Impact of Chromatin Structure on DNA Replication

Van Andel Research Institute, Grand Rapids MI

Investigators

Abstract

During each cell division cycle the genetic material must be precisely duplicated in a process called DNA replication. In eukaryotic cells, DNA replication initiates at hundreds to thousands of replication origins dispersed throughout the genome. Within cells, DNA is complexed with histone proteins as chromatin, which effectively compacts and organizes the DNA into functional regions. Importantly, the chromatin structure surrounding replication origins strongly influences the initiation of DNA replication through mechanisms that are not well understood. The long-term goal of this research is to determine how chromatin structure impacts the initiation of DNA replication and to define the chromatin-modifying proteins that act at replication origins. The specific goal of this project is to determine how chromatin modifications and/or nucleosome positioning regulates assembly of the pre-Replicative Complex (pre-RC). The pre-RC is a multi-protein complex that assembles at replication origins in G1 phase and is required for initiating DNA synthesis in S-phase. Interestingly, loss of the Sir2 histone deacetylase (HDAC) in the budding yeast allows genome-wide DNA replication when pre-RC assembly is compromised. Sir2 is the founding member of a conserved family of proteins called "Sirtuins" that are critical regulators of aging, heterochromatin formation, cell cycle control and tumor suppression in multiple organisms. This data linking Sir2 with replication initiation indicates that Sir2 has a previously uncharacterized role throughout the genome that inhibits DNA replication at a substantial number of replication origins. Although little is known about the exact mechanism, Sir2 acts through specific inhibitory elements within nucleosomes that are directly adjacent to the site of pre-RC assembly. The broad goal of this project is to determine how the chromatin environment at replication origins regulates the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Defects in pre-RC assembly can cause genome instability because either too few or too many replication origins are activated in a particular S-phase. Therefore, understanding how chromatin structure influences pre-RC assembly is important for understanding how genetic stability is maintained. The program will provide training opportunities for both undergraduate and graduate students, including populations that are typically underrepresented in science. In addition, yeast genetics and molecular biology are wonderful tools for training young scientists in the fundamentals of doing critical science.