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 MutS1 and MutS2 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.
Inherited defects in mismatch repair are responsible for the most common form of inherited colon cancer and defects in mismatch repair are found in many other cancers. Oxidative damage is one of the most common forms of damage to DNA and has been implicated in aging, cancer, and other diseases. Understanding how oxidative damage causes mutations, and the repair processes that act on it, such as mismatch repair, is extremely important in developing diagnostics and treatments.
|Crouse, Gray F (2016) Non-canonical actions of mismatch repair. DNA Repair (Amst) 38:102-9|
|Flood, Carrie L; Rodriguez, Gina P; Bao, Gaobin et al. (2015) Replicative DNA polymerase ? but not ? proofreads errors in Cis and in Trans. PLoS Genet 11:e1005049|
|Romanova, Nina V; Crouse, Gray F (2013) Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast. PLoS Genet 9:e1003920|
|Shockley, Arthur H; Doo, David W; Rodriguez, Gina P et al. (2013) Oxidative damage and mutagenesis in Saccharomyces cerevisiae: genetic studies of pathways affecting replication fidelity of 8-oxoguanine. Genetics 195:359-67|
|Rodriguez, Gina P; Song, Joseph B; Crouse, Gray F (2013) In vivo bypass of 8-oxodG. PLoS Genet 9:e1003682|
|Rodriguez, Gina P; Song, Joseph B; Crouse, Gray F (2012) Transformation with oligonucleotides creating clustered changes in the yeast genome. PLoS One 7:e42905|
|Rodriguez, Gina P; Romanova, Nina V; Bao, Gaobin et al. (2012) Mismatch repair-dependent mutagenesis in nondividing cells. Proc Natl Acad Sci U S A 109:6153-8|
|Crouse, Gray F (2010) An end for mismatch repair. Proc Natl Acad Sci U S A 107:20851-2|