DNA rearrangements occur in response to many genomic insults including natural radiation as well as radiation administered to fight cancer. Unfortunately, these radiation-induced rearrangements can often stimulate new neoplasias. Cells ranging from simple organisms such as yeast and bacteria up to humans respond to induced DNA damage by localizing checkpoint, repair and recombination proteins to distinct foci. These foci also form spontaneously during DNA synthesis and segregation suggesting that cells deal with DNA damage during normal cell cycles. These spontaneous foci likely represent attempts to repair replication errors and other natural insults to chromosomal DNA. Cells also "adapt" to persistent DNA damage and attempt to divide even in the absence of successful repair. We have been examining the genetic control of these processes by studying the formation and disassembly of repair/recombination foci using Saccharomyces cerevisiae as a model system. We will continue to examine these processes in living yeast cells using time-lapse microscopy of multi-labeled cellular components. We will use reagents that we have engineered to dissect the genetic pathways responsible for focus formation. We will take advantage of high-throughput methods that we recently developed that allow us to rapidly screen the non-essential yeast genes and identify those that regulate both assembly and disassembly of DNA repair foci. We will apply these same methods to discover how kinetochore components interface with the DNA damage response to affect foci both after damage as well as during a unique asymmetric cell division after spores are formed. Our studies will permit a further glimpse into the in vivo action of key components of the cellular response to both ionizing radiation as well as spontaneous lesions.
Our specific aims are as follows: (1) Continue our molecular and genetic dissection of checkpoint and repair foci using fluorescent protein tags to specifically label chromosomal double-strand break sites and repair proteins to explore the composition of foci, their dynamics of assembly/disassembly by using live cell imaging and time-lapse microscopy. (2) Determine the order of events that take place as the cell begins to repair a double-strand break to address how the cell makes the decision to form a focus and identify the locus to be used for repair. (3) Use the yeast gene disruption library to find genes affecting the timing, progression, assembly and disassembly of DNA repair centers using high throughput screening capabilities comprised of a novel mating approach, called selective ploidy ablation along with a new imaging platform. (4) Examine the effects of the spindle assembly checkpoint and, more broadly, the kinetochore on the DNA damage response. The approaches outlined in this renewal are general. The insights that we gain and the methods we develop will not be confined to yeast alone, but will be applicable to many cellular systems. We purposely centered this proposal on the genetics and cell biology of the DNA damage response as we are one of the few labs committed to examining the relationship between repair centers (foci) and the DNA damage response (repair).
DNA damage not only causes cancer, it is used extensively to treat it using radiation therapy and/or chemotherapeutic drugs. Model systems have provided a glimpse into the mechanisms responsible for the repair of DNA damage. In this proposal, we will explore the genetic and cell biological control of DNA repair processes.
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