The long-term goal of this proposed research is to understand how eukaryotic cells maintain genome stability when challenged by DNA damage. Genome stability requires fidelity in the processes involved in genome replication, repair, and segregation, and this is achieved by coordinated action of repair pathways and cell cycle controls including the cell cycle checkpoints. The DNA Damage Checkpoint (DDC) triggers G2/M arrest when DNA is damaged after replication, and the maintenance of this arrest requires the Spindle Assembly Checkpoint (SAC). In animals, SAC proteins have been found at some kinetochores upon DNA damage, however how SAC is controlled in DNA damage response is unknown.
This research aims to uncover the mechanism of crosstalk between two highly- conserved cell cycle checkpoints, the SAC and the DDC, using the budding yeast Saccharomyces cerevisiae. The goal of Aim 1 is to identify how the SAC responds to signals from DNA double- stranded breaks (DSBs) by: (1) determining whether an unrepairable DSB recruits SAC proteins to kinetochores through disruption of kinetochore-microtubule attachment, (2) identifying factors that affect SAC activity following DSB formation. Effects of DSB on kinetochore-microtubule attachment and SAC complex recruitment to kinetochores on the broken chromosome will be examined using live cell (4-D) microscopy. In parallel, the pattern of SAC activity relative to DDC activity following DSB formation will be analyzed using molecular markers for SAC activity (Mad1 phosphorylation) and for DDC activity (Rad53 and Chk1 phosphorylation). Knowledge about when SAC becomes important during DSB-induced G2/M arrest and factors affecting this timing will constrain the possible mechanisms of SAC regulation after DSB formation.
Aim 2 is designed to identify proteins that act through the SAC in the DNA damage response. The first approach will be a targeted genetic analysis of DDC and SAC genes that will reveal whether DDC proteins control SAC proteins during DSB- induced G2/M arrest. In contrast, the second, unbiased, approach aims to identify novel factors involved in SAC regulation upon DNA damage. Mutations in genes required to maintain DSB-induced G2/M arrest will be selected by generating a DSB in a supernumerary chromosome so that DSB- induced chromosome loss will not cause lethality during screening. By applying strategies used in Aim 1 to characterize SAC regulators identified in Aim 2, the proposed research will uncover the mechanism of SAC control during the DNA damage response. Since DDC and SAC misregulation occurs in many cancers, understanding their regulatory interactions will not only advance our understanding of how eukaryotic cells prevent proliferation when challenged by DNA damage, but will also highlight potential diagnostic and therapeutic targets.
This proposed research aims to elucidate the mechanism of crosstalk between two major cell cycle checkpoints, the DNA Damage Checkpoint (DDC) and the Spindle Assembly Checkpoint (SAC). Since DDC and SAC misregulation occurs in many cancers, understanding their regulatory interactions will not only advance our understanding of how eukaryotic cells prevent proliferation when challenged by DNA damage, but will also highlight potential diagnostic and therapeutic targets.
|Nakajima, Yuko; Haber, James E (2016) Chromosomes at loose ends. Nat Cell Biol 18:257-9|