CAREER: Biochemical Analysis of the DNA Damage Checkpoint

Harvard University, Cambridge MA

Investigators

Abstract

Transitions between different phases of the cell division cycle are controlled by cell cycle checkpoints. Checkpoints are regulatory systems that monitor cellular processes and ensure that a given process has been satisfactorily completed before transition into the next phase of the cell cycle is allowed. When chromosomal DNA suffers damage, through exposure to mutagens, the DNA damage checkpoint arrests the cell cycle so that damage repair systems have an opportunity to repair the damage prior to transition into the next phase of the cell cycle. This activity is especially important when damage is incurred during S phase of the cell cycle, as lesions on chromosomes must be repaired before the damaged DNA is used as a template during DNA replication. The long-term goal of this research program is to use Xenopus egg extracts to biochemically address the problem of how damaged DNA is detected by the damage checkpoint. Our knowledge of how damage is sensed is currently quite limited. To correct this, a series of experiments will be performed that aim to answer basic mechanistic questions about how DNA damage is sensed. The Xenopus extract system is ideally suited for this type of analysis, and recent advances in the preparation of these extracts will be incorporated into these studies. The problems to be addressed will include the role that DNA replication forks play in sensing damage, the role that DNA repair pathways play in activating the checkpoint, and the effect that the checkpoint has on DNA replication. Once these questions have been answered, then the identity of the individual proteins that function to sense damage can be addressed. Because this problem has obvious connections to cancer, interest in the subject is high amongst undergraduates who are preparing for careers in medicine or laboratory research. To accommodate and encourage this interest, a lecture course has been developed, to be taught chiefly to undergraduates, that focuses on cell cycle control. This lecture course and the Research Plan described in this proposal are tightly connected in that, over the five-year funding period for the proposal, students taking the course will annually receive a copy of the Research Plan, they will be updated on the progress that has been made, and they will participate directly in the work by performing some of the experiments as part of a laboratory section that is attached to the class. This exercise not only teaches students about the cell cycle, but will also enable students to observe how science is practiced, from the conception of an idea (as outlined in the proposal), to the performance of an experiment to the interpretation of data.