Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated de novo telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of de novo telomere addition. Treatment of CAF-I-or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.

SummaryThe repair of spontaneous or induced DNA damage by homologous recombination (HR) in Saccharomyces cerevisiae will suppress chromosome rearrangements. Alternative chromosome healing pathways can result in chromosomal instability. One of these pathways is de novo telomere addition where the end of a broken chromosome is stabilized by telomerasedependent addition of telomeres at non-telomeric sites. De novo telomere addition requires the recruitment of telomerase to chromosomal targets. Subsequently, annealing of the telomerase reverse transcriptase RNA-template (guide RNA) at short regions of homology is followed by extension of the nascent 3 ¢ -end of the broken chromosome to copy a short region of the telomerase guide RNA; multiple cycles of this process yield the new telomere. Proteins including Pif1 helicase, the single-stranded DNA-binding protein Cdc13 and the Ku heterocomplex are known to participate in native telomere functions and also regulate the de novo telomere addition reaction. Studies of the sequences added at de novo telomeres have lead to a detailed description of the annealing-extension-dissociation cycles that copy the telomerase guide RNA, which can explain the heterogeneity of telomeric repeats at de novo and native telomeres in S. cerevisiae .

In telomerase-deficient Saccharomyces cerevisiae, telomeres are maintained by recombination. Here we used a S. cerevisiae assay for characterizing gross chromosomal rearrangements (GCRs) to analyze genome instability in post-senescent telomerase-deficient cells. Telomerase-deficient tlc1 and est2 mutants did not have increased GCR rates, but their telomeres could be joined to other DNAs resulting in chromosome fusions. Inactivation of Tel1 or either the Rad51 or Rad59 recombination pathways in telomerase-deficient cells increased the GCR rate, even though telomeres were maintained. The GCRs were translocations and chromosome fusions formed by nonhomologous end joining. We observed chromosome fusions only in mutant strains expressing Rad51 and Rad55 or when Tel1 was inactivated. In contrast, inactivation of Mec1 resulted in more inversion translocations such as the isochromosomes seen in human tumors. These inversion translocations seemed to be formed by recombination after replication of broken chromosomes.Telomeres function in replication and maintenance of chromosome ends, to prevent DNA ends from being inappropriately joined to each other and to prevent chromosome ends from activating checkpoints 1,2 . Telomeres are maintained by telomerase, which consists of the Est2 catalytic subunit, the Tlc1 RNA and other subunits 2 .Telomere maintenance also requires other proteins. These include the Tel1 protein kinase that functions in telomere protection and length regulation and proteins such as Cdc13 and Ku that target telomerase to telomeres and protect telomeres from degradation 2 . Proteins such as Pif1 help regulate telomere length 3 and prevent telomerase from adding telomeres to broken DNAs 3,4 . In telomerase-deficient S. cerevisiae cells telomeres are maintained by recombination 5,6 . Most mammalian cells lack telomerase 7 and have a limited lifespan. Immortalization and cancer progression require increased telomere maintenance capacity, either through upregulation of telomerase activity 7 or through the alternative lengthening of telomere pathway 8 .Recombination and the Tel1 and Mec1 checkpoints differentially effect genome rearrangements driven by telomere dysfunction in yeast L E T T E R S 612VOLUME 36 | NUMBER 6 | JUNE 2004 NATURE GENETICS RDKY5233 is a tlc1∆ type II strain. Additional relevant GCR rates include the tlc1∆ type I strain, RDKY5232 (3.1 × 10 -10 (0.9)); lig4∆ strain, RDKY3641 (1.6 × 10 -9 (9); ref. 10); tel1∆ lig4∆ strain, RDKY5238 (4.2 × 10 -9 (12)); and tel1∆ lig4∆ est2∆ strain, RDKY5240 (3.5 × 10 -9 (10)). ND, not determined.

Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerasedefective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae.The development and progression of cancer are correlated with genetic instability. These genomic changes are associated with either a microsatellite instability (MSI) phenotype, involving defects in mismatch repair and a dramatic increase in base substitution and insertion/deletion mutation rates, or a chromosomal instability (CIN) phenotype, involving changes in chromosome number and structure (reviewed in reference 55); however, some tumors have been observed with both MSI and CIN. A causal role for CIN in tumorigenesis is still debated; however, most cancers are associated with dramatic changes to the chromosomal complement (68), and several hereditary cancer predisposition syndromes are closely linked to CIN (49). Using the mutator hypothesis (61), it has been argued that changes in gene dosage, a loss of heterozygosity, a deregulation of gene expression, and the generation of gain-of-function protein chimeras due to CIN are sufficient to drive tumorigenesis in many cases, even without mutations in oncogenes or tumor suppressor genes (29). Thus, understanding CIN is likely to provide some insight into the mechanisms of tumorigenesis.One of the major barriers to understanding the CIN phenotype, even for model organisms such as the yeast Saccharomyces cerevisiae, is that there are very few genetic systems capable of systematic characterizations of genome rearrangements (49, 105). In principle, rearrangements can arise through both homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways. In practice, the lack of a detailed understanding of the genetic and biochemical mechanisms that underlie these types of rearrangements in vivo has remained a bottleneck to understanding the generation of genome rearrangements in cancer and other human diseases.An assay designed to analyze the formation of translocations and other gross chromosomal rearrangements (GCRs) was developed wit...

BackgroundThe gross chromosomal rearrangements (GCRs) observed in S. cerevisiae mutants with increased rates of accumulating GCRs include predicted dicentric GCRs such as translocations, chromosome fusions and isoduplications. These GCRs resemble the genome rearrangements found as mutations underlying inherited diseases as well as in the karyotypes of many cancers exhibiting ongoing genome instabilityMethodology/Principal FindingsThe structures of predicted dicentric GCRs were analyzed using multiple strategies including array-comparative genomic hybridization, pulse field gel electrophoresis, PCR amplification of predicted breakpoints and sequencing. The dicentric GCRs were found to be unstable and to have undergone secondary rearrangements to produce stable monocentric GCRs. The types of secondary rearrangements observed included: non-homologous end joining (NHEJ)-dependent intramolecular deletion of centromeres; chromosome breakage followed by NHEJ-mediated circularization or broken-end fusion to another chromosome telomere; and homologous recombination (HR)-dependent non-reciprocal translocations apparently mediated by break-induced replication. A number of these GCRs appeared to have undergone multiple bridge-fusion-breakage cycles. We also observed examples of chromosomes with extensive ongoing end decay in mec1 tlc1 mutants, suggesting that Mec1 protects chromosome ends from degradation and contributes to telomere maintenance by HR.Conclusions/SignificanceHR between repeated sequences resulting in secondary rearrangements was the most prevalent pathway for resolution of dicentric GCRs regardless of the structure of the initial dicentric GCR, although at least three other resolution mechanisms were observed. The resolution of dicentric GCRs to stable rearranged chromosomes could in part account for the complex karyotypes seen in some cancers.

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Retinoblastoma (Rb) protein promotes cell survival after DNA damage. We show here that the LxCxE binding site in Rb mediates both cell survival and cell-cycle arrest after DNA damage. Replication factor C (RF-C) complex plays an important role in DNA replication. We describe a novel function of the large subunit of RF-C in promoting cell survival after DNA damage. RF-Cp145 contains an LxCxE motif, and mutation of this motif abolishes the protective effect of RF-Cp145. The inability of wild-type RF-Cp145 to promote cell survival in Rb-null cells is rescued by Rb but not by Rb mutants defective in binding LxCxE proteins. RF-C thus enhances cell survival after DNA damage in an Rb-dependent manner.

Broken chromosomes healed by de novo addition of a telomere are a major class of genome rearrangements seen in Saccharomyces cerevisiae and similar to rearrangements seen in human tumors. We have analyzed the sequences of 534 independent de novo telomere additions within a 12-kb region of chromosome V. The distribution of events mirrored that of four-base sequences consisting of the GG, GT, and TG dinucleotides, suggesting that de novo telomere additions occur at short regions of homology to the telomerase guide RNA. These chromosomal sequences restrict potential registrations of the added telomere sequence. The first 11 nucleotides of the addition sequences fell into common families that included 91% of the breakpoints. The observed registrations suggest that the 3 end of the TLC1 guide RNA is involved in annealing but not as a template for synthesis. Some families of added sequences can be accounted for by one cycle of annealing and extension, whereas others require a minimum of two. The same pattern emerges for sequences added onto the most common addition sequence, indicating that de novo telomeres are added and extended by the same process. Together, these data indicate that annealing is central to telomerase registration, which limits telomere heterogeneity and resolves the problem of synthesizing Rap1 binding sites by a nonprocessive telomerase with a lowcomplexity guide RNA sequence.