This project aims to identify alternately spliced transcripts from the DNA-repair genes from skin tissue thatperturb the endogenous DNA-repair mechanism. A number of alternately spliced transcripts of the DNA-repairgenes have been identified from adult mouse and human tissue. Interestingly, these variants disruptDNA repair in a model system, even in the presence of the wild-type protein. To extend this study, an mRNAsplicing screen of the genes responsible for error-free DNA repair in mammalian cells will be undertakenusing Cores A & C. Both normal and tumor (adenocarcinoma) tissue will be screened using a splice-specificRT-PCR protocol. Using this procedure all of the alternately spliced DNA-repair transcripts from thesetissues can be characterized. Typically mutations in the DNA-repair genes are lethal to the cell. However,exciting preliminary data shows that alternatively-spliced transcripts can encode novel proteins, which modifythe DNA-repair activity of the cell. To identify splice variants that disrupt DNA repair, a rapid functionalscreen using a yeast model system will be performed. Yeast is used as the DNA-repair paradigm foreukaryotes and its DNA-repair activities are functionally conserved with mammals. Select variants thatdominantly disrupt DNA-repair in yeast will then be tested in mammalian cell culture to ensure that theeffects seen in yeast are conserved in human cells, with the aid of Core B. In this way, splice variants of theDNA-repair genes that dominantly disrupt DNA repair and potentially lead to tumorigenesis will beidentified.The alternately spliced transcripts identified in this screen will provide candidate biomarkers fortumorigenesis. Based upon the data from this study, it is anticipated that a larger screen of clinical sampleswill be conducted to establish a direct association with a specific tumor type. Such a screen is beyond thescope of this application. By focusing upon splicing, a new class of biological regulation may be uncoveredthat profoundly affects gene function, but is normally overlooked in other clinical screens.ProfessorRothstein, who is an Established Investigator with expertise in DNA repair and no previous work in skindiseases, qualifies as Catergory #2 in the guidelines. Cores A, B, C and D are essential for this proposal.
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