Callahan, Julie L, et al.Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility.”. Mol Cell Biol 23.21 (2003): , 23, 21, 7849-60. Print.Abstract
Expansion of trinucleotide repeats (TNRs) is the causative mutation in several human genetic diseases. Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in lagging-strand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/helicase Dna2, RNase HI, DNA ligase, polymerase delta, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.
Alexander, Mary Kate, and Virginia A Zakian. “Rap1p telomere association is not required for mitotic stability of a C(3)TA(2) telomere in yeast.”. EMBO J 22.7 (2003): , 22, 7, 1688-96. Web.Abstract
Telomeric DNA usually consists of a repetitive sequence: C(1-3)A/TG(1-3) in yeast, and C(3)TA(2)/T(2)AG(3) in vertebrates. In yeast, the sequence-specific DNA- binding protein Rap1p is thought to be essential for telomere function. In a tlc1h mutant, the templating region of the telomerase RNA gene is altered so that telomerase adds the vertebrate telomere sequence instead of the yeast sequence to the chromosome end. A tlc1h strain has short but stable telomeres and no growth defect. We show here that Rap1p and the Rap1p-associated Rif2p did not bind to a telomere that contains purely vertebrate repeats, while the TG(1-3) single-stranded DNA binding protein Cdc13p and the normally non-telomeric protein Tbf1p did bind this telomere. A chromosome with one entirely vertebrate-sequence telomere had a wild-type loss rate, and the telomere was maintained at a short but stable length. However, this telomere was unable to silence a telomere-adjacent URA3 gene, and the strain carrying this telomere had a severe defect in meiosis. We conclude that Rap1p localization to a C(3)TA(2) telomere is not required for its essential mitotic functions.
Taggart, Andrew KP, and Virginia A Zakian. “Telomerase: what are the Est proteins doing?”. Curr Opin Cell Biol 15.3 (2003): , 15, 3, 275-80. Print.Abstract
Saccharomyces cerevisiae has proven to be a useful model organism for the study of telomerase, a specialized cellular reverse transcriptase that helps maintain genomic stability by adding telomeric DNA repeats to the ends of chromosomes. Yeast telomerase is thought to be a holoenzyme containing Est2p and TLC1 RNA, the catalytic subunit and its intrinsic template, respectively, as well as the TLC1-RNA-associated factors Est1p and Est3p. Cdc13p, a sequence-specific telomere-DNA-binding protein, is also required for action in vivo. A current model for telomerase regulation is that telomere-associated Cdc13p binds Est1p, thereby recruiting telomerase. However, recent chromatin immunoprecipitation experiments suggest an alternate role for Est1p in activating Est2p-TLC1-RNA that is already bound to the telomere. Three models for Est1p activation are presented.
Ivessa, Andreas S, et al.The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes.”. Mol Cell 12.6 (2003): , 12, 6, 1525-36. Print.Abstract
The Saccharomyces cerevisiae RRM3 gene encodes a 5' to 3' DNA helicase. While replication of most of the yeast genome was not dependent upon Rrm3p, in its absence, replication forks paused and often broke at an estimated 1400 discrete sites, including tRNA genes, centromeres, inactive replication origins, and transcriptional silencers. These replication defects were associated with activation of the intra-S phase checkpoint. Activation of the checkpoint was critical for viability of rrm3Delta cells, especially at low temperatures. Each site whose replication was affected by Rrm3p is assembled into a nonnucleosomal protein-DNA complex. At tRNA genes and the silent mating type loci, disruption of these complexes eliminated dependence upon Rrm3p. These data indicate that the Rrm3p DNA helicase helps replication forks traverse protein-DNA complexes, naturally occurring impediments that are encountered in each S phase.
Taggart, Andrew KP, Shu-Chun Teng, and Virginia A Zakian. “Est1p as a cell cycle-regulated activator of telomere-bound telomerase.”. Science 297.5583 (2002): , 297, 5583, 1023-6. Web.Abstract
In Saccharomyces cerevisiae, the telomerase components Est2p, TLC1 RNA, Est1p, and Est3p are thought to form a complex that acts late during chromosome replication (S phase) upon recruitment by Cdc13p, a telomeric DNA binding protein. Consistent with this model, we show that Est1p, Est2p, and Cdc13p are telomere-associated at this time. However, Est2p, but not Est1p, also binds telomeres before late S phase. The cdc13-2 allele has been proposed to be defective in recruitment, yet Est1p and Est2p telomere association persists in cdc13-2 cells. These findings suggest a model in which Est1p binds telomeres late in S phase and interacts with Cdc13p to convert inactive, telomere-bound Est2p to an active form.
Ivessa, Andreas S, et al.Saccharomyces Rrm3p, a 5' to 3' DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA.”. Genes Dev 16.11 (2002): , 16, 11, 1383-96. Web.Abstract
In wild-type Saccharomyces cerevisiae, replication forks slowed during their passage through telomeric C(1-3)A/TG(1-3) tracts. This slowing was greatly exacerbated in the absence of RRM3, shown here to encode a 5' to 3' DNA helicase. Rrm3p-dependent fork progression was seen at a modified Chromosome VII-L telomere, at the natural X-bearing Chromosome III-L telomere, and at Y'-bearing telomeres. Loss of Rrm3p also resulted in replication fork pausing at specific sites in subtelomeric DNA, such as at inactive replication origins, and at internal tracts of C(1-3)A/TG(1-3) DNA. The ATPase/helicase activity of Rrm3p was required for its role in telomeric and subtelomeric DNA replication. Because Rrm3p was telomere-associated in vivo, it likely has a direct role in telomere replication.
Zhou, Jin-Qiu, et al.Schizosaccharomyces pombe pfh1+ encodes an essential 5' to 3' DNA helicase that is a member of the PIF1 subfamily of DNA helicases.”. Mol Biol Cell 13.6 (2002): , 13, 6, 2180-91. Print.Abstract
The Saccharomyces cerevisiae Pif1p DNA helicase is the prototype member of a helicase subfamily conserved from yeast to humans. S. cerevisiae has two PIF1-like genes, PIF1 itself and RRM3, that have roles in maintenance of telomeric, ribosomal, and mitochondrial DNA. Here we describe the isolation and characterization of pfh1+, a Schizosaccharomyces pombe gene that encodes a Pif1-like protein. Pfh1p was the only S. pombe protein with high identity to Saccharomyces Pif1p. Unlike the two S. cerevisiae Pif1 subfamily proteins, the S. pombe Pfh1p was essential. Like Saccharomyces Pif1p, a truncated form of the S. pombe protein had 5' to 3' DNA helicase activity. Point mutations in an invariant lysine residue in the ATP binding pocket of Pfh1p had the same phenotype as deleting pfh1+, demonstrating that the ATPase/helicase activity of Pfh1p was essential. Although mutant spores depleted for Pfh1p proceeded through S phase, they arrested with a terminal cellular phenotype consistent with a postinitiation defect in DNA replication. Telomeric DNA was modestly shortened in the absence of Pfh1p. However, genetic analysis demonstrated that maintenance of telomeric DNA was not the sole essential function of S. pombe Pfh1p.
Ivessa, Andreas S, and Virginia A Zakian. “To fire or not to fire: origin activation in Saccharomyces cerevisiae ribosomal DNA.”. Genes Dev 16.19 (2002): , 16, 19, 2459-64. Web.
Tham, Wai-Hong, and Virginia A Zakian. “Transcriptional silencing at Saccharomyces telomeres: implications for other organisms.”. Oncogene 21.4 (2002): , 21, 4, 512-21. Web.Abstract
Telomeres are the natural ends of eukaryotic chromosomes. In most organisms, telomeres consist of simple, repeated DNA with the strand running 5' to 3' towards the end of the chromosome being rich in G residues. In cases where the very end of the chromosome has been examined, the G-strand is extended to form a short, single stranded tail. The chromatin structure of telomeric regions often has features that distinguish them from other parts of the genome. Because telomeres protect chromosome ends from degradation and end-to-end fusions and prevent the loss of terminal DNA by serving as a substrate for telomerase, they are essential for the stable maintenance of eukaryotic chromosomes. In addition to their essential functions, telomeres in diverse organisms are specialized sites for gene expression. Transcription of genes located next to telomeres is repressed, a phenomenon termed telomere position effect (TPE). TPE is best characterized in the yeast Saccharomyces cerevisiae. This article will focus on the silencing properties of Saccharomyces telomeres and end with speculation on the role of TPE in yeasts and other organisms.
Tham, WH, et al.Localization of yeast telomeres to the nuclear periphery is separable from transcriptional repression and telomere stability functions.”. Mol Cell 81 (2001): , 8, 1, 189-99. Print.Abstract
The left telomere of Saccharomyces chromosome VII was often localized near the nuclear periphery, even in cells lacking the silencing proteins Sir3 or Hdf1. This association was lost in late mitotic cells and when transcription was induced through the telomeric tract. Although in silencing competent cells there was no correlation between the fraction of cells in which a telomeric gene was repressed and the fraction of cells in which it was localized to the periphery, no condition was found where the telomere was both silenced and away from the periphery. We conclude that localization of a telomere to the nuclear periphery is not sufficient for transcriptional repression nor does it affect the stability function of yeast telomeres.
Alexander, MK, BD Bourns, and VA Zakian. “One-hybrid systems for detecting protein-DNA interactions.”. Methods Mol Biol 177 (2001): , 177, 241-59. Web.
Bessler, JB, JZ Torres, and VA Zakian. “The Pif1p subfamily of helicases: region-specific DNA helicases?”. Trends Cell Biol 11.2 (2001): , 11, 2, 60-5. Print.Abstract
DNA helicases are required for DNA replication, recombination and repair. Despite a common enzymatic function - the ability to unwind duplex DNA - most helicases share only limited amino acid sequence similarity. Helicases that have significant sequence similarity define a subfamily. It remains unclear, however, how this sequence similarity relates to helicase function. The Saccharomyces cerevisiae Pif1p helicase is the prototype member of a helicase subfamily that is conserved from yeasts to humans. As the two Pif1p subfamily members studied to date affect the same DNA sequences, the amino acid similarity that defines this subfamily might reflect common substrates.
Tsukamoto, Y, AK Taggart, and VA Zakian. “The role of the Mre11-Rad50-Xrs2 complex in telomerase- mediated lengthening of Saccharomyces cerevisiae telomeres.”. Curr Biol 11.17 (2001): , 11, 17, 1328-35. Print.Abstract
BACKGROUND: The Saccharomyces Mre11p, Rad50p, and Xrs2p proteins form a complex, called the MRX complex, that is required to maintain telomere length. Cells lacking any one of the three MRX proteins and Mec1p, an ATM-like protein kinase, undergo telomere shortening and ultimately die, phenotypes characteristic of cells lacking telomerase. The other ATM-like yeast kinase, Tel1p, appears to act in the same pathway as MRX: mec1 tel1 cells have telomere phenotypes similar to those of telomerase-deficient cells, whereas the phenotypes of tel1 cells are not exacerbated by the loss of a MRX protein. RESULTS: The nuclease activity of Mre11p was found to be dispensable for the telomerase-promoting activity of the MRX complex. The association of the single-stranded TG1-3 DNA binding protein Cdc13p with yeast telomeres occurred efficiently in the absence of Tel1p, Mre11p, Rad50p, or Xrs2p. Targeting of catalytically active telomerase to the telomere suppressed the senescence phenotype of mec1 mrx or mec1 tel1 cells. Moreover, when telomerase was targeted to telomeres, telomere lengthening was robust in mec1 mrx and mec1 tel1 cells. CONCLUSIONS: These data rule out models in which the MRX complex is necessary for Cdc13p binding to telomeres or in which the MRX complex is necessary for the catalytic activity of telomerase. Rather, the data suggest that the MRX complex is involved in recruiting telomerase activity to yeast telomeres.
Mangahas, JL, et al.Repair of chromosome ends after telomere loss in Saccharomyces.”. Mol Biol Cell 12.12 (2001): , 12, 12, 4078-89. Print.Abstract
Removal of a telomere from yeast chromosome VII in a strain having two copies of this chromosome often results in its loss. Here we show that there are three pathways that can stabilize this broken chromosome: homologous recombination, nonhomologous end joining, and de novo telomere addition. Both in a wild-type and a recombination deficient rad52 strain, most stabilization events were due to homologous recombination, whereas nonhomologous end joining was exceptionally rare. De novo telomere addition was relatively rare, stabilizing <0.1% of broken chromosomes. Telomere addition took place at a very limited number of sites on chromosome VII, most occurring close to a 35-base pair stretch of telomere-like DNA that is normally approximately 50 kb from the left telomere of chromosome VII. In the absence of the Pif1p DNA helicase, telomere addition events were much more frequent and were not concentrated near the 35-base pair tract of telomere-like DNA. We propose that internal tracts of telomere-like sequence recruit telomerase by binding its anchor site and that Pif1p inhibits telomerase by dissociating DNA primer-telomerase RNA interactions. These data also show that telomeric DNA is essential for the stable maintenance of linear chromosomes in yeast.
Balakumaran, BS, CH Freudenreich, and VA Zakian. “CGG/CCG repeats exhibit orientation-dependent instability and orientation-independent fragility in Saccharomyces cerevisiae.”. Hum Mol Genet 91 (2000): , 9, 1, 93-100. Print.Abstract
An expansion to >200 CGG/CCG repeats (hereafter called CGG) in the 5' region of the FMR1 gene causes fragile X syndrome, and this locus becomes a folate-sensitive fragile site. We used Saccharomyces cerevisiae as a model system to study the stability and fragility of CGG repeats. Tracts of (CGG)(81)and (CGG)(160)were integrated onto a yeast chromosome in both orientations relative to the nearest replication origin. Tracts of this length are pre-mutation alleles in humans, with a high probability of expansion in future generations. The CGG tracts in yeast colonies showed a length-dependent instability with longer tracts being more prone to contraction than shorter tracts. In addition, there was an orientation bias for tract stability with tracts having fewer contractions when the CCG strand was the template for lagging strand synthesis. Expansions of the CGG tracts also occurred in an orientation-dependent manner, although at a lower frequency than contractions. To determine whether CGG tracts are fragile sites in yeast, the CGG tracts were flanked by direct repeats, and the rate of recombination between the repeats determined. Strains carrying the (CGG)(160)tract in either orientation had a large increase in their rate of recombination compared with a no-tract control strain. Because this increase was dependent on genes involved in double-strand break repair, recombination was likely to be initiated by CGG tract-induced breakage between the direct repeats. The observation of orientation-dependent instability and orientation-independent fragility suggests that at least some aspects of their underlying mechanisms are different.
Zhou, J, et al.Pif1p helicase, a catalytic inhibitor of telomerase in yeast.”. Science 289.5480 (2000): , 289, 5480, 771-4. Print.Abstract
Mutations in the yeast Saccharomyces cerevisiae PIF1 gene, which encodes a 5'-to-3' DNA helicase, cause telomere lengthening and a large increase in the formation rate of new telomeres. Here, we show that Pif1p acts by inhibiting telomerase rather than telomere-telomere recombination, and this inhibition requires the helicase activity of Pif1p. Overexpression of enzymatically active Pif1p causes telomere shortening. Thus, Pif1p is a catalytic inhibitor of telomerase-mediated telomere lengthening. Because Pif1p is associated with telomeric DNA in vivo, its effects on telomeres are likely direct. Pif1p-like helicases are found in diverse organisms, including humans. We propose that Pif1p-mediated inhibition of telomerase promotes genetic stability by suppressing telomerase-mediated healing of double-strand breaks.
Ivessa, AS, JQ Zhou, and VA Zakian. “The Saccharomyces Pif1p DNA helicase and the highly related Rrm3p have opposite effects on replication fork progression in ribosomal DNA.”. Cell 100.4 (2000): , 100, 4, 479-89. Print.Abstract
Replication of Saccharomyces ribosomal DNA (rDNA) proceeds bidirectionally from origins in a subset of the approximately 150 tandem repeats, but the leftward-moving fork stops when it encounters the replication fork barrier (RFB). The Pif1p helicase and the highly related Rrm3p were rDNA associated in vivo. Both proteins affected rDNA replication but had opposing effects on fork progression. Pif1p helped maintain the RFB. Rrm3p appears to be the replicative helicase for rDNA as it acted catalytically to promote fork progression throughout the rDNA. Loss of Rrm3p increased rDNA breakage and accumulation of rDNA circles, whereas breakage and circles were less common in pif1 cells. These data support a model in which replication fork pausing causes breakage and recombination in the rDNA.
Qi, H, and VA Zakian. “The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase alpha and the telomerase-associated est1 protein.”. Genes Dev 14.14 (2000): , 14, 14, 1777-88. Print.Abstract
Saccharomyces telomeres consist of approximately 350 bp of C(1-3)A/TG(1-3) DNA. Most of this approximately 350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C(1-3)A strand is degraded to generate a long single strand TG(1-3) tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG(1-3) DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase alpha, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 or POL1 that reduced the Cdc13p-Pol1p interaction resulted in telomerase mediated telomere lengthening. Over-expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG(1-3) strand lengthening by telomerase and its interaction with Pol1p promotes C(1-3)A strand resynthesis by DNA polymerase alpha.
Teng, SC, et al.Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process.”. Mol Cell 64 (2000): , 6, 4, 947-52. Print.Abstract
Type II survivors arise in Saccharomyces cells lacking telomerase by a recombinational pathway that results in very long and heterogeneous length telomeres. Here we show that type II telomeres appeared abruptly in a population of cells with very short telomeres. Once established, these long telomeres progressively shortened. Short telomeres were substrates for rare, one-step lengthening events. The generation of type II survivors was absolutely Rad50p dependent. In a telomerase-proficient cell, the telomere-binding Rif proteins inhibited telomerase lengthening of telomeres. In a telomerase-deficient strain, Rif proteins, especially Rif2p, inhibited type II recombination. These data argue that only short telomeres are substrates for type II recombination and suggest that the donor for this recombination is not a chromosomal telomere.
de Bruin, D, et al.Telomere folding is required for the stable maintenance of telomere position effects in yeast.”. Mol Cell Biol 20.21 (2000): , 20, 21, 7991-8000. Print.Abstract
Yeast telomeres reversibly repress the transcription of adjacent genes, a phenomenon called telomere position effect (TPE). TPE is thought to result from Rap1 and Sir protein-mediated spreading of heterochromatin-like structures from the telomeric DNA inwards. Because Rap1p is associated with subtelomeric chromatin as well as with telomeric DNA, yeast telomeres are proposed to form fold-back or looped structures. TPE can be eliminated in trans by deleting SIR genes or in cis by transcribing through the C(1-3)A/TG(1-3) tract of a telomere. We show that the promoter of a telomere-linked URA3 gene was inaccessible to restriction enzymes and that accessibility increased both in a sir3 strain and upon telomere transcription. We also show that subtelomeric chromatin was hypoacetylated at histone H3 and at each of the four acetylatable lysines in histone H4 and that histone acetylation increased both in a sir3 strain and when the telomere was transcribed. When transcription through the telomeric tract occurred in G(1)-arrested cells, TPE was lost, demonstrating that activation of a silenced telomeric gene can occur in the absence of DNA replication. The loss of TPE that accompanied telomere transcription resulted in the rapid and efficient loss of subtelomeric Rap1p. We propose that telomere transcription disrupts core heterochromatin by eliminating Rap1p-mediated telomere looping. This interpretation suggests that telomere looping is critical for maintaining TPE.