Thermal blockage of viruslike particle formation for the yeast retrotransposon Ty3 reveals differences in the cellular stress response

Summary.  The long terminal repeat (LTR) retrotransposons of the yeast Saccharomyces cerevisiae are similar in their structures and life cycles to animal retroviruses. The yeast LTR retrotransposon Ty3 does not transpose under conditions where the cellular stress response is activated. During stress, mature Ty3 proteins, indicative of the formation of intracellular Ty3 viruslike particles (VLPs), do not accumulate. In order to examine the role of stress proteins in Ty3 transposition, a sensitive genetic assay was developed to measure VLP formation. The assay employs a Ty3 element marked with a mutant allele of the yeast HIS3 gene (his3AI). To create a stable His⁺ phenotype, Ty3 must form VLPs, reverse transcribe Ty3 RNA into cDNA, and then insert the cDNA into either chromosomal or plasmid DNA. Using this assay, thermal inhibition of Ty3 transposition was evident at temperatures as low as 30 °C. The level of production of mature Ty3 proteins parallels the transposition frequency. Although overexpression of the yeast UBP3 gene allows VLPs to form and transposition to occur in the constitutively stressed ssa1 ssa2 strain, it does not alleviate the inhibition of these processes during stress induced by heat or ethanol. This suggests that the genetic and physical modes of stress response induction are not equivalent. Accepted April 11, 2001 Received February 2, 2001     

De novo telomere formation is suppressed by the Mec1-dependent inhibition of Cdc13 accumulation at DNA breaks

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability, since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. Here we report that Mec1, the ATR ortholog, couples the detection of DNA ends with the inhibition of telomerase. Mec1 inhibits telomere healing by phosphorylating Cdc13 on its S306 residue, a phosphorylation event that suppresses Cdc13 accumulation at DSBs. Conversely, telomere addition at accidental breaks is promoted by Pph3, the yeast protein phosphatase 4 (PP4). Pph3 is itself modulated by Rrd1, an activator of PP2A family phosphatases. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DNA breaks. These studies therefore identify a mechanism by which the ATR family of kinases enforces genome integrity, and a process that underscores the contribution of Cdc13 to the fate of DNA ends.     

MECP2 duplications in six patients with complex sex chromosome rearrangements

Duplications of the Xq28 chromosome region resulting in functional disomy are associated with a distinct clinical phenotype characterized by infantile hypotonia, severe developmental delay, progressive neurological impairment, absent speech, and proneness to infections. Increased expression of the dosage-sensitive MECP2 gene is considered responsible for the severe neurological impairments observed in affected individuals. Although cytogenetically visible duplications of Xq28 are well documented in the published literature, recent advances using array comparative genomic hybridization (CGH) led to the detection of an increasing number of microduplications spanning MECP2. In rare cases, duplication results from intrachromosomal rearrangement between the X and Y chromosomes. We report six cases with sex chromosome rearrangements involving duplication of MECP2. Cases 1-4 are unbalanced rearrangements between X and Y, resulting in MECP2 duplication. The additional Xq material was translocated to Yp in three cases (cases 1-3), and to the heterochromatic region of Yq12 in one case (case 4). Cases 5 and 6 were identified by array CGH to have a loss in copy number at Xp and a gain in copy number at Xq28 involving the MECP2 gene. In both cases, fluorescent in situ hybridization (FISH) analysis revealed a recombinant X chromosome containing the duplicated material from Xq28 on Xp, resulting from a maternal pericentric inversion. These cases add to a growing number of MECP2 duplications that have been detected by array CGH, while demonstrating the value of confirmatory chromosome and FISH studies for the localization of the duplicated material and the identification of complex rearrangements.     

TY3 GAG3 protein forms ordered particles in Escherichia coli

The yeast retrovirus-like element Ty3 GAG3 gene encodes a Gag3 polyprotein analogous to retroviral Gag. Gag3 lacks matrix, but contains capsid, spacer, and nucleocapsid domains. Expression of a Ty3 Gag3 or capsid domain optimized for expression in Escherichia coli was sufficient for Ty3 particle assembly. Virus-like ordered particles assembled from Gag3 were similar in size to immature particles from yeast and contained nucleic acid. However, particles assembled from the CA domain were variable in size and displayed much less organization than native particles. These results indicate that assembly can be driven through interactions among capsid subunits in the particle, but that the nucleocapsid domain, likely in association with RNA, confers order upon this process.     

Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery

DNA double-strand breaks (DSBs) are among the most deleterious forms of DNA lesions in cells. Here we induced site-specific DSBs in yeast cells and monitored chromatin dynamics surrounding the DSB using Chromosome Conformation Capture (3C). We find that formation of a DSB within G1 cells is not sufficient to alter chromosome dynamics. However, DSBs formed within an asynchronous cell population result in large decreases in both intra- and interchromosomal interactions. Using live cell microscopy, we find that changes in chromosome dynamics correlate with relocalization of the DSB to the nuclear periphery. Sequestration to the periphery requires the nuclear envelope protein, Mps3p, and Mps3p-dependent tethering delays recombinational repair of a DSB and enhances gross chromosomal rearrangements. Furthermore, we show that components of the telomerase machinery are recruited to a DSB and that telomerase recruitment is required for its peripheral localization. Based on these findings, we propose that sequestration of unrepaired or slowly repaired DSBs to the nuclear periphery reflects a competition between alternative repair pathways.     

Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains

Wine yeast strains show a high level of chromosome length polymorphism. This polymorphism is mainly generated by illegitimate recombination mediated by Ty transposons or subtelomeric repeated sequences. We have found, however, that the SSU1-R allele, which confers sulfite resistance to yeast cells, is the product of a reciprocal translocation between chromosomes VIII and XVI due to unequal crossing-over mediated by microhomology between very short sequences on the 5' upstream regions of the SSU1 and ECM34 genes. We also show that this translocation is only present in wine yeast strains, suggesting that the use for millennia of sulfite as a preservative in wine production could have favored its selection. This is the first time that a gross chromosomal rearrangement is shown to be involved in the adaptive evolution of Saccharomyces cerevisiae.     

Genomic complexity and chromosomal rearrangements in wine-laboratory yeast hybrids

In this report we describe the genomic complexity of a number of Saccharomyces yeast strains isolated from sherry wine (flor yeasts), and the genomic stability of a yeast hybrid derived from one of these and a laboratory strain. Flor yeast strains largely differed in their DNA content, but showed very few variations their molecular karyotype. These strains contained a large number of Ty2 sequences, but lacking the Ty1 elements commonly found in laboratory strains. The genetic analysis of a flor-laboratory hybrid indicated that flor yeasts were aneuploid. Hybridization patterns obtained with Ty1 and Ty2 probes in the meiotic progeny of this hybrid suggested that recombination may occur not only among homologous chromosomes of similar length, but also among polymorphic partners with different sizes. New chromosomal variants were frequently observed in the meiotic products, suggesting that polymorphism in chromosome length may itself be a major source of karyotypic variation. The genetic analysis of such variants indicated that recombinational events leading to new chromosomal forms may occur both mitotically and meiotically. Received: 2 April / 26 May 1996     

Low-copy repeats at the human VIPR2 gene predispose to recurrent and nonrecurrent rearrangements

Submicroscopic structural variations, including deletions, duplications, inversions and more complex rearrangements, are widespread in normal human genomes. Inverted segmental duplications or highly identical low-copy repeat (LCR) sequences can mediate the formation of inversions and more complex structural rearrangements through non-allelic homologous recombination. In a patient with 7q36 inverted duplication/terminal deletion, we demonstrated the central role of a pair of short inverted LCRs in the vasoactive intestinal peptide receptor gene (VIPR2)-LCRs in generating the rearrangement. We also revealed a relatively common VIPR2-LCR-associated inversion polymorphism disrupting the gene in almost 1% of healthy subjects, and a small number of complex duplications/triplications. In genome-wide studies of several thousand patients, a significant association of rare microduplications with variable size, all involving VIPR2, with schizophrenia was recently described, suggesting that altered vasoactive intestinal peptide signaling is likely implicated in the pathogenesis of schizophrenia. Genetic testing for VIPR2-LCR-associated inversions should be performed on available cohorts of psychiatric patients to evaluate their potential pathogenic role.     

Localization of the 17q breakpoint of a constitutional 1;17 translocation in a patient with neuroblastoma within a 25-kb segment located between the ACCN1 and TLK2 genes and near the distal breakpoints of two microdeletions in neurofibromatosis type 1 patients

We have constructed a 1.4-Mb P1 artificial chromosome/bacterial artificial chromosome (PAC/BAC) contig spanning the 17q breakpoint of a constitutional translocation t(1;17)(p36.2;q11.2) in a patient with neuroblastoma. Three 17q breakpoint-overlapping cosmids were identified and sequenced. No coding sequences were found in the immediate proximity of the 17q breakpoint. The PAC/BAC contig covers the region between the proximally located ACCN1 gene and the distally located TLK2 gene and SCYA chemokine gene cluster. The observation that the 17q breakpoint region could not be detected in any of the screened yeast artificial chromosome libraries and the localization of the 17q breakpoint in the vicinity of the distal breakpoints of two microdeletions in patients with neurofibromatosis type 1 suggest that this chromosomal region is genetically unstable and prone to rearrangements.