Regulation of DNA Replication Timing by Origin Recognition Complex (ORC) Phosphorylation

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PROJECT SUMMARY Successful duplication of the genome requires the accurate replication of billions of bases. Failure to do so results in genomic instability and ultimately a host of diseases. To mitigate against this, DNA replication is a coordinated and tightly regulated process; however, this regulation must also be flexible as DNA replication is subject to change through development and differentiation. Much still remains unknown regarding exactly how DNA replication is regulated and how this regulation changes through development. One such regulatory process where much remains to be discovered is the replication timing program, which controls when certain segments of the genome are replicated during S phase. Replication timing (RT) is a highly conserved phenomenon, found in species ranging from yeast to humans, yet the precise mechanisms that govern replication timing remains unknown. RT has also been implicated in mutation rates and the development of drug resistance in certain cancers. Rif1, a conserved active regulator of RT provides a key to unlock the underlying molecular mechanisms controlling RT. Rif1 is thought to delay the activation of replicative helicases that lie within certain sections of the genome, causing these sections to replicate later in S phase. How Rif1 is targeted to these genomic regions to cause delayed replication remains unclear. We sought to determine how Rif1 controls RT by carrying out immunoprecipitations of Rif1 followed by high-resolution mass spectrometry. We discovered that Rif1 co- immunoprecipated with the origin recognition complex (ORC). ORC is the first complex to bind to DNA during the initiation of DNA replication and controls where across the genome DNA replication will start. In this proposal, we seek to characterize the interaction between Rif1 and ORC. To achieve this, we will determine the molecular relevance of this interaction, characterize where Rif1 and ORC bind to the genome through the cell cycle, and use in vitro approaches to determine if the Rif1/ORC interaction is direct. The findings from this work could provide a new mechanism paradigm for how Rif1 controls RT, which will likely be applicable to other species as Rif1 and ORC are functionally conserved. This work will also provide insight into the regulation driving replication timing and DNA replication, both of which critical for maintaining genomic stability and integrity. Furthermore, this proposal will provide the opportunity to learn bioinformatic and biochemical skills widely applicable to careers in science, enhance critical thinking and problem-solving skills, and improve scientific communication skills by formally presenting this work and national conferences and university meetings. I will work with my mentors and the ASPIRE program through the office of career development office at Vanderbilt to identify career development opportunities. Lastly, I will continue to develop mentorship skills by training undergraduates at Vanderbilt and seek opportunities for training in data analytics so that I may become a successful leader in health data science.