Book of Abstracts: Albany 2003
June 17-21 2003
Resolution of Undistorted Symmetric Immobile DNA Junctions by Vaccinia Topoisomerase I
Holliday junctions are central intermediates in genetic recombination. They consist of four strands of DNA that flank a branch point. In natural systems, their sequences have twofold (homologous) sequence symmetry. This sequence symmetry enables the molecules to undergo an isomerization, known as branch migration, that relocates the site of the branch point. Branch migration can lead to polydispersity which makes it difficult to characterize both Holliday junction substrate behavior and the physical properties that arise from the sequence flanking the branch point. Previous studies have reported two symmetric immobile junctions: One that is immobilized by coupling to an asymmetric junction in a double helical context (1), and a second that is based on molecules containing 5', 5' and 3', 3' linkages (2). Both are flawed by distorting the structure of the symmetric junction from its natural conformation. Here, we report an undistorted symmetric immobile junction based on the use of DNA parallelogram structures. In the drawing below, I represents immobile junctions and SI represents symmetric immobile. The immobile junctions hold the undistorted symmetric immobile junction in its designated position.
The crossover points are separated by one and a half turns of double helical DNA. We have demonstrated by hydroxyl radical autofootprinting that the system is immobile. We have used a series of these junctions to characterize the preferred site at which Vaccinia virus topoisomerase I recombines symmetric Holliday junctions. We have moved the crossover position over five different sites in the vicinity of the symmetric immobile junction, on both the crossover and the non-crossover (helical) strands. We find that the preferred position is two nucleotides 5' to the branch point on the helical strand. Covalent intermediates appear to be precursors to recombinant products in all reactions; the ratios of intermediates to products shows an inverse qualitative relationship with reaction rates and product accumulation. The data suggest that the intermediates in the least productive complexes are poorly formed, or perhaps are formed only on one strand.
This research supported by NIGMS, ONR, DARPA/AFOSR, and NSF.
1Department of Chemistry