In Vivo DNA Binding Properties of Wild-type and Mutant p53 Proteins in MammaryCell Lines During the Course of Cell Cycle

摘要

Using ligation mediated PCR genomic footprinting, we observed striking p53dependent protection of the mdm2 gene's putative p53 response elements (REs) as well as the adjacent TATA box. Interestingly, this protection pattern differed considerably from that observed with purified p53 on naked DNA. Indirect Southern blot analysis revealed constitutive nuclease-hypersensitivity at both the p53 independent (P1) and P2 promoters, indicating that both promoters are located in altered chromatin conformations that are most likely nucleosome free. We are also actively pursuing control of p53 function throughout the cell cycle. We carry out these studies using centrifugal elutriation of ML-1 cells in order to separate the cell cycle populations. We have determined that p53 resembles cyclins in that in exponentially growing ML-1 cells p53 is present only during discrete cell cycle time points. In healthy cells, the p53 level peaks at the G1/S border, however after treatment of the cells with camptothecin a surprising increase of p53 occurs only during G1 and G2/M while no increase is observed during the S phase of the cell cycle. Interestingly, only the activated p53 in the G2/M cell cycle fractions is able to bind to the gadd45 p53 DNA binding site.