Retroviruses are currently under intensive study because they can elicit malignant tumors and cause AIDS. At least 0.1-0.6% of the human genome is composed of endogenous retroviruses and the related elements, retrotransposons. The life-cycle of these elements includes integration into host cell DNA. In some cases, the integrated retrovirus causes cancer by activating a nearby cellular proto-oncogene. Likewise, insertions of yeast Ty1 and mouse IAP retrotransposons have been shown to activate nearby cellular genes and alter their regulation. The long- term goal of this proposal is to determine how host genes and proteins influence the frequency of retroviral and retrotransposon integration and the choice of integration sites. These results should stimulate the development of new antiviral treatments that target these genes and their functions. The yeast retrotransposon Ty1 will be used as a model to study host factors that influence retrotransposon and retroviral integration. The principal investigator has shown that mutations in the well-studied DNA repair gene, rad6, cause a 100-fold increase in Ty1 transposition and eliminate the Ty1 insertion site bias for the 5' region of the CAN1 target gene. Lesions in rad6 are the only known host mutations that increase the rate, or alter the target-site specificity, of retrotransposition. RAD6 encodes a ubiquitin-conjugating enzyme capable of polyubiquitinating histones in vitro. The high conservation of both RAD6 and retrotransposons makes it likely that the rad6 effect on Ty1 transposition in yeast will have relevance to higher eucaryotes. Ty1 target-site distributions will be examined at several loci in isogenic rad6 mutant and wild-type strains to determine if the altered distribution caused by rad6 at the CAN1 locus is a general effect. Independent mutations will be isolated at LYS2, URA3 and tRNA(Gly). The polymerase chain reaction will be used to identify those mutations caused by Ty1 insertions and to determine the sites of integration. The effect of altering transcription levels of the target sequence on Ty1 target-site preferences will also be determined. The hypothesis that rad6 mutations alter chromatin structure will be tested. DNA from rad6 mutant and isogenic wild-type strains will be examined for in vivo accessibility to methylation, supercoiling of plasmids, and positions of micrococcal nuclease sensitive sites. If rad6 alters any of these properties, then the hypothesis that chromatin structure is critical for target-site specificity would be supported. The role of the RAD6 poly-acidic tail in the control of Ty1 transposition will be determined. This tail is essential for sporulation but not for DNA repair. Other host genes that influence the rate of Ty1 transposition will be sought by examining mutations in known genes involved in DNA repair, ubiquitin conjugation, or chromatin structure. Mutations in new loci will be isolated from a screen of mutagenized cells for an increased rate of transposition.
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