c-Jun stimulates origin-dependent DNA unwinding by polyomavirus large T antigen

Abstract

The AP1 protein c-Jun has previously been shown to stimulate polyomavirus (Py) DNA replication in vivo. In order to define the mechanism, we added purified c-Jun protein to the origin-dependent and large T antigen (LT)-dependent in vitro DNA unwinding assay. c-Jun protein was found to stimulate by ~ 5-fold the unwinding of a 290 bp linear DNA fragment containing both the Py origin and the AP1 recognition sequence to which c-Jun binds. Efficient levels of stimulation were specifically observed at limiting concentrations of LT for unwinding. Under similar conditions, Py DNA replication was stimulated to a comparable extent by AP1 in a purified in vitro replication assay. Mobility shift and DNase I footprinting assays showed that c-Jun stimulates the ATP-dependent binding of LT to the origin core by ~ 7-fold. Furthermore, c-Jun was found to interact directly with LT, but not with replication protein A. The activities of c-Jun to stimulate unwinding and origin binding of LT were found to be harbored within the N-terminal region of c-Jun, which is distinct from the DNA binding domain. We speculate that certain transcription factors may possess specific DNA replication domains that function to stimulate the loading of replication factors at the origin during the initiation of DNA synthesis.