Reactive oxygen species (ROS) are known to create various types of damage to DNA that can lead to mutation. However, it is becoming increasingly clear that ROS can also damage free nucleotides in the cell and these damaged nucleotides can be misincorporated into DNA and cause mutation. There is much less known about this mutagenic pathway but there are indications that it may be as important to mutagenesis as direct DNA damage. One indication of the danger of oxidized nucleotides is that there are enzymes whose role is to cleanse the nucleotide pool of such damaged nucleotides and the absence of these cleansing enzymes can lead to cancer. Our colleague Dave Lambeth has recently found that 60-70% of early human colon cancers overexpress the NADPH-oxidase Nox1 and as a result have increased levels of ROS. He has developed a mouse intestinal tumor model system to study the association of Nox1-derived ROS and cancer. Our ultimate objective is to determine whether the increased ROS observed in these cells creates mutagenically important oxidized nucleotides. In order to speed our progress toward this objective, we have developed a set of tools in the yeast S. cerevisiae that will allow us to study activities associated with oxidized nucleotide cleansing, incorporation, and extension with much more speed, precision, and efficiency than the use of mammalian cells. We will then translate the results of our yeast experiments to our study of this mouse model system, using cell lines created in the mouse core of this program project. Specifically we propose to: 1) Determine the effect of oxidized nucleotide cleansing enzymes on both nuclear and mitochondria! mutagenesis in yeast, 2) Determine the effect of yeast replication and repair genes on the incorporation of oxidatively damaged nucleotides, and 3) Determine the effect of Nox1-derived ROS species on nucleotide pools in a mouse intestinal tumor model system.
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