1,1,1-Trifluoro-2,2-dichloroethane (HCFC-123) and 1,1,1-trifluoro-2-bromo-2-chloroethane (halothane) cause similar biochemical effects in rats exposed by inhalation for five days

Control of p53 turnover is critical to p53 function. E1A binding to p300/CBP translates into enhanced p53 stability, implying that these coactivator proteins normally operate in p53 turnover control. In this regard, the p300 C/H1 region serves as a specific in vivo binding site for both p53 and MDM2, a naturally occurring p53 destabilizer. Moreover, most of the endogenous MDM2 is bound to p300, and genetic analysis implies that specific interactions of p53 and MDM2 with p300 C/H1 are important steps in the MDM2-directed turnover of p53. A specific role for p300 in endogenous p53 degradation is underscored by the p53-stabilizing effect of overproducing the p300 C/H1 domain. Taken together, the data indicate that specific interactions between p300/CBP C/H1, p53, and MDM2 are intimately involved in the MDM2-mediated control of p53 abundance.     

Wild-type but not mutant p53 activates the hepatocyte growth factor/scatter factor promoter

p53 transactivates the expression of a variety of genes by binding to specific DNA sequences within the promoter. We have investigated the ability of wild-type p53 and a non-DNA binding p53 mutant to activate the hepatocyte growth factor/scatter factor (HGF/SF) promoter using chloramphenicol acetyltransferase reporter constructs. We also used deletion sequences of the HGF/SF promoter to identify which regions, if any, were responsible for p53 binding. Our results show that wild-type but not mutant p53 activates the HGF/SF promoter when using -3000 and -755 bp upstream of the HGF/SF gene. This activation is lost when promoter sequences covering -365 and -239 bp are used. Analysis of the DNA sequence between -365 and -755 bp shows one putative p53 half-site with 80% homology to the consensus sequence and another half-site 3 bases downstream of this with 100% homology to the consensus sequence. In contrast to previously identified p53 binding DNA sequences, the downstream half-site is inverted. We propose that the HGF/SF promoter can be activated by wild-type p53 in vivo and that this could be as a result of a novel form of sequence-specific DNA binding.     

High affinity insertion/deletion lesion binding by p53. Evidence for a role of the p53 central domain

In addition to binding DNA in a sequence-specific manner, p53 can interact with nucleic acids in a sequence-independent manner. p53 can bind short single-stranded DNA and double-stranded DNA containing nucleotide loops; these diverse associations may be critical for p53 signal transduction. In this study, we analyzed p53 binding to DNA fragments containing insertion/deletion mismatches (IDLs). p53 required an intact central domain and dimerization domain for high affinity complex formation with IDLs. In fact, the C terminus of p53 (amino acids 293-393) was functionally replaceable with a foreign dimerization domain in IDL binding assays. From saturation binding studies we determined that the KD of p53 binding to IDLs was 45 pM as compared with a KD of 31 pM for p53 binding to DNA fragments containing a consensus binding site. Consistent with these dissociation constants, p53-IDL complexes were dissociated with relatively low concentrations of competitor consensus site-containing DNA. Although p53 has a higher affinity for DNA with a consensus site as compared with IDLs, the relative number and availability of each form of DNA in a cell immediately after DNA damage may promote p53 interaction with DNA lesions. Understanding how the sequence-specific and nonspecific DNA binding activities of p53 are integrated will contribute to our knowledge of how signaling cascades are initiated after DNA damage.