Genome instability is common to all tumors and is thought to drive the alteration of oncogenes, tumor suppressor genes as well as expression and epigenetic changes. Chromosomal instability resulting from recombination repair (RR) defects have been linked to a variety of human cancers including hereditary breast cancer (BRCA1/2) as well as hematopoietic and other solid tumors (Ataxia telangiectasia mutated, ATM;Nijmegen Breakage syndrome, NBS;Fanconia Anemia, FANC;Bloom's syndrome, BLM), among others. Chromosomes are a complex assortment of proteins and DNA (chromatin). Nucleosomes are the fundamental unit of chromatin and contain ~146 bp DNA that surrounds an H2A, H2B, H3, and H4 histone octamer. Chromosome structure and maintenance depends on the temporal regulation of an expanding repertoire of histone post-translational modifications (PTMs). Remarkably, there is very little quantitative data detailing the biophysical events associated with DSB repair on chromatin? This is largely because the reactions have not been reconstituted with well define and physiologically relevant chromatin DNA. Unlike many repair pathways, RR engenders a complex cascade of responses that include cellular signaling integrated with the physical processes of DSB repair. The DSB repair reaction itself involves a complex cascade of enzymatic reactions that must manage the chromatin composite on the broken donor DNA in order to search and pair with the assembled chromatin of a homologous acceptor DNA. Deficiencies in any one of the multitudes of steps will affect the outcome of the RR process and ultimately affect genome stability. Several innovative real time single molecule visualization systems wil be used to quantitatively resolve RR mechanisms that cannot be determined by traditional bulk biochemical studies. We propose to examine fundamental DSB repair processes on well-defined chromatin containing physiologically relevant histone PTMs to: 1.) Determine the kinetics and mechanism of the RAD51 homology search, strand exchange and remodeling function(s) within chromatin, and 2.) Determine the ensemble interplay between RAD51-paralogs, INO80, and PRMT5 on the Rad51-RAD54 homology search, strand exchange, and remodeling function(s) within chromatin. These studies will detail the complex interactions of DSB repair components with chromatin and will provide additional insight into protein mechanics on chromosomes, chromosomal maintenance and ultimately rational therapeutic approaches to cancer and radiation therapies.
We propose to determine the detailed biophysical processes of the homology search, pairing, strand exchange, and remodeling of chromatin containing histones with defined physiologically relevant post- translational modifications associated with homologous recombination repair. We will use state-of-the-art time- resolved fluorescence resonance energy transfer (FRET), single molecule magnetic tweezers, and single molecule total internal reflection fluorescence (TIRF) to quantitatively analyze the kinetics and mechanism of these processes. The results of these studies should close several significant gaps in our knowledge of DSB repair and identify useful targets for cancer and radiation therapeutics.
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