Mechanisms and Regulation of Papovavirus DNA Replication
Papovaviruses are a group of small DNA viruses that infect animals and can potentially cause tumors in both the animals they infect and cell culture systems. Due to the relative simplicity of their genomes, papovaviruses rely upon the host replication machinery to facilitate their replication. Since only one or two viral gene products are required for replication of the viral genome, papovaviruses behave at the biochemical level in a manner very similar to the host cell. Papovaviruses are one of the most easily manipulated and well-established models known to study mechanisms of various viral and host eukaryotic systems. Using both human papillomavirus type 11 (HPV-11) and the polyomavirus simian virus 40 (SV40), several mechanisms of DNA replication regulation were investigated. The interaction between two replication proteins that are essential during HPV-11 replication are determined. Then, the effect of an inhibitory human transcription factor on HPV replication is presented. Finally, using the well-established SV40 in vitro replication assay, the poorly understood DNA damage induced intra-S phase checkpoint is further characterized. These studies together, using papovaviruses as powerful models, will extend our knowledge of the processes by which both viruses and their human hosts regulate the processes involved in DNA replication and repair, which will also further the field in the context of cancer progression in humans.
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Active ATR is recruited to SV40 replicons in the absence
Figure M1 Chromatographic separation of 293 extract
Preliminary findings and future directions
The two major Sphase checkpoint pathways of ATMATR signaling
Phosphorylation of papovavirus replication proteins by purified
Phosphorylation of RPA during SV40 DNA replication
An overall model for ATR mediated inhibition of in vitro
Mixing of MO59J extract with checkpoint competent 293 extract
293 extract activate ATR activation of ATR addition of DNA ATR activity ATR cofactors ATR(kd baculovirus buffer CDP binding cell extracts cell line cellular extracts Chk1 claspin cofactors compare lanes cyto/nucleosolic extracts data not shown DMSO DNA damage DNA damage response DNA fragments DNA synthesis DNA-fragment-induced DNA-PK dsDNA E1 and E2 E2 protein elution fractions fragment-induced gel filtration genes genome helicase human cells immunoblotting immunoprecipitated incubated infection inhibition of SV40 interaction investigated kinase activity large T-antigen Materials and Methods MO59J extract NaCl Nbs1 origin of replication panel papillomavirus phosphorylation phosphorylation of GST-Rad17(SQ2 phosphorylation of RPA phosphorylation of TAg PI3K PIKK plasmid polyomavirus presence purified DNA-PK purified FLAG-ATR reaction assembly reconstituted replication assay replication fork replication in vitro replication proteins RPA32 phosphorylation SDS-PAGE ss/dsDNA ssDNA subunit SV40 DNA replication SV40 large T-antigen SV40 replication reaction topoisomerase vitro checkpoint response vitro DNA replication vitro SV40 vivo wortmannin