) Genetic instability is a critical factor in carcinogenesis and tumor progression. Recent work from the laboratory of Thomas Shenk has revealed that the human cytomegalovirus (CMV) proteins IE1 and IE2 can mediate cellular transformation in a manner associated with the induction of mutagenesis in the host cell genome. In this proposal, the nature and mechanism of the IE1- and IE2- induced genetic instability will be investigated. The induced mutation patterns will be characterized using a series of mutation reporter assays, with emphasis on the use of a chromosomally integrated lambda phage shuttle vector carrying the supF mutation reporter gene. The possibility that the proteins generated a hypermutable state will be tested by examining mutagen-induced as well as spontaneous. These studies will be facilitated by exploiting techniques for high efficiency gene transfer and for isolation of successfully transfected cells, so that the full effect of transient viral protein expression can be examined in bulk cell populations. Using reporter constructs developed by Dr. Glazer, the ability of IE1 and IE2 to promote recombination will also be determined as another measure of genetic instability. The influence of IE1 and IE2 expression on DNA repair will be investigated using assays for reactivation of damaged expression vector DNA, including lesions subject to nucleotide excision repair, base excision repair, and mismatch repair. Direct measurements of lesion removal from cellular DNA will also be performed, with analysis of both bulk DNA and actively transcribed regions. Possible protein-protein interactions between IE1 and IE2 and selected repair factors will be examined. A series of deletion mutants of IE1 and IE2 will be tested to identify protein domains associated with mutagenicity, for comparison with domains associated with the transcriptional regulatory and anti- apoptotic activities of the proteins. Pathways associated with IE1- and IE2-induced mutagenesis will be explored by testing mutagenesis in p53, c-fos, and c-jun knock out cells and by using genome array analyses to detect genes up- and down-regulated by the proteins. This work will build on Dr. Glazer's experience in the area of mutagenesis and DNA repair, and will benefit from a matrix of ongoing studies to examine other genetic and epigenetic causes of genetic instability in cancer. The studies will help to elucidate a novel pathway of viral protein- induced mutagenesis and transformation, and should help to identify fundamental cellular pathways contributing to genetic instability. As such, this project will serve as a direct bridge between the Dimaio and Sweasy projects.
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