Faithful chromosome segregation is essential to the life of a eukaryotic cell. This project will characterize novel connections between DNA replication and chromosome segregation, using fission yeast as a model eukaryotic system. It suggests that proteins involved in the initiation of DNA replication may have linked functions in assembling active centromeres and segregation-competent chromosomes during each cell cycle. This analysis builds on prior work suggesting that replication proteins affect several stages of centromere function, including heterochromatin assembly at the centromere, chromosome condensation, and regulation of centromere attachment. The proposal hypothesizes that DNA replication proteins are required to remodel various components of centromere function during each cell cycle. The first aim analyzes physical interactions between replication proteins and the heterochromatin protein HP1/Swi6. The second aim investigates whether Swi6 influences assembly or activation of replication complexes and replication timing in the centromere region. The final aim screens for additional mutants that define genes that link replication and chromosome segregation. The ultimate goal is to understand the network linking these events. This project investigates a fundamental question of cell division by investigating how the events of DNA replication affect accurate chromosome segregation in all eukaryotic cells. Its ultimate goal is to identify a genetic network that regulates these process that can be used as a framework for more mechanistic studies in the future. It will promote teaching, training, and learning by actively involving PhD students and undergraduates in carrying out the research, ensuring an educational impact in addition to scientific advancement.
This project examined cellular mechanisms that contribute to the function of the centromere. This is a specialized region of our chromosomes (DNA) that the cell uses to pull the duplicated chromosomes apart during cell division. Faithful cell division is essential for life in all organisms, so that studying how it occurs is a fundamental question in biology. In this project, we used a simple single celled organism, the fission yeast Schizosaccharomyces pombe, as a model to study this process. Fission yeast chromosomes are organized very similarly to human chromosomes, with the added advantage of existing in a cell that easy to grow, inexpensive, and very easily manipulated using genetic and molecular tools. The project started with the hypothesis that the process of DNA replication is particularly important for maintenance of specialized regions called "heterochromatin" that are part the centromere. Heterochromatin occurs when particular chemical modifications occur in regions of the chromosome that affect the packaging or gene expression of the DNA. This is an example of "epigenetic modification" , in which the function of our genes is affected by marks placed "on top" of the DNA sequence. It is only in the 10-20 years that we have realized how important these marks are in the normal function of our genome. The heterochromatin in the centromere of the fission yeast has developed as an important model for the heterochromatin in other organisms, including humans. We used a variety of methods including genetics and molecular biology. Of particular note, we used advanced microscopy including imaging of live cells to study the behavior of heterochromatin-associated proteins. We showed that at least one such protein, called Swi6/HP1, interacts specifically with a protein involved in DNA replication, called Cdc18/Cdc6. We also found that the timing of replication in the heterochromatin region is affected by the presence of some, but not all of the heterochromatin-associated proteins. Finally, we discovered that defects in heterochromatin proteins allows the DNA in this region to undergo inappropriate rearrangements, and this effect is enhanced if we also destabilize the replication fork by mutations or DNA damaging chemicals. Our conclusion is that DNA replication proteins function synergistically with heterochromatin proteins to maintain the structure of the heterochromatin, and the appropriate function of the centromere. Future experiments will examine the contribution of additional proteins to this stabilization function. This project was carried out by graduate and undergraduate students, so it has a broad impact on student education. The data were also used as part of the Principal Investigator’s teaching of a class in epigenetics. The data are shared through publications and the materials shared with other academic researchers upon request.
