1, causes accumulation of single-stranded TG(1-3) DNA near telomeres along with a G(2)/M cell cycle arrest at non-permissive temperatures. We report here that when the single-stranded TG(1-3) DNA is masked by its binding proteins, such as S. cerevisiae Gbp2p or Schizosaccharomyces pombe Tcg1, the growth arrest phenotype of cdc13-1 is rescued. Mutations on Gbp2p that disrupt its binding to the single-stranded TG(1-3) DNA render the protein unable to complement the defects of cdc13-1. These results indicate that the presence of a single-stranded TG(1-3) tail in cdc13-1 cells serves as the signal for the cell cycle checkpoint. Moreover, the binding activity of Gbp2p to single-stranded TG(1-3) DNA appears to be associated with its ability to restore the telomere-lengthening phenotype in cdc13-1 cells. These results indicate that Gbp2p is involved in modulating telomere length.     

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.     

Saccharomyces cerevisiae RAD5 influences the excision repair of DNA minor groove adducts

Nucleotide excision repair (NER) is the primary pathway for the removal of DNA adducts that distort the double helix. In the yeast Saccharomyces cerevisiae the RAD6 epistasis group defines a more poorly characterized set of DNA damage response pathways, believed to be distinct from NER. Here we show that the elimination of the DNA minor groove adducts formed by an important class of anticancer antibiotic (CC-1065 family) requires NER factors in S. cerevisiae. We also demonstrate that the elimination of this class of minor groove adduct from the active MFA2 gene depends upon functional Rad18 and Rad6. This is most clear for the repair of adducts on the transcribed strand, where an absolute requirement for Rad6 and Rad18 was seen. Further experiments revealed that a specific RAD6-RAD18-controlled subpathway, the RAD5 branch, mediates these events. Cells disrupted for rad5 are highly sensitive to this minor groove binding agent, and rad5 cells exhibit an in vivo adduct elimination defect indistinguishable from that seen in rad6 and rad18 cells as well as in NER-defective cells. Our results indicate that the RAD5 subpathway may interact with NER factors during the repair of certain DNA adducts.     

Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae

We have examined the roles of eukaryotic DNA topoisomerases I and II in DNA replication by the use of a set of four isogenic strains of Saccharomyces cerevisiae that are TOP1+ TOP2+, TOP1+ top2 ts, delta top1 TOP2+, and delta top1 top2 ts. Cells synchronized by treatment with the alpha-mating factor, or by cycles of feeding and starvation, were released from cell-cycle arrest, and the size distribution of DNA chains that were synthesized after the cells reentered the S-phase was determined as a function of time. The results indicate that synthesis of short DNA chains several thousand nucleotides in length can initiate in the absence of both topoisomerases, but their further elongation requires at least one of the two topoisomerases. Inactivation of DNA topoisomerase II does not alter significantly the time dependence of the patterns of nascent DNA chain synthesis, which is consistent with the notion that the requirement of this enzyme for viability is due to its essential role during mitosis, when pairs of intertwined newly replicated chromosomes are being segregated. The absence of DNA topoisomerase I leads to a temporary delay in the extension of the short DNA chains; this delay in chain elongation is also reflected in the rate of total DNA synthesis in the delta top1 mutant during the early S-phase. Thus, in wild-type cells, DNA topoisomerase I is probably the major replication swivel. The patterns of DNA synthesis in asynchronously grown delta top1 top2 ts cells at permissive and non-permissive temperatures are also consistent with the above conclusions.