Reactive oxygen species generated by ionizing radiation and normal cellular respiration damages DNA and contributes to a variety of human disorders including tumor promotion. A major product of that DNA damage is the formation of 8-oxoguanine, which is a frequently occurring, highly mutagenic form of base modification produced by oxidative stress. The repair of this lesion is therefore of the utmost importance for preserving the stability of the genome. In this regard, we have cloned a Drosophila gene S3 whose encoded product apparently repairs 8-oxoguanine. This conclusion is based upon tests utilizing 5' end-labeled DNA fragments that were specifically cleaved by S3 at sites of 8-oxoguanine as revealed on DNA sequencing gels. This and other results support the notion that S3 contains N-glycosylase activity for the removal of 8-oxoguanine, although this has not been directly proven. To establish the nature of 8-oxoguanine removal by S3, defined DNA substrates that allow for the monitoring of N-glycosylase activity will be utilized. Other tests are described that should identify the types of nuclease activities associated with S3 when presented with a variety of modified 3' and 5' termini. Beyond a detailed biochemical characterization of S3, this proposal also describes experiments that will attempt to reconcile the rather bewildering array of roles apparently carried out by S3. For example, S3 has been shown to be a ribosomal protein with functions in protein synthesis. Notably, it is also a member of 6 ribosomal genes whose mRNA s are elevated in colorectal cancers. As a means of solving this conundrum, the entire gene and associated promoter will be cloned, its organization evaluated, and regulation in response to a variety of DNA damaging agents assessed. Lastly, S3 has been mapped to a region on the third chromosome that encodes a lethal Minute mutation. There are four alleles at this locus, all of which will be sequenced from heterozygotes to determine the DNA alteration. Along with some predictions from a yeast nuclease gene whose encoded product contains a domain similar to S3, we will begin to break down the S3 gene sequence by deletion mutagenesis to determine which areas are vital for its role as a DNA repair protein. Taken together, these experiments are aimed at determining the various domains utilized by a multifunctional protein to carry out its individual roles that perhaps will lead to an increased understanding of the phenotypic variability of human diseases with compromised systems of DNA repair.
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