Abstract
The DNA damage response is a highly conserved and coordinated system that responds to and facilitates repair of DNA damage to maintain genomic integrity. Double-stranded breaks (DSB)s are the most deleterious form of DNA damage that, if left unrepaired, can lead to cell death or the development of cancer. When DSB is detected, the DNA damage checkpoint (DDC) arrests cells at the metaphase to anaphase barrier to allow the cells an opportunity to repair the DSB. Homologous recombination is one of the 2 branches of DSB repair and utilizes a donor with homology to the DSB-site as a template for repair. Previous work has focused on the establishment of checkpoint arrest and the repair of DSB once a donor has been found, but it is still not well understood what is required for the maintenance of checkpoint arrest or how the DSB finds a donor sequence through homology search. Using budding yeast, S. cerevisiae, I use the HO-endonuclease to make site-specific DSBs to study the requirements for the maintenance of the DNA damage checkpoint and the role of the spindle assembly checkpoint in DSB-induced checkpoint arrest. I found that most proteins required for the activation of the DNA damage checkpoint are also required for the maintenance of arrest. While the spindle assembly checkpoint is not required for the establishment of arrest, it contributes later to extend the length of arrest or is able to fully take over arrest from the DNA damage checkpoint. In addition, I found that the actin nucleator Arp2/3, which was recently found to be involved in DSB mobility in Drosophila and in human tissue culture samples, has an evolutionarily conserved role in the mobility of DSBs. Arp2/3 and the nucleation promoting factors (Las17, Vrp1, and the type-I myosins Myo3 and Myo5) are required for the initiation of long-range resection as well as increasing chromatin mobility on the local level near the DSB and globally on undamaged chromosomes. I also show that chromatin remodelers and endonucleases that affect the rate of resection have an impact on DSB mobility. Collectively, these results show the complexity of the DNA damage response and homology search.