Homologous Recombination and Repair Functions Required for Mutagenicity during Yeast Meiosis.

Several meiotic events reshape the genome prior to its transfer (via gametes) to the next generation. The occurrence of new meiotic mutations is tightly linked to homologous recombination (HR) and firmly depends on Spo11-induced DNA breaks. To gain insight into the molecular mechanisms governing mutagenicity during meiosis, we examined the timing of mutation and recombination events in cells deficient in various DNA HR-repair genes, which represent distinct functions along the meiotic recombination process.

Mutagenicity in haploid yeast meiosis resulting from repair of DSBs by the sister chromatid.

Mutations in diploid budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. Such meiotic mutations are thought to result from the repair of double-strand breaks (DSBs) in meiosis, during the process of recombination. Here, we report studies of mutagenicity in haploid strains that may undergo meiosis due to the expression of both mating-type alleles, MATa and MATα.

Timing of appearance of new mutations during yeast meiosis and their association with recombination.

Mutations in budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. In contrast to mutations that occur in somatic cells, meiotic mutations have a special, long-range impact on evolution, because they are transferred to the following generations through the gametes. Understanding the mechanistic basis of meiotic mutagenicity is still lacking, however.

Imaging Cell Cycle Phases and Transitions of Living Cells from Yeast to Woman.

The eukaryotic cell cycle is comprised of different phases that take place sequentially once, and normally only once, every division cycle. Such a dynamic process is best viewed in real time in living dividing cells. The insights that can be gained from such methods are considerably larger than any alternative technique that only generates snapshots.

Phosphorylation and dephosphorylation regulate APC/C(Cdh1) substrate degradation.

The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/C(Cdh1) mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1(m11) mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity.

Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.

Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets.

Separation of roles of Zip1 in meiosis revealed in heterozygous mutants of Saccharomyces cerevisiae.

Synapsis of homologs during meiotic prophase I is associated with a protein complex built along the bivalents--the synaptonemal complex (SC). Mutations in the SC-component gene ZIP1 diminish SC formation, leading to reduced recombination levels and low spore viability. Here we show that in SK1 strains heterozygous for a deletion of ZIP1 in certain regions meiotic interference are impaired with no decrease in recombination levels.

APC/CCdh1 specific degradation of Hsl1 and Clb2 is required for proper stress responses of S. cerevisiae.

Cdh1 activates the Anaphase Promoting Complex/Cyclosome (APC/C(Cdh1)) throughout G(1) to degrade key cell cycle proteins. Cdh1 is not essential for cell proliferation, in spite of the fact that overexpression of some its degradation substrates is highly toxic. We report here that cdh1Delta cells are sensitive to stresses that activate the CWI (Cell Wall Integrity) and Hog1 MAP kinase pathways.

Functional conservation of the yeast and Arabidopsis RAD54-like genes.

The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions.

Spore germination in Saccharomyces cerevisiae: global gene expression patterns and cell cycle landmarks.

Background: Spore germination in the yeast Saccharomyces cerevisiae is a process in which non-dividing haploid spores re-enter the mitotic cell cycle and resume vegetative growth. To study the signals and pathways underlying spore germination we examined the global changes in gene expression and followed cell-cycle and germination markers during this process.

Results: We find that the germination process can be divided into two distinct stages.

Four linked genes participate in controlling sporulation efficiency in budding yeast.

Quantitative traits are conditioned by several genetic determinants. Since such genes influence many important complex traits in various organisms, the identification of quantitative trait loci (QTLs) is of major interest, but still encounters serious difficulties. We detected four linked genes within one QTL, which participate in controlling sporulation efficiency in Saccharomyces cerevisiae.

Modulation of the transcription regulatory program in yeast cells committed to sporulation.

Background: Meiosis in budding yeast is coupled to the process of sporulation, where the four haploid nuclei are packaged into a gamete. This differentiation process is characterized by a point of transition, termed commitment, when it becomes independent of the environment. Not much is known about the mechanisms underlying commitment, but it is often assumed that positive feedback loops stabilize the underlying gene-expression cascade.

Application of SNPs for assessing biodiversity and phylogeny among yeast strains.

We examined the efficacy of single-nucleotide polymorphism (SNP) markers for the assessment of the phylogeny and biodiversity of Saccharomyces strains. Each of 32 Saccharomyces cerevisiae strains was genotyped at 30 SNP loci discovered by sequence alignment of the S. cerevisiae laboratory strain SK1 to the database sequence of strain S288c.

Involvement of Sir2/4 in silencing of DNA breakage and recombination on mouse YACs during yeast meiosis.

Yeast artificial chromosomes (YACs) that contain human DNA backbone undergo DNA double-strand breaks (DSBs) and recombination during yeast meiosis at rates similar to the yeast native chromosomes. Surprisingly, YACs containing DNA covering a recombination hot spot in the mouse major histocompatibility complex class III region do not show meiotic DSBs and undergo meiotic recombination at reduced levels. Moreover, segregation of these YACs during meiosis is seriously compromised.

Mammalian meiosis involves DNA double-strand breaks with 3' overhangs.

Meiotic recombination in yeast is initiated at DNA double-strand breaks (DSBs), processed into 3' single-strand overhangs that are active in homology search, repair and formation of recombinant molecules. Are 3' overhangs recombination intermediaries in mouse germ cells too? To answer this question we developed a novel approach based on the properties of the Klenow enzyme. We carried out two different, successive in situ Klenow enzyme-based reactions on sectioned preparations of testicular tubules.

DNA motif associated with meiotic double-strand break regions in Saccharomyces cerevisiae.

Meiotic recombination in yeast is initiated by DNA double-strand breaks (DSBs) that occur at preferred sites, distributed along the chromosomes. These DSB sites undergo changes in chromatin structure early in meiosis, but their common features at the level of DNA sequence have not been defined until now. Alignment of 1 kb sequences flanking six well-mapped DSBs has allowed us to define a flexible sequence motif, the CoHR profile, which predicts the great majority of meiotic DSB locations.

Double-strand breaks on artificial chromosomes in yeast.

Yeast artificial chromosomes composed primarily of bacteriophage gamma DNA exhibit very low levels of meiotic crossing over compared with similarly sized intervals of natural yeast DNA. When these recombinationally quiet chromosomes were augmented with a 12.5 kb insert of sequences from yeast chromosome VIII, genetic studies demonstrated that the artificial chromosomes had acquired recombination properties characteristic of this region of chromosome VIII.

Meiotic double-strand breaks in Schizosaccharomyces pombe.

Meiotic DNA double-strand breaks (DSBs) are associated with recombination hot spots in the yeast Saccharomyces cerevisiae and are believed to initiate the process of recombination. Until now, meiosis-induced breaks have not been shown to occur regularly in other organisms. Here we show, by pulsed-field gel electrophoresis of DNA, that meiotic DSBs occur transiently in all three chromosomes of the fission yeast Schizosaccharomyces pombe.

Increased instability of human CTG repeat tracts on yeast artificial chromosomes during gametogenesis.

Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable.

Sister chromatid-based DNA repair is mediated by RAD54, not by DMC1 or TID1.

In the mitotic cell cycle of the yeast Saccharomyces cerevisiae, the sister chromatid is preferred over the homologous chromosome (non-sister chromatid) as a substrate for DNA double-strand break repair. However, no genes have yet been shown to be preferentially involved in sister chromatid-mediated repair. We developed a novel method to identify genes that are required for repair by the sister chromatid, using a haploid strain that can embark on meiosis.

Switching yeast from meiosis to mitosis: double-strand break repair, recombination and synaptonemal complex.

Background: When Saccharomyces cerevisiae cells that have begun meiosis are transferred to mitotic growth conditions ('return-to-growth', RTG), they can complete recombination at high meiotic frequencies, but undergo mitotic cell division and remain diploid. It was not known how meiotic recombination intermediates are repaired following RTG. Using molecular and cytological methods, we investigated whether the usual meiotic apparatus could repair meiotically induced DSBs during RTG, or whether other mechanisms are invoked when the developmental context changes.

Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes.

The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts. Each chromosome had strong and weak double-strand break (DSB) sites. On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites.

Double-strand breaks on YACs during yeast meiosis may reflect meiotic recombination in the human genome.

Meiotic recombination in the yeast Saccharomyces cerevisiae is initiated at double-strand breaks (DSBs), which occur preferentially at specific locations. Genetically mapped regions of elevated meiotic recombination ('hotspots') coincide with meiotic DSB sites, which can be identified on chromosome blots of meiotic DNA (refs 4,5; S.K.

A versatile method for efficient YAC transfer between any two strains.

The ability to transfer yeast artificial chromosome (YAC) clones among yeast hosts greatly enhances their utility as cloned DNAs by increasing the range of methods available for experimental manipulation. An effective method for the transfer of YACs between strains in Kar1- matings is described in the accompanying paper (F. Spencer et al.

A short chromosomal region with major roles in yeast chromosome III meiotic disjunction, recombination and double strand breaks.

A multicopy plasmid was isolated from a yeast genomic library, whose presence resulted in a twofold increase in meiotic nondisjunction of chromosome III. The plasmid contains a 7.5-kb insert from the middle of the right arm of chromosome III, including the gene THR4.