LOCAL AND GLOBAL STRUCTURES OF SUPERCOILED DNA

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Description:(Verbatim from the applicant's abstract) DNA supercoiling plays a critical role in virtually all genetic processes, yet our understanding of the relationship between the structure of supercoiled DNA and its specific roles in biology is still rather limited. One important feature of supercoiling is that it dramatically increases the probability of interaction of distantly separated DNA segments. Many genetic events require communication between proteins bound to distant sites on DNA. Examples include DNA replication, control of gene expression, site-specific recombination and other genome rearrangements. Another fundamental property of supercoiled DNA is that negative supercoiling facilitates local structural transitions in DNA. Certain short DNA regions can adopt alternative conformations and, in some cases specific enzymes or regulatory proteins target these regions to exert their regulatory effects. This grant will test the idea that the local and global structures of supercoiled DNA are linked, such that local structural transitions can have a significant impact on the overall geometry or topography of supercoiled DNA. One hypothesis to be tested is that local alternative conformations can define the shape of DNA molecule in a way that may effect the juxtaposition of two sites along a DNA molecule. We will test the influence of the formation of cruciforms, intramolecular triplex DNA (H-DNA), and left-handed Z-DNA on plasmid topography. We will also determine if proteins that have a strong affinity for alternative structures, including the Z-alpha domain from a human RNA editing enzyme, participate in changing of the overall geometry of the molecule. In addition, we will test a new biological idea - that is, we will test the hypothesis that the topographical or positional localization of a DNA sequence within a superhelix can modulate DNA structural transitions. This is an important and novel concept in that it provides another level of regulation of DNA secondary structural transitions that are clearly involved in biological processes. This will be determined by positioning inverted repeats at an apical or non-apical position in a plasmid. The studies outlined above will utilize atomic force microscopy (AFM), 2-dimensional agarose gel electrophoresis, and chemical probe analysis for structural studies of DNA and nucleoprotein complexes.