The In Vivo DNA Binding Properties of Wild-type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.
Annual rept. 1 Jun 96-31 May 97,
HUNTER COLL NEW YORK
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Using ligation mediated PCR genomic footprinting we observed striking p53 dependent protection of the mdm2 genes 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 G1S border, however after treatment of the cells with camptothecin a surprising increase of p53 occurs only during G1 and G2M while no increase is observed during the S phase of the cell cycle. Interestingly, only the activated p53 in the G2M cell cycle fractions is able to bind to the gadd45 p53 DNA binding site.
- Anatomy and Physiology