The long term goal of my laboratory is to study the cellular responses to, and consequences of, irreparable DNA damage. Yeast presented with irreparable damage initially arrest at a cell cycle checkpoint and attempt repair, but eventually forgo this attempt and proceed through cell division (checkpoint adaptation). Previously, we identified mutants unable to undergo this adaptation process. In yeast, double stranded breaks are repaired almost exclusively using homologous recombination. When this form of repair is not possible, cells will lose the broken chromosome or repair it using some alternative error-prone method. Unpublished experiments have shown that adaptation precedes, and is required for, chromosome loss and some forms of error-prone repair, thereby promoting genomic instability. We will test this further by comparing the rates with which adaptation-proficient and adaptation-deficient strains undergo three different events; chromosome loss, chromosome truncation, and loss of heterozygosity. The identification of one of our adaptation-defective mutants as an allele of CDC5 suggests mechanisms by which checkpoint adaptation may occur. CDC5 encodes a highly conserved protein kinase that promotes mitosis, in part by activating ubiquitin conjugation of B type cyclins. We will determine whether B cyclins are the critical target of CDC5 during checkpoint adaptation and examine whether CDC5p is modified or activated during an adaptation time-course. We will also identify the downstream targets of the adaptation pathway. To this end, we will determine if checkpoint protein phosphorylations known to occur during a checkpoint arrest are reversed during adaptation. CDC20 interacts genetically with CDC5 and is implicated in the checkpoint pathway. We will examine whether CDC20p is modified or present in a novel complex during either checkpoint arrest or adaptation. Like yeast, mammalian cells also encounter damage that cannot be repaired by standard methods. Loss of at least two different DNA repair pathways (recombination and mismatch repair) predisposes individuals to cancer. Checkpoint adaptation may occur in such situations.
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