To enhance their ability to repair double-strand chromosome breaks (DSBs), eukaryotic cells invoke a DNA damage checkpoint, causing cells to arrest at the G2/M boundary of the cell cycle, prior to mitosis. Failures of checkpoint regulation are a hallmark of cancer cells, and chromosomal rearrangements arising by the failure to repair broken chromosomes in an accurate fashion are also evident in late-stage cancers. Although many proteins, including a cascade of protein kinases, have been implicated in this checkpoint process, it has not been established what proteins directly identify the damage and how cells recover and resume growth when that damage has been repaired. Saccharomyces cerevisiae cells that suffer unrepairable DNA damage arrest for a long time, but are capable of adaptation and the resumption of growth. Work from this laboratory has established that the ability of cells to adapt depends on the extent of single-stranded DNA (ssDNA) produced by 5' to 3' exonuclease resection of DSB ends. When the amount of ssDNA increases, cells arrest permanently. A mutation in the ssDNA-binding complex, RPA, suppresses this permanent arrest.
One aim of this project is to determine how adaptation occurs. Experiments are proposed to determine if adapting cells down-regulate the rate of DNA resection or the persistence of ssDNA, or if cells become insensitive to the continued presence of ssDNA. The turnover and dephosphorylation of checkpoint protein kinases will be measured. Several new adaptation-defective mutations have recently been identified by this laboratory, and more mutations will be sought. These will be characterized in terms of their effect on the formation of ssDNA and their interactions with RPA. Proteins that interact with a mutant RPA subunit in monitoring the extent of ssDNA will be identified by a high-copy suppressor screen.
A second aim will be to assess the role of DNA-damage inducible genes in the efficiency of DNA repair. A third objective is to understand the relationship between adaptation and """"""""recovery,"""""""" when DNA damage can be repaired. A newly-designed wild type strain will be used, in which a single DSB can be repaired, but only after 6 hr, by which time cells have arrested in G2/M. By spreading out the time between the induction of damage and repair, it will be possible to assess the contributions of many checkpoint proteins.
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