DNA binding specificity of proteins derived from alternatively spliced mouse p53 mRNAs.

The mouse p53 gene generates two alternative splice products encoding p53 protein and a naturally occurring protein (p53as) with changes at the C-terminus. In p53as the negative regulatory region for DNA binding and PAb421 antibody binding site are replaced, and p53as is constitutively active for sequence-specific DNA binding. Using the technique of randomized synthetic oligonucleotide in cyclic amplification and selection of targets, we have found that p53as and p53 proteins have the same DNA binding specificities but that these specificities frequently diverge from the consensus of two copies of PuPuPuCATGPyPyPy. The importance of tetranucleotide CATG was confirmed but there was a less rigorous requirement for patterns of flanking or intervening sequences. In particular, the three purines upstream and three pyrimidines downstream of CATG are not required for p53 or p53as binding, 29 or more intervening nucleotides are tolerated, and one CATG is sufficient where adjacent nucleotides contain a region of homology with certain previously reported non-consensus p53 binding sequences. These results suggested further definition of the non-consensus motifs, and database searches with these uncovered additional candidate genes for p53 protein binding. We conclude that p53as and perhaps other activated forms of p53 exert their effects on the same genes and that differential activities of p53 protein forms are not due to inherently different sequence selectivities of DNA binding.     

Effect of HMG protein 17 on the thermal stability of control and acetylated HeLa oligonucleosomes.

Many studies have implicated histone acetylation and HMG proteins 14 and 17 in the structure of active chromatin. Studies of the binding of HMG 14 and 17 to chromatin core particles have shown that there are two binding sites for HMG 14 or 17 located within 20-25 bp of the DNA ends of the core particles [13-15]. Such binding sites may result from the free DNA ends in the core particle being available for the binding of HMG 14 and 17. We have studied the effects of the binding of HMG 17 on the thermal denaturation of DNA in mono, di and trinucleosomes. In each case the binding of 1 HMG 17 molecule per nucleosome reduces the DNA premelt region by 50%, while the binding of 2 HMG 17 molecules per nucleosome abolishes the premelt region. From this it is concluded that there are two HMG 17 binding sites per nucleosome which are located between the entry and exit points to the nucleosome and the strongly complexed central DNA region. Highly acetylated mono, di and trinucleosomes have been isolated from butyrate treated HeLa S3 cells. For this series of acetylated oligonucleosomes, it has been found that there are also two HMG 17 binding sites per acetylated nucleosome.