Chromosomes are copied by a complex holoenzyme called the replisome. Obstacles are routinely negotiated by the replisome with auxiliary mechanisms that ensure genomic integrity, aberrance of which can lead to chromosome instability and a broad range of diseases including cancer. The candidate?s long term goal is to understand the molecular basis for genetic and epigenetic fidelity, with the potential to improve the treatment and/or prevention of disease. In this proposal, the candidate will use a fully functional replisome reconstituted from over 30 pure polypeptides to study how replisomes bypass obstacles that regularly occur in the genome while enforcing genetic and epigenetic integrity across generations. In the first specific aim the candidate?s current work on the molecular mechanisms of lesion bypass by the replisome will be elaborated, with a focus on how checkpoint kinases Mec1 and Rad53 and the Mrc1/Tof1/Csm3 (MTC) complex modify the activity of the replisome while regulating lesion bypass. Interactions between several key replisome components and the MTC complex will be probed by microscale thermophoresis (MST) and cross-linking mass spectrometry (XL- MS). In addition to biochemical experiments, single-molecule approaches will be used to probe the mechanism of replisome regulation by the MTC complex, resolving replisome components with fluorescence during active replication on DNA. Single-molecule FRET experiments will be used in the independent phase to probe how MTC affects the structural dynamics of the replisome. In the second aim, the candidate will investigate nucleosome bypass by the replisome, focusing on the post-replication fate of histones during the mentored phase. Using histones enriched for fluorescence, replication-coupled histone deposition will be tracked by a first-of-its-kind attempt at spatially resolving leading vs. lagging strand products with a combination of optical trapping, fluorescence, and flow. Along with bead-based biochemical experiments, the results will help differentiate between models of epigenetic inheritance. In the independent phase, interactions between the replisome and FACT, a histone chaperone, will be determined with MST and XL-MS. The molecular mechanisms of various chaperones will be probed using single-molecule FRET experiments monitoring the spatiokinetics of chromatin remodeling in real time, also determining the role of histone modifications in remodeling. The mentored phase of the project will be conducted in the laboratories of Dr. Michael O?Donnell (mentor) and Dr. Shixin Liu (co-mentor) at Rockefeller University, a world-class research environment. The success of the candidate?s proposed research depends critically on using advanced integrative single-molecule techniques, as well as XL-MS. Thus, the candidate seeks intensive training with manipulation and detection of individual molecules in the co-mentor?s state-of-the-art facilities, in addition to XL-MS with Drs. Brian Chait and Yi Shi (collaborators). The candidate also has also planned activities to improve mentoring, lab management, scientific communication, and professional skills, enabling a successful transition to an independent career.
Chromosomes are duplicated by complex molecular machines called replisomes, which frequently encounter obstacles during DNA replication such as DNA damage, errors in the genetic code itself, as well as nucleosomes, the basic units of chromatin that preserve epigenetic marks across generations. Replisomes stall at these obstacles, necessitating additional molecular bypass mechanisms to avoid cellular catastrophe and to negotiate faithful and efficient duplication of genetic and epigenetic information. Failure to preserve this genomic information as it is copied is well understood to lead to a broad array of human pathologies; therefore, the candidate proposes to elucidate the molecular mechanisms that allow bypass of DNA damage and nucleosomes while strictly enforcing genomic and epigenetic fidelity, providing critical insight into cellular processes that underlie reliable and accurate chromosomal heritage.