The overall goal of this project is to determine how cells communicate chromosome break signals across large chromosomal distances. DNA double-strand breaks (DSBs) are dangerous insults to genome integrity because of their potential to cause chromosome rearrangements and chromosome instability, both of which are strongly associated with cancer progression as well as birth defects. The risk of genome instability is dramatically amplified in situations where multiple DSBs occur at the same time, as is the case with radiotherapy and many forms of chemotherapy. However, at least under certain circumstances, cells are able to efficiently orchestrate the repair of multiple concurrent DSBs. The most prominent example is meiosis, when germ cells introduce hundreds of programmed DSBs across most of their genomes. A key feature of meiotic DSB repair is that it is coordinated at a chromosomal level, such that repair decisions at one DSB are transmitted in a chromosome- autonomous way to DSBs that occurred a large distance away on the same chromosome. The mechanism by which such communication occurs is essentially unknown, but would provide important new insights into how cells cope with massive chromosomal insults. Preliminary analysis of meiotic DNA damage signaling in the sexually reproducing yeast Saccharomyces cerevisiae revealed several signals that appeared to visibly propagate along meiotic chromosomes following meiotic DSB formation. We hypothesize that these signals form part of the communication apparatus that allows meiotic cells to communicate DSB repair decisions. The signals take several different forms, including propagation of protein phosphorylation and changes in chromosome structure, and exhibit temporal and spatial differences, suggesting that they may communicate different aspects of the meiotic DSB repair process. To determine the meiotic roles of these signals, the dynamics of chromosomal signaling and DSB repair will be analyzed by genetics and super resolution microscopy, taking advantage of a novel conditional nuclear depletion approach that allows stage-specific knock-downs of the often pleiotropic repair factors. In addition, signal integration will be analyzed usig cytology, biochemistry, and physical analysis of repair intermediates. Finally, the proposal will close a major technological gap with the development of a method to map DSB repair intermediates across the entire genome. Together, these analyses will provide first insights into the mechanisms of chromosomal signal propagation controlling DNA repair, and open new avenues for understanding the errors in DSB repair that cause birth defects and cancer.
Chromosome breaks are major source of genome rearrangements associated with cancers and a variety of birth defects. By defining the break response mechanisms in the context of meiotic chromosome reshuffling in the model organism Saccharomyces cerevisiae, this project will provide insight into the protection of chromosomal integrity during sperm and egg cell production and will serve as a general framework for the study of birth defects and cancer-associated chromosome rearrangements in humans.
