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Chromosome Structure, Integrity, and Evolution
Summary: Douglas Koshland hopes to elucidate the underlying molecular mechanisms that modulate genome integrity in the context of speciation and cancer progression and that allow cellular survival in response to extreme desiccation.
Genome integrity, the ability of cells to maintain proper chromosome number and integrity, impacts cell viability, speciation, birth defects, and human disease. Similarly, desiccation tolerance, the ability of cells to survive extreme changes in available water, influences cell survival, crop productivity, and wound recovery. However, the molecular bases for desiccation tolerance and genome integrity remain major unsolved problems of cell biology. By studying these two processes in the simple unicellular budding yeast by genetic, biochemical, and cell biological approaches, we hope to uncover mechanisms that enlighten our understanding of genome and water homeostasis in all eukaryotes.
Chromosome Structure
Different types of higher-order chromosome organization are mediated by divergent Smc (structural maintenance of chromosome) complexes: sister chromatid cohesion by cohesin, condensation by condensin, global transcription control by the dosage compensation complex, and DNA repair by the Smc5/6 and MRX/N complexes. These Smc complexes provide a powerful platform to study both the biological function and molecular basis for different types of higher-order chromosome structure.
My laboratory has shown that different types of higher-order chromosome organization are coordinated because the functions of Smc complexes are highly interdependent. Different Smc complexes interact in at least three distinct ways to mediate proper chromosome morphogenesis in mitosis, meiosis, and DNA repair. They cooperate to build other chromosomal structures, such as synaptonemal complexes, to regulate each other's assembly, and to regulate each other's disassembly.
We have also studied cohesin as a prototype to understand how a Smc complex tethers two strands of chromatin to achieve higher-order folding. Cohesin tethers the two replicated copies of a chromosome together to facilitate chromosome segregation and to protect cells from DNA damage, both at the site of the lesion and genome-wide. We have shown that cohesins bind chromatin and then become cohesive. We have also shown that this conversion to the cohesive state is regulated by complex post-translational modifications in response to cell cycle and DNA damage. Our understanding of cohesin regulation allows us to lock cohesin in either the noncohesive or cohesive-competent state, providing critical new tools to assess specificity of in vitro activities and to generate sufficient quantities of active complex for in-depth biochemical and structural studies.
Chromosome Integrity
Dissecting the molecular basis for chromosome integrity is challenging because changes such as GCRs in chromosome integrity are usually rare and often either lethal or genetically silent. Factors that prevent GCRs have been identified in budding yeast from a candidate list, using an elegant but labor-intensive genetic assay. Although important, these studies have likely missed processes that protect cells from GCRs because the candidate list included mostly DNA repair and replication factors. To identify novel processes that prevent GCR, we developed a high-throughput assay to follow GCR, using a yeast artificial chromosome (YAC). We used this assay to screen mutant yeast deleted for each of 5,000 nonessential genes. Our studies identified novel proteins required to promote chromosome integrity. We find, for example, that factors required for general mRNA metabolism play an active role in sequestering newly formed RNA molecules to prevent them from hybridizing back to DNA. When RNA:DNA hybrids do form, RNase H acts as a surveillance mechanism for their removal, thereby safeguarding the genome. We are currently investigating how RNA:DNA hybrids lead to GCR.
Genome Evolution
Genome evolution, both in speciation and cancer, often involves changes in chromosome number and GCRs. GCRs occur frequently through nonallelic homologous recombination (NAHR) between repetitive elements at distinct chromosomal positions. In collaboration with Yixian Zheng (HHMI, Carnegie Institution) and Maitreya Dunham (University of Washington), we would like to understand the NAHR mechanism and the contribution of GCRs to genome and organism fitness in the context of purebred and hybrid genomes. Yeast is uniquely suited to analyze the NAHR mechanism because of its sophisticated genetics and the knowledge of the position and sequence of every repeat in the large repetitive family of Ty retrotranspons. Because of its short generation time, yeast is also ideally suited for the study of the impact of GCR on genome fitness through chemostat evolution experiments.
Our analyses of NAHR in purebred and hybrid yeast has generated surprising observations that challenge widely accepted models for homologous recombination, suggesting exciting alternative mechanisms by which homologous recombination shapes genomes. We also showed that purebred and hybrid yeast evolve through a different spectrum of GCRs when exposed to the same selective pressure for 100–300 generations. We plan to ask how this evolution changes for a novel family of repetitive elements in which we manipulate the sequence divergence and chromatin structure of the repetitive elements.
Desiccation Tolerance
Desiccation tolerance has been correlated with a number of stress response pathways, including heat shock, osmotic shock, DNA damage, or high salt. We showed, however, through targeted mutational analysis, that all of these pathways are dispensable for desiccation tolerance. Given that these prior correlations were uninformative, we screened for desiccation sensitivity in mutants deleted for each of the 5,000 nonessential genes. We observed extreme desiccation sensitivity (10,000-fold reduction in viability) only when respiration is compromised. Experiments exploiting temporal inactivation of respiration show that desiccation tolerance requires respiration during cell growth prior to desiccation but not during the actual process of desiccation. We hypothesized that entry into the respiration state induces gene products or metabolites that protect cells from subsequent desiccation. We have identified loss-of-function second-site suppressors that restore nearly wild-type levels of desiccation tolerance to respiration-defective cells. One second-site suppressor is a transcriptional repressor, implying that desiccation tolerance is controlled at least in part by a transcriptional program. These are the first mutants discovered in any species that have a significant effect on desiccation tolerance, and the identification of a transcriptional program sets the stage for the elucidation of the actual effectors of desiccation tolerance.
Last updated October 05, 2009
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