In dividing cells, newly replicated chromosomes are non-covalently linked during S-phase, a property called sister chromatid cohesion (SCC), until the onset of cell division. SCC is executed by a protein complex called Cohesin, and is essential for bi-allelic attachment of chromosomes to the mitotic spindle. In the absence of cohesin or the proteins required for its proper assembly on chromosomes (collectively referred to as cohesion establishment factors or CEFs), chromosome mis-segregation may occur, resulting in aneuploidy, a hallmark of many genetic disorders including cancer. Knowledge at the molecular level of how SCC is executed by CEFs will not only shed light on a vital cellular mechanism to avert chromosome mis-segregation, but may also reveal critical targets of genome surveillance pathways which function to prevent mitotic progression in the presence of defective chromosomes. Although current models propose that CEFs function to coordinate DNA replication with cohesion establishment, the mechanism by which the process is executed, and the precise role of each CEF is unclear. The focus of this project is on elucidating the role of the CEF Ctf18-RFC, which shares activities similar to the DNA replisome component RFC. A requirement for the RFC-like activities of Ctf18-RFC for SCC will be investigated by defining the domains of Ctf18-RFC required for its RFC-like function in vitro, and then correlating loss of its RFC-like function with impaired SCC in vivo.
The very basic aspect of genetic inheritance addressed by this project is also the vehicle for our K-12 and undergraduate science education goals, which seek to broaden the base and depth of scientific knowledge acquired by students, and increase the number of students who choose careers in basic science research.
Your DNA exists in cells as 46 discreet, unique units called chromosomes. During each stage of the cell cycle (G1, S, G2, and M-phases), an exquisitely orchestrated series of events guarantees proper inheritance of each chromosome (diagram below, light blue line), and begins with two different, but coupled/linked processes that occur within S-phase; (1) the duplication of each chromosome- called DNA replication (top, highlighted in gray-blue), and (2) the tethering/bundling (small brown ovals) of chromosome duplicates (light and gray-blue lines), called sister chromatid/chromosome cohesion (SCC, bottom, highlighted in brown). Cohesion allows the cell to easily keep track of duplicates, and ensures that when a cell undergoes M-phase (cell division), each new cell receives only one of each chromosome. Incomplete chromosome duplication or cohesion has been shown to contribute to the underpinnings of numerous genetic disorders, and our goal has been to understand how these processes work and how they are coupled. Our studies on one protein complex common to both processes, called Ctf18-RFC, have yielded what we believe are significant advances toward our goals. It is well established that the linking (much putting on an ankle bracelet) of a ring-shaped protein called PCNA (green ring in diagram) around chromosomes by a protein called RFC is essential for their duplication. Equally important is its removal from chromosomes as sections are replicated, and duplication of the entire chromosome is completed. However, evidence for the latter function has been lacking until now. Based on our studies we propose that Ctf18-RFC (dark blue cartoon) functions to remove PCNA from chromosomes in vivo, and that this function is critical for the late stage of S-phase and completion of DNA replication. Moreover, we discovered that without Ctf18-RFC, cells fail to activate an important quality control pathway, and as a consequence, they bypass both completion of DNA replication and cohesion construction, and undergoes cell division with defective/faulty chromosomes. Routinely, cells keep track of the accuracy and integrity of processes using proteins that function much like an "inspector#13", who assesses and verifies the integrity of a product before it makes it way to the consumer. In this novel quality control pathway, Ctf18-RFC functions as an "inspector 13" who verifies that chromosomes are completely duplicated before advancing to the next process- chromosome condensation. If they are not, Ctf18-RFC tells the cell to delay subsequent events (red T-arrow in diagram) until chromosomes are ready to move on. Because faulty chromosomes from cells lacking this Ctf18-RFC-dependent quality control pathway are predicted to mimic those associated with developmental disorders such as Fragile-X syndrome, knowledge of all pathway components and mechanism may provide a deeper understanding of the genesis of such disorders, and yield new bio-markers for development of better detection and therapeutic methodologies. What is Ctf18-RFC's function in cohesion? Previous studies had shown that Ctf18-RFC was important for the function of an enzyme that works to build cohesion between chromosomes called Eco1 (purple cartoon), but the reason why left unexplained. Our research suggests that Ctf18-RFC's cohesion function is related to its function in chromosome duplication, and this insight, we believe, is the first clue to the mechanism by which these processes are coupled. Groundbreaking in this project was the development of an assay for the biochemical function of Eco1 in vitro. Using this tool, we made the unanticipated discovery and insight into the mechanism of coupling chromosome duplication and cohesion: Eco1 is held in an inactive state (non-functional "off" state in cartoon) when bound to PCNA, and becomes active (functional "on" state depicted in yellow) when un-bound or free from PCNA. This revelation explains the requirement of Ctf18-RFC for Eco1 function- Ctf18-RFC's proposed PCNA un-linking function during replication releases PCNA from both chromosomes and Eco1, promoting Eco1 activation. Thus, Ctf18-RFC functions as a molecular switch (follow the yellow arrows…), turning on chromosome cohesion as it functions to complete DNA replication. We believe this "Ctf18-RFC switch" is a fundamental component of the mechanism of coupling these processes, and provides a foundation to biochemically dissect and reassemble the entire coupling process. Beyond the intellectual merit of a deepened understanding of how initial events in chromosome inheritance are coordinated and quality controlled, my research program provided inquiring minds from 8th grade on up research opportunities. I opened my laboratory to and shared my science with numerous high school, undergraduate and graduate students, from all socioeconomic and cultural backgrounds, particularly under-represented groups. The fruits or broader impacts of my science pedagogy- K-12 students inspired to become cancer researchers, undergraduate students attending doctoral programs, and graduate students choosing science education at the K-12 level- are as equally rewarding as the discoveries made at the bench. Much like my experience, I anticipate their efforts will similarly motivate within their respective communities youth participation in Science Technology Engineering and Mathematics (STEM) programs and careers in science.
