Analysis of RAD9 functions: Roles in the checkpoint response, DNA damage processing, and prevention of genomic instability.

摘要

In the 15 years since Rad9's discovery, we have come to understand a great about Rad9 biology, yet numerous questions still remain. Especially intriguing questions include: (a) How does Rad9 get localized and/or recognize DNA damage? (b) How does Rad9 activate downstream checkpoint proteins? and (c) Does Rad9 play additional roles in recognizing and/or repairing DNA damage that have yet to be discovered?; In an effort to try to answer some of these questions, I analyzed the effects of various RAD9 mutations. To assess the contribution of RAD9 to inhibiting DNA degradation and its role in the cell cycle arrest and DNA damage repair responses, I performed a pentapeptide mutagenesis screen in order to obtain RAD9 separation-of-function mutants that were proficient for one known phenotype and deficient in the other. I was able to obtain 2 such mutants that were hypomorphic in their ability to prevent DNA degradation and completely proficient for arresting the cell cycle in the presence of DNA damage and for repairing such damage. Despite many efforts, I was unable to enhance the hypomorphic dysfuntion of these mutants in preventing DNA degradation such that a suppressor screen to identify other genes in this pathway could be performed.; In another effort to try to understand the role of the BRCT domains for RAD9 functions, I analyzed the effects of other various RAD9 mutants. By deletion analysis, I was able to determine that the predominant function of RAD9's BRCT domains is to mediate concentration of the Rad9 protein for function by two means: (1) by conferring stability to the Rad9 protein, and (2) by homodimerizing Rad9 to increase its local concentration to enable interactions with downstream checkpoint components.