P53 INHIBITS DNA-REPLICATION IN-VITRO IN A DNA-BINDING-DEPENDENT MANNER

The p53 tumor suppressor gene product is a sequence-specific DNA-bindi ng protein that is necessary for the G(1) arrest of many cell types, C onsistent with its role as a cell cycle checkpoint factor, p53 has bee n shown to be capable of both transcriptional activation and repressio n.;Here we show a new potential role for p53 as a DNA-binding-dependen t regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the lat e side of the polyomavirus origin were used in in vitro replication as says. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a m utant version of the RGC site was not affected by p53. Several tumor-d erived mutant p53 proteins were unable to inhibit replication of the c onstruct with wild-type RGC sites, Additionally, the transactivator GA L4-VP16 was unable to inhibit replication of a construct containing GA L4 binding sites adjacent to the polyomavirus origin, We also show tha t the inhibition by p53 can occur from sites cloned as far as 600 bp f rom the origin, Preincubation experiments suggest that p53 inhibits re plication at a step mediated by ATP, possibly by inhibiting the bindin g of polyomavirus T antigen to the core origin, The presence of an end ogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition.