Showing papers by "Nancy Kleckner published in 1999"

Meiotic chromosomes: integrating structure and function.

Abstract: ▪ Abstract Meiotic chromosomes have been studied for many years, in part because of the fundamental life processes they represent, but also because meiosis involves the formation of homolog pairs, ...

Functional specificity of MutL homologs in yeast: Evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction

Abstract: The yeast genome encodes four proteins (Pms1 and Mlh1–3) homologous to the bacterial mismatch repair component, MutL. Using two hybrid-interaction and coimmunoprecipitation studies, we show that these proteins can form only three types of complexes in vivo. Mlh1 is the common component of all three complexes, interacting with Pms1, Mlh2, and Mlh3, presumptively as heterodimers. The phenotypes of single deletion mutants reveal distinct functions for the three heterodimers during meiosis: in a pms1 mutant, frequent postmeiotic segregation indicates a defect in the correction of heteroduplex DNA, whereas the frequency of crossing-over is normal. Conversely, crossing-over in the mlh3 mutant is reduced to ≈70% of wild-type levels but correction of heteroduplex is normal. In a mlh2 mutant, crossing-over is normal and postmeiotic segregation is not observed but non-Mendelian segregation is elevated and altered with respect to parity. Finally, to a first approximation, the mlh1 mutant represents the combined single mutant phenotypes. Taken together, these data imply modulation of a basic Mlh1 function via combination with the three other MutL homologs and suggest specifically that Mlh1 combines with Mlh3 to promote meiotic crossing-over.

Somatic pairing of homologs in budding yeast: existence and modulation

Abstract: FISH analysis of well-spread chromosomes reveals that homologs are paired in vegetatively growing budding yeast diploid cells, via multiple interstitial interactions, and independent of recA homologs and mating type heterozygosity. Pairing is present during G1 and G2, and in cells arrested at G1 by mating pheromone, but is disrupted during S phase. Thus, somatic pairing is qualitatively analogous to premeiotic and early meiotic pairing. S-phase pairing disruption occurs by a complex intranuclear program involving regional, nucleus-wide, and temporal determinants. Pairing is also disrupted in two G2-arrest conditions (cdc13ts and nocodazole). Together these findings suggest that cell cycle signals may provoke pairing disruption by modulating underlying chromosome and/or chromatin structure. Whether the cell chooses to disrupt pairing contacts or not (e.g., S phase and G2 arrest, but not G1 arrest or normal G1 or G2), could be dictated by functional considerations involving homolog/sister discrimination.

Collisions between yeast chromosomal loci in vivo are governed by three layers of organization.

Abstract: The relative probabilities that different pairs of chromosomal loci will collide with one another in vegetatively growing diploid yeast cells have been assessed using a genetic assay for Cre/loxP site-specific recombination. Recombination rates have been determined for 18 different pairs of loxP sites representing diverse pairs of positions within the genome. Overall, relative collision probabilities vary over an eightfold range. Within this range, a hierarchy comprising three levels of organization can be discerned. First, collisions between loci on nonhomologous chromosomes are governed by nonspecific centromere clustering. Second, a sequence is closer to allelic or nearby sequences on its homolog than to sequences on nonhomologous chromosomes, an effect most simply attributed to homolog pairing. Third, a sequence can be closer to other sequences nearby on the same chromosome than to sequences on other chromosomes. These findings provide a framework for assessing the role of chromosome disposition in cellular processes such as DNA repair and gene expression. Also the possibility is raised that genome-wide coalignment of homologs is not the fundamental raison d’etre of the somatic pairing process. We suggest instead that pairing may exist to promote juxtaposition of homologous regions within irregular genome complements.

GCN5-dependent histone H3 acetylation and RPD3-dependent histone H4 deacetylation have distinct, opposing effects on IME2 transcription, during meiosis and during vegetative growth, in budding yeast

Abstract: Diploid yeast undergo meiosis under certain conditions of nutrient limitation, which trigger a transcriptional cascade involving two key regulatory genes. IME1 is a positive activator of IME2, which activates downstream genes. We report that Gcn5, a histone H3 acetylase, plays a central role in initiation of meiosis via effects on IME2 expression. An allele, gcn5–21, was isolated as a mutant defective in spore formation. gcn5–21 fails to carry out meiotic DNA replication, recombination, or meiotic divisions. This mutant also fails to induce IME2 transcription; IME1 transcription, however, is essentially normal. Further investigation shows that during wild-type meiosis the IME2 promoter undergoes an increase in the level of bound acetylated histone H3. This increase is contemporaneous with meiotic induction of IME2 transcription and is absent in gcn5–21. In contrast, the RPD3 gene, which encodes a histone H4 deacetylase and is known to be required for repression of basal IME2 transcription in growing yeast cells, is not involved in induction of IME2 transcription or IME2 histone acetlyation during meiosis. These and other results suggest that Gcn5 and Rpd3 play distinct roles, modulating transcription initiation in opposite directions under two different cellular conditions. These roles are implemented via opposing effects of the two gene products on acetylation of two different histones. Finally, we find that gcn5 and rpd3 single mutants are not defective in meiosis if acetate is absent and respiration is promoted by a metabolically unrelated carbon source. Perhaps intracellular acetate levels regulate meiosis by controlling histone acetylation patterns.