We report on several specific projects that are related to the DNA sequence-dependent structural characteristics, important for interactions with proteins (including human p53 and bacterial gal-repressor), higher order self-organization of genomic DNA (formations of nucleosomes and DNA loops), and gene regulation.? ? ? p53-induced activation and repression of transcription.? In response to DNA damage and other types of cellular stress, the p53 protein becomes activated and binds DNA sequence-specifically, functioning as a transcriptional factor. p53 is unique in regulating a wide spectrum of genes: thousands of human genes are either activated or repressed by p53. Normally, the p53 tetramer binds to DNA response elements, consisting of two decamers RRRCWWGYYY (half-sites) separated by a spacer. (The length of the spacer, S, varies from 0 to 14 bp in the known functional binding sites, but in most cases S=0 or 1.) How many putative p53 binding sites, consistent with this scheme, are there in the human genome? What is the distribution of the spacer lengths? ? ? Recent sequencing of the human genome allows us to answer these questions. The distribution of spacers proves to be extremely nonuniform in all human chromosomes, with strong peaks in the profile, exceeding the average background 3-4 fold. The peaks at S=0 and 10 bp, and the gap at 4-5 bp are consistent with our earlier computer modeling and electrophoresis measurements, indicating the lateral positioning of the p53 core domains on the outer side of the DNA loop. In general, these data agree with the idea that the p53 tetramer can bind DNA wrapped in nucleosomes in the course of transcriptional activation of the chromatin-assembled genes. ? ? Currently, we are exploring localization of the putative p53 sites with respect to the starts of transcription (in collaboration with C. Harris, NCI). Our database includes more than 1500 human genes, proven to be up- and down-regulated by p53 under various conditions. The results of computer analysis indicate statistically significance difference between the up- and down-regulated genes in terms of distribution of the p53 sites in the vicinity of genes. The up-regulated genes are characterized by a high occurrence of the p53 sites with spacer S=0. On the other hand, the down-regulated genes reveal a higher fraction of the p53 sites with """"""""unusual"""""""" spacer S=3 bp, shown earlier to repress transcription of several genes. Based on these observations, we suggested a three-dimensional model accounting for the functional difference between the tetrameric p53 sites with spacers S=0 and 3 base pairs (the model is currently being tested). ? ? If confirmed experimentally, this novel concept opens exciting prospects for predicting the p53-activated and repressed genes. ? ? ? DNA packaging and distribution of the A- and G-tracts in pro- and eukaryotes? Periodic positioning of the A- and G-tracts in DNA causes DNA curvature in solution and facilitates its bending in the complexes with proteins. Here, we analyzed distribution of these sequences in the pro- and eukaryotic genomes. ? ? Distribution of the strongly bent A-tracts (4-7 bp) in the prokaryotic genomes reveals a remarkable periodicity of 10-11 bp. Such a periodicity may reflect the intrinsic propensity of prokaryotic DNA for bending and forming the loop-shaped structures. Based on these data and by analogy with the """"""""gene repression"""""""" gal- and lac-loops in E. coli, we hypothesize that the loop folds with the structural period of 100 bp may be elementary units of the prokaryotic nucleoid packaging. This hypothesis was tested by the micrococcal nuclease digestion of bacterial nucleoids (in collaboration with S. Adhya, NCI). The results show that the 100 bp DNA fragments are highly overrepresented in digestion products, thereby implying a highly specific nucleoid packaging, with the DNA structural period of 100 bp.?
