Grant: $61,738 - National Institutes of Health - Sep. 9, 2009
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Award Description: Cellular Responses to Radiation and Other Types of Damage Radiation is responsible for mutation and genomic instability as a result of DNA strand nicks and breaks and misreplication of damaged DNA. This damage if not correctly repaired is responsible for numerous health effects, including aging and cancer. Much of the damage to DNA is created by oxidative damage as a result of reactive oxygen species generated directly and indirectly by ionizing radiation. The experiments in this proposal are designed to increase understanding of the mechanisms by which oxidative damage to DNA is repaired, and mutation prevented, in eukaryotic cells. The proposed studies utilize S. cerevisiae as a model organism as it has proven to be spectacularly successful in yielding information about various repair pathways, DNA replication, and translesion synthesis that is applicable in all eukaryotes, including humans. There have been many elegant in vitro studies of the biochemistry of these pathways; however, it has proven difficult to study defined mismatches and damaged bases in a the context of normal chromosomal replication. New assays have been developed that will now make such experiments possible for the first time. One assay uses mutations in a TRP5 gene located near a defined origin of replication; each mutation can revert only via a specific base pair mismatch. Another assay uses transformation of yeast with single-stranded oligonucleotides, thereby placing a defined base-base mismatch in the genome at a given location, with the possibility of an introduced base having a specific type of damage. In addition, we have developed strains that are partially deficient in DNA proofreading so that we can study proofreading in a context in which other repair processes such as mismatch repair are still intact. In Aim 1, we will establish how the recognition and processing of mispairs is affected by the replication strand, mismatch repair, and translesion synthesis. In Aim 2, we will make use of the assays and results from Aim 1 to study the fate of oxidative damage on base pair mutagenesis. We have developed conditions in which we can study the effect of increased oxidative damage on a variety of different bases, and have also shown we can study the effect of oxidative damage introduced on synthetic oligonucleotides. This will make possible the study of defined DNA damage in the most natural in vivo context yet available. In Aim 3, we will begin the study of oxidative damage and frameshift mutagenesis by studying the mechanism of frameshift mutagenesis, with a particular focus on the interactions of mismatch repair and primer versus template strand slippage loops in the DNA. We are already using these assays to uncover fundamental information about these pathways, such as the unequal distribution of MutSalpha and MutSbeta activity on replication strands, and an independent verification of the importance of mismatch repair in preventing mutagenesis due to oxidative damage, and are now set to achieve the proposed aims.
Project Description: This request for Recovery Act Administrative Supplement Funds was for equipment that will significantly enhance the quality and efficiency of the research sponsored by the parent grant. In each case the requested item of equipment was either not available or would significantly enhance the capabilities of existing equipment . Purchase of the equipment will provide an immediate stimulus to the economy and will facilitate better quality of research data and increased amount of research to be done. This research that is being done will help our understanding of mutagenesis due to radiation and the frequently accompanying oxidative damage and the repair processes that prevent such mutations. These processes underlie the cause of many diseases including cancer, as well as aging. To date, over half of the proposed equipment has been ordered.
Infrastructure Description: N/A
Jobs Summary: Although these funds have been used to purchase equipment rather than for salaries, they will have a clear positive impact for the firms making the equipment and the firms selling the equipment. (Total jobs reported: 0)
Project Status: More than 50% Completed
This award's data was last updated on Sep. 9, 2009. Help expand these official descriptions using the wiki below.