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.
Background: The focus of this 3-year award was to learn more about how chromatin structure affects the initiation of DNA replication. DNA replication occurs every time a cell divides. This highly coordinated and involved process involves the precise copying of the genetic material on chromosomes, which are subsequently partitioned equally between the mother and daughter cells. Errors in DNA replication can have catastrophic effects for the cell leading to cell death. We are studying this process in the model organism, Saccharomyces cerevisiae, which is better known as bakers’ yeast. All genes are made up of DNA but DNA is packaged and compacted together with various proteins in the cell to form "chromatin." Particular genes or control regions can exist within "open" or "closed" forms of chromatin and this variable accessibility determines whether the gene is "on" or "off." DNA replication is no exception to this control and it is known that chromatin can either inhibit or facilitate replication in a dynamic way. DNA replication can be divided into roughly three phases: initiation, elongation and termination. We study how DNA replication initiates - or begins - and this occurs at discrete locations along each chromosome called "replication origins." There are perhaps 10,000 replication origins in the human genome but only ~400 in yeast. One of the reasons we are studying this process in yeast is because the location of the relatively small number of yeast origins are quite precisely known. Intellectual Merit: To date we have mapped the DNA sequence elements present at six replication origins and found that these elements had a flexibility and redundancy built into the sequence. Furthermore, although replication origins generally exist in chromatin "free" regions this is not always the case. The presence of nearby nucleosomes (a basic unit of chromatin – like beads on the string of DNA) can interfere with the binding of key proteins required to initiate DNA replication. The modification state of those nucleosomes likely influences initiation since the Sir2 histone deacetylase, a protein that modifies chromatin, negatively regulates several origins we mapped. We have also identified multiple residues within histones (a core component of chromatin) that negatively or positively influence DNA replication such as on histone H3. Several residues we identified have been previously unlinked to DNA replication control. Lastly, we have undertaken a genome-wide approach to understand how the Sir2 protein controls DNA replication at multiple origins. This is leading us to understand new aspects of DNA replication control and also perhaps new roles for Sir2 in the cell. Broader Impacts: During the course of this project we have mentored three women from minority groups that are underrepresented in science, including high school students and college undergraduates. These students have worked full-time in the laboratory over the summer to gain hands-on laboratory experience on yeast genetics projects. The students leave this experience with a great enthusiasm for science and this generally plays a big role in their career trajectory. The high school students are recruited through a local program to promote science career opportunities among disadvantaged students. This has been a rewarding experience both for our lab and the students.
