Human papillomaviruses (HPVs) are the causative agent of papillomas (warts) and are responsible for nearly all cervical and most anogenital and oral/oropharyngeal cancers. In addition to being important human pathogens, HPVs also serve as excellent models for eukaryotic DNA replication. Other than the PV replication proteins E1 and E2 (the viral replicative helicase and the transcriptional regulator protein, respectively), HPVs rely entirely on host proteins to replicate their genomes. Preliminary data demonstrates a direct interaction and a functional relationship between HPV replication proteins and pol , which result in increased DNA synthesis activity of pol . Though it has been demonstrated in prokaryotic systems, a functional coupling between helicase and leading strand polymerase has never been described in the HPV replication system. The scope of this proposed work is two-fold; it will help clarify the mechanisms and interactions behind eukaryotic DNA replication fork elongation and will also serve to identify essential interactions between viral replication protein and host replication factors that may provide potential targets for pharmacological intervention. Therefore, the aims of this project are to characterize the functional interactions between HPV replication proteins (E1 and E2) and cellular DNA polymerase (pol ). Through the use of various DNA templates, the DNA synthesis activities of pol , including processivity, chain elongation efficiency, polymerase recycling, and polymerase loading will be analyzed in the presence and absence of E1 (and E2) and known polymerase co-factors. Conversely, the activities of E1 will be analyzed in the presence and absence of the polymerase complex to determine if interaction with pol stimulates E1 function. E1 activity will be analyzed using functional assays examining helicase unwinding and helicase ATPase activity. E2 will also be assessed for its ability to bind DNA in the presence of pol , as well as its ability to form a complex with pol , through the use of electrophoretic mobility shift assays (EMSAs).
Given the biological requirement for the faithful duplication of the genetic material and the link to cancer in the event of its mismanagement, DNA replication is one of the most critical processes to human health. Human papillomavirus (HPV) is one of the greatest causes of cancer worldwide. Since there are currently no anti-virals against HPV, characterization of the functional interactions between viral replication proteins and cellular DNA polymerase ? may identify an interaction that is essential for HPV replication and would therefore be a potential target for pharmacological inhibition.
