This research program is concerned with unusual structures of DNA and their role in biological processes, such as recombination or repair. For many years, we have bulit unusual DNA motifs that have been used to characterize the physical chemistry of Holliday junctions. A generalization of the Holliday junction is a structure known as PX DNA. This is an inter-wrapped structure of two double helices. A prediction of this structure, is that the presence of homology in a supercoiled plasmid should lead to the relaxation of the supercoiling. During the current project period, we have verified this prediction, both by 2D gel electrophoresis and by atomic force microscopy. We have also shown that PX-homology, a lower extent of homology consistent with the PX structure, produces the same effect. In this application, we propose experiments to characterize this effect further, and to establish whether it is indeed due to a PX structure.
The specific aims i nvolving this phenomenon are: to (i) establish the parameters that affect the relaxation;(ii.1) to complete the data on the PX homology;(ii.2) to extablish whether the structure entails a wrapping of the two homologous double helices around each other;(ii.3) to obtain independent structural data that establish the nature of the homology strutcure;(iii.1) to perform crosslinking experiments within bacterial cells to determine whether this effect occurs in vivo;(iii.2) to seek proteins that interact with the structure;and to determine whether PX homology can function as regular homology in recombination. Another specific aim involves using a DNA device, developed during the current project period, that measures the load against which a DNA-distorting protein can work when it binds to DNA. We will perform these experiments with the various mis-paired sites recognized by the MutS DNA repair protein. In a third aim, we will develop a series of different potential substrates from DNA parallelogram-based structures, that will be tested as possible substrates for human and yeast topoisomerase II.
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