When purified from healthy individuals, the regulatory regions of most human polyomaviruses contain single enhancers. However, when isolated from productively infected cells from immune- compromised individuals, the genomes of polyomaviruses contain duplicated enhancers that promote viral DNA replication and subsequent pathogenesis. The mechanism(s) that give rise to the duplicated enhancers in pathogenic polyomaviruses are, however, not known. To address this issue, we proposed a model for the duplication of the polyomavirus enhancers that is based upon advances in our understanding of; 1) the initiation of polyomavirus DNA replication, 2) the formation of “long flaps” via displacement synthesis o f O k a z a k i f r a g m e n t s and 3) the subsequent steps needed for double stranded break repair. We now wish to test key features of the “Replication Dependent Enhancer Duplication” (RDED) model. Regarding the assays that will be employed to test this model, previous experiments established that the enhancer duplications can be detected in CV1 cells following transfection of SV40 virus. Thus, this is a viable assay for testing the RDED model. However, we wish to test the hypothesis that the enhancer duplications will also occur in more versatile plasmid-based replication assays. Once the optimal cell type and assays (e.g., PCR) are established, we will use Next-generation sequencing (NGS) to quantitate the frequency of the enhancer duplications and to identify the sequences of all the products formed in the reactions. In addition, we will test the RDED- based hypothesis that the enhancer duplications will be promoted by the aberrant expression of factors needed for Okazaki fragment formation. For instance, a cell line containing a doxycycline inducible shRNA targeting FEN-1 will be used to test the hypothesis that the resulting “long flap” containing nascent DNA will promote the enhancer duplications. In related experiments, we will test the hypothesis that hairpins designed to block FEN1 loading will also promote the enhancer duplications. Additional experiments will test the RDED-based hypothesis that nicks are generated on the template strand in the vicinity of the viral origin. Moreover, we will test the theory that nucleosomes play a major role in limiting the enhancer duplications to the regulatory region. Finally, non-viral enhancers are also subjected to duplications and deletions and these alterations significantly impact the activity of disease genes, including oncogenes and tumor suppressors. Therefore, our studies of the formation of the enhancer duplications in polyomaviruses will have an important role in establishing the mechanism(s) needed to form the enhancer duplications associated with a wide variety of human diseases.