Physical Chemistry of Recombinational intermediates

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Recombination is a universal process in all species that affords the flexibility necessary for adaptation to a changing environment. We seek to construct and characterize synthetic nucleic acid model systems that mimic structures involved in recombination, particularly the Holliday junction, which is a four-stranded branched intermediate. In the presence of Mg2+, the Holliday junction forms a stacked-X structure, and in its absence its structure is open; both structures appear relevant to cellular processes. We propose to examine both the individual Holliday junction and complex motifs composed of several Holliday junctions to determine features of the structure, its transformations and its resolution. We will also characterize a protein-nucleic acid interaction using a Holliday-derived construct. The specific aims are: [1] To use parallelograms made from four Holliday junctions as tools to characterize Holliday junctions: We aim to examine the swivel angles between the helices in the stacked-X structure. We will use a parallelogram-based system to make undistorted symmetric immobile junctions, that fix their branch points; this system will enable us to characterize both the thermodynamics and resolution of junctions with otherwise mobile homologous sequences. Parallelograms also offer a window on the mechanical potential of the swivel angles. [2] We will follow the transition between the open and stacked structures by means of synchrotron-generated hydroxyl radicals. [3] We plan to use ligation-closure experiments to characterize quantitatively the flexibility of Holliday junctions and double crossover molecules, a key factor in their functioning. [4] A new motif, paranemic crossover DNA has properties that suggest it may act as an intermediate in homology recognition in homologous recombination. We will establish whether its formation can be driven by supercoiling, and we will use the molecule as a system to measure the free energy difference between a parallel crossover structure and two parallel juxtaposed helices. [5] We will use a Holliday-derived system to test the unwinding of SoxR regulatory protein when it binds to DNA. These studies are designed to test key hypotheses and make central observations on DNA structures and processes that are involved in genetic recombination.