Nancy M. Hollingsworth - US grants

DESCRIPTION (provided by applicant): Meiosis is a specialized cell division used by sexually reproducing organisms to produce haploid gametes from diploid cells. This reduction in chromosome number is accomplished by having two rounds of chromosome segregation follow a single round of chromosome duplication. Failures in meiotic chromosome segregation in humans lead to infertility and birth defects such as Trisomy 21, the leading cause of mental retardation in the United States. In addition, meiotic chromosome segregation requires the repair of programmed double strand breaks (DSBs). DSB repair in mitotically dividing cells is critical for genome integrity and the prevention of cancer. Budding yeast is an excellent model system for studying meiosis. Mice and yeast mutated for orthologous meiotic genes are phenotypically similar, demonstrating that studies of meiosis in yeast are likely to be illuminating with regard to meiosis in humans. The first meiotic division (MI) is unique in that sister chromatids segregate to the same pole (called reductional segregation). Proper MI segregation requires that homologous chromosomes be connected to each other by crossovers between non-sister chromatids in combination with sister chromatid cohesion. Recent work has shown that crossing over between non-sister chromatids during meiosis is promoted in part by active suppression of DSB repair between sister chromatids. This suppression is mediated by Mek1, a meiosis-specific serine/threonine kinase. Understanding the molecular mechanism by which Mek1 inhibits meiotic sister chromatid repair requires identification of the substrates phosphorylated by this kinase. We will use novel biochemical and genetic strategies to identify Mek1 targets and characterize phosphorylation- defective versions of these proteins elucidate this process. In addition we will test a specific model for how cohesins may be involved in suppressing meiotic intersister DSB repair. Repair of meiotic DSBs utilizes two recombinases for strand invasion: Dmc1, which is meiosis-specific and Rad51, the major recombinase in vegetative cells. How the action of these two strand exchange proteins is coordinated during meiotic recombination not yet understood. Recent biochemical experiments have suggested a novel mechanism for regulating Rad51 activity mediated by Mek1 phosphorylation of an accessory protein, Rad54. One goal is to determine whether this mechanism functions in meiotic cells. PUBLIC HEALTH RELEVANCE: Failures in the evolutionarily conserved cell division of meiosis result in infertility and birth defects such as Down syndrome. Proper meiotic chromosome segregation requires double strand break repair-a process also required to maintain genome integrity and prevent cancer. This grant is focused on understanding the molecular basis for proper chromosome behavior during meiosis-knowledge that may ultimately lead to the diagnosis and/or prevention of certain types of infertility, birth defects and even cancer.

Eukaryotic organisms utilize two types of cell division. Mitosis creates genetically identical daughter cells, thereby providing the raw material for growth and differentiation. Meiosis, in contrast, divides the chromosome number of cells in half, producing haploid gametes containing new combinations of alleles. This reduction is essential in keeping the chromosome number constant when two gametes fuse at fertilization. Mitosis and meiosis share many features in common: for example, chromosomes are segregated using microtubule based spindles, sister chromatids are held together by cohesins, and destruction of cohesins occurs via the same proteolytic machinery. Several meiosis-specific processes have evolved, however, to allow two divisions to occur after a single round of DNA replication such that homologous chromosomes, instead of sister chromatids, disjoin to opposite poles at Meiosis I. These include the connection of homologous chromosomes by a combination of crossing over and cohesion, the temporally distinct two step removal of cohesins at Meiosis I and Meiosis II and the mono-orientation of sister kinetochores at Meiosis I. Recent work has shown that these meiosis-specific processes result from the interplay between meiosis-specific proteins and mitotic cell cycle kinases such as CDK, Cdc5 and Cdc7. Using an analog sensitive conditional allele of CDC7, cdc7-as, my lab has shown that CDC7 is essential for meiotic recombination, mono-orientation of sister kinetochores and meiotic progression. The purpose of this grant is to use biochemical, genetic and genomic approaches to understand how Cdc7 regulates meiotic processes at the molecular level. In Aim 1, we will identify meiotic substrates of Cdc7 using novel strategies recently developed for use with analog sensitive kinases. In Aim 2, we will investigate how Cdc7 regulates the expression of NDTSO, a meiosis-specific transcription factor that acts a molecular switch to allow exit from pachytene, meiotic progression and differentiation of haploid products into spores.

PROJECT SUMMARY Sexual reproduction requires the creation of haploid gametes from diploid cells. This two-fold reduction in chromosome number is carried out by a specialized, evolutionarily conserved, cell division called meiosis. In humans, failures in meiosis result in infertility and birth defects such as Trisomy 21 or Down syndrome. Unique to meiosis is the first meiotic division, in which homologous pairs of sister chromatids segregate to opposite poles. To promote proper orientation at Metaphase I of meiosis, homologous chromosomes are physically connected by a combination of sister chromatid cohesion and reciprocal crossovers. Crossovers result from meiotic recombination, which is initiated by double strand breaks (DSBs). Repair of these DSBs brings homologs together to form a structure called the synaptonemal complex (SC). Meiotic DSB repair is highly regulated to ensure that each homolog pair gets at least one crossover and that chromosome segregation is delayed until all DSBs have been repaired. An important element of this regulation in budding yeast involves phosphorylation by the meiosis-specific kinase, Mek1, as well as the conserved cell cycle kinases, CDK (cyclin-dependent kinase), DDK (Cdc7-Dbf4) and polo-like kinase (Cdc5). The goal of this grant is to understand how phosphorylation regulates meiotic recombination and chromosome synapsis. The first aim tests a specific mechanistic hypothesis for how meiotic DSB repair is coordinated with meiotic progression through Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80. The second aim addresses the role that phosphorylation of a conserved region of an SC protein called Zip1 plays in regulating in the crossover/noncrossover decision by enabling the creation of a specific class of crossovers that are distributed throughout the genome. The third aim takes advantage of a major resource we developed in the last grant period?namely a dataset containing thousands of phosphorylated amino acids on proteins arrested in meiotic prophase. Phosphosites on proteins important for meiotic recombination and synapsis will be mutated and phenotypically characterized to discover the functional role of the phosphorylation. Two examples are provided for proteins we plan to pursue in the near future: Ecm11?a SUMOylated protein that is necessary for SC formation and Red1, whose degradation is mediated by Cdc5, resulting in SC disassembly and inactivation of Mek1 to allow repair of residual DSBs prior to the onset of the first meiotic division.