Cell Cycle Manipulation in Breast Cancer: Implications for Improved Therapy.
Annual summary rept. 1 Jul 97-30 Jun 98,
VANDERBILT UNIV MEDICAL CENTER NASHVILLE TN
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The role of DNA damage intermediates and the network of signaling pathways by which cells activate p53 in the overall response to DNA damage is not well defined. It is known that p53 binds DNA in a sequence-specific manner as well as DNA containing insertiondeletion mismatches IDLs. In the funded studies, we determined that p53 requires intact central and dimerization domains to complex with IDLs and that the KD of p53 binding to IDLs is 45 pM as compared to a KD of 31 pM for p53 binding to DNA fragments containing a consensus binding site. The picomolar dissociation constant that we observed for p53 binding to IDLs suggests that p53 would bind to IDLs in the cell. To investigate this possibility, oligonucleotide duplexes containing an IDL lesion were transfected into cells and the cells crosslinked to trap p53DNA complexes. Our crosslinking-based technique has also allowed us to investigate the kinetics of p53 binding to downstream target gene promoters e.g. p21waf1, mdm2, and PIG3 and determine whether specific post-translational modifications of p53 are required for transactivation. Understanding how the sequence-specific and sequence-nonspecific DNA binding activities of p53 are integrated will contribute to our knowledge of how human cells respond to a wide range of DNA lesions.
- Medicine and Medical Research