DNA Deformations and Interactions with Proteins

人类DNA测序的完成给基因组功能的结构方面带来了新的重点。在后基因组时代，了解基因调控的分子机制（通常通过蛋白质-核酸相互作用实现）变得更加重要。在大多数情况下，调节性核蛋白复合物的结构分析需要使用非常规的方法，因为它们对于核磁共振和x射线晶体学来说太大了。下面提出的项目旨在将我们最近开发的新方法应用于三个具体问题：原核抑制体的组装；转录延伸复合物的稳定与调控p53与DNA的序列特异性结合。

Completion of the human DNA sequencing brings new emphasis on the structural aspects of the genome functioning. In the post-genomic era, it becomes more important to understand the molecular mechanisms of gene regulation (usually achieved through the protein-nucleic acid interactions). In most cases, the structural analysis of the regulatory nucleoprotein complexes requires usage of unconventional approaches, as they are too large for NMR and X-ray crystallography. The project presented below is aimed at application of the new methods, developed by us recently, to three specific problems: assembly of prokaryotic repressosomes; stabilization and regulation of the transription elongation complexes; sequence-specific binding of p53 to DNA. (1) DNA looping in prokaryotes Stabilization of the multi-subunit protein-DNA complexes is facilitated by DNA looping, which brings the proteins close to each other. One of the best characterized is the gal loop in E. coli. To visualize such a complex structure, we used an innovative approach. First, genetic (sequence variability) data were used to determine the overall orientation of the gal repressors in a tetrameric structure. Next, the empirically based computer modeling developed by us earlier was employed to determine the optimal trajectory of the DNA loop. In particular, we found that the antiparallel gal loop is energetically more favorable than the parallel one. The same trend was found for the DNA loop formed upon binding of the lac repressors to DNA. The atomic force microscopy data support the antiparallel DNA looping, both with the galand lac repressors (in preparation). These results imply that the antiparallel DNA looping may be a general feature of the condensed bacterial nucleoid, as opposed to the parallel DNA wrapping around histones in eukaryotic chromatin. The hypothetical regular DNA folding in prokaryotes is consistent with the periodic distribution of the curved A-tracts in bacterial genomes, observed by us recently. Based on these findings, we designed detailed experiments to elucidate further the 3D organization of the DNA loops in bacteria (in collaboration with S. Adhya, NCI). (2) Elongation transcription complexes Another new method, enabling visualization of the DNA and RNA strands in the nucleoprotein complexes, is the iodine-125 radioprobing. This is based on analysis of the DNA strand breaks produced by the decay of an electron-emitting radioisotope, 125 I, incorporated in the C5 position of cytosine. The weaker the DNA break, the larger the distance from the radioisotope to the cleavage site. In this way, it is possible to reconstruct the 3D trajectories of nucleic acids in large nucleoprotein complexes. Using radioprobing, we detected for the first time the DNA-RNA heteroduplex outside the polymerase (in collaboration with R. Neumann and I. Panyutin, Clinical Center, NIH). Among other applications, this result suggests a novel mechanism for stabilization and regulation of the elongation complex. In addition to radioprobing and computer modeling, now we are studying the transcription complexes with time-resolved fluorescence, to visualize all the three DNA/RNA strands in real time. Our preliminary data imply different conformational behavior of the DNA strands during initiation and elongation (in collaboration with T. Jovin, MPI, Goettingen, Germany). We anticipate that this combined approach will give us a better understanding of those molecular mechanisms, which are responsible for the fidelity of RNA synthesis and are involved in recognition of the DNA mismatches in the course of transcription. (3) Sequence-specific p53-DNA binding The tetrameric p53-DNA complex plays the key role in tumor suppression. Therefore, it is important to elucidate the p53-DNA complex structure, as this could lead to understanding how formation of the tetrameric complex modifies binding sites on the p53 surface (which, in turn, serve as the signals for other members of the multi-protein cascade involved in the tumor supression pathway). Our early gel electrophoresis data revealed the DNA bending and increase in its twisting upon binding to p53, although the stereochemical details remained unclear. To determine the precise locations of the DNA bending and twisting in the tetrameric p53-DNA complex, we used the iodine-125 radioprobing described above. The DNA bending into the major groove was detected in the CATG tetramers in the centers of the two 10-mers (half-sites of the consensus 20-mer). Importantly, the internal CA:TG dimers (located closer to the center of the 20-mer) were distorted stronger than the external CA:TG (located closer to the ends of the p53 binding sites). This finding is consistent with the sequence variability observed for the known p53 response elements (in preparation). A next step is to analyze the effects of the p53 mutations and substitutions at the DNA response elements on the conformation and stability of the p53-DNA complex. Availability of the human DNA sequence allows determination of all potential p53 binding sites in the genome. We observed highly nonrandom distribution of such sites in all human chromosomes, reflecting the lateral p53 binding to the DNA loops, consistent with our electrophoresis measurements. We are currently exploring localization of the p53 sites with respect to the starts of transcription. The observed genome-wide distribution of the p53 sites also suggests further experiments regarding the p53 binding to nucleosomal DNA, and its possible role in gene regulation (in progress). The expansion of structural databases for nucleic acids, proteins and nucleoprotein complexes, as well as continued progress in elucidating the sequence-dependent structural properties of DNA and RNA, will improve the accuracy with which large nucleoprotein complexes can be characterized by the methods described above.