Mechanisms of programmed chromosome breakage

程序总结/文摘

Program Summary/Abstract The overall goal of this research is to understand how organisms safely break their own DNA. DNA double- strand breaks (DSBs) are highly hazardous lesions whose improper repair can cause loss of heterozygosity and copy-number variations, leading to numerous psychiatric and developmental disorders, as well as cancers. Despite these risks, most eukaryotes introduce programmed DSBs into their genomes at one or more points in their life cycle. These breaks occur in the soma and the germ line and function to create genetic diversity, remove unwanted DNA, and support adaptation to changing environments. Cells go to great lengths to keep programmed DSBs safe. They control the location and timing of DSBs, promote correct repair-template choice, and use surveillance mechanisms to coordinate DSB formation with other cellular processes, including cell-cycle progression. Defects in any of these layers of control leave organisms with a higher risk of genome instability, and thus provide key insights into the genome instability associated with cancers and the chromosomal abnormalities that lead to birth defects and infertility. To investigate safe DSB formation, the proposed work focuses on two highly conserved instances of developmentally induced DNA breakage: (1) meiotic recombination, which involves hundreds of DSBs per meiotic germ cell, and (2) programmed copy-number changes in the ribosomal DNA (rDNA), the most highly expressed gene locus in eukaryotes. The proposed work uses genetics, molecular biology, and genomics to investigate these processes. Most of the work is conducted in the yeast Saccharomyces cerevisiae, but conservation of rDNA copy-number control is also tested in human cell lines. To investigate meiotic DSBs, research over the next 5 years will build on results of a phospho-proteomics screen to dissect the surveillance network that coordinates many meiotic processes with DSB formation. Experiments will also define the role of chromosome architecture in making DSB hotspots hot, and a genome- wide approach will be developed to measure meiotic repair-template choice across the genome. To analyze copy-number dynamics in the rDNA, research will focus on the mechanisms that drive re- expansion of critically short rDNA clusters and investigate the role of a novel DNA repair intermediate in this process. In addition, the proposed work will investigate the spreading of genetic variants among repeats of an rDNA cluster over evolutionary time scales and upon selection in the laboratory. Together, these analyses will provide fundamental insights into the dynamics of developmentally induced DSBs, open avenues for understanding how these endogenous processes contribute to genomic plasticity in health and disease.