We will continue and extend our analysis of the DNA damage checkpoint in the model organism, Saccharomyces cerevisiae. In yeast, as well as in human cells, the DNA damage checkpoint acts to prevent mitosis until DNA has been repaired. Failure of the checkpoint in mammalian cells leads to chromosome losses and chromosome translocations that are the hallmark of human cancers. This grant focuses its attention on the cellular responses induced by an unrepaired, site-specific single double-strand break (DSB) that causes ATR and ATM- dependent chromatin modification and pre-anaphase cell-cycle arrest. Of great interest is how cells maintain the checkpoint and how they adapt to the presence of an unrepaired DSB. The project also investigates how adaptation differs from the process of recovery, where cells repair the DSB and then turn off the DNA damage checkpoint in a concerted fashion, leading to the removal of phosphorylated histone H2AX (3-H2AX) from the 100-kb region surrounding the DSB.
The first Aim i s to examine checkpoint-mediated chromatin modification. One goal is to study how induction of a DSB triggers the activation of nearby, dormant origins of DNA replication. Experiments are designed to determine over what distance origin activation occurs and how activation is related to the DNA damage checkpoint. A second goal will be to determine how the 3-H2AX chromatin modification around a DSB is regulated, to discover whether the modification is confined to sequences along the same DNA molecule or can also be found on nearby non-covalently linked DNA. The effect of changing the balance between histones H2AX (which can be phosphorylated) and H2A (which cannot) also will be investigated. Mutations that change the size of the modified domain will be sought.
A second Aim i s to understand how the DNA damage checkpoint is maintained. Specifically, research will test whether the cell monitors oligodeoxynucleotides as a signal or continuing 5'to 3'degradation of DSB ends or whether maintenance depends on turnover of the 9-1-1 clamp complex that binds at the degraded DNA end. A second goal of this AIM is to clarify the synergistic relationship between the DNA damage checkpoint and the spindle activation checkpoint.
A third Aim i s to analyze the processes of adaptation and recovery. It is important to understand in detail how cells that have adapted - either to a single unrepaired DSB or to the damage caused at telomeres when the Cdc13 protein activity is compromised - respond to a newly generated DSB. The consequences of simultaneously deleting phosphatase genes PTC2, PTC3 and PPH3 on checkpoint functions and especially their unanticipated role in DSB repair will be examined. The final goal is to learn how cells lacking two histone H3-H4 chaperones, Asf1 and CAF-1, fail to recover after repair of a specific DSB and how this is linked to chromatin re- assembly after DSB repair.
Chromosomal double-strand breaks threaten genome integrity. The DNA damage checkpoint acts to prevent cells from segregating broken chromosomes in mitosis as the highly recombinagenic ends are associated with the formation of chromosome translocations and aneuploidy that are characteristic of human cancers. Not only are the core components of the DNA damage checkpoint conserved from yeast to humans, but ability of cells carrying persistent DNA damage to resume cell-cycle progression (termed adaptation) has also been recently shown to occur in human cells as it has been well documented in yeast. Hence, it is important to study the DCC in budding yeast, where the fate of broken DNA and the recruitment of DNA damage-responsive proteins can be studied in great detail.
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