Graduate Thesis Or Dissertation
 

Sequence dependent conformational variations in DNA holliday junctions

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  • Four-stranded DNA junctions (also known as Holliday junctions) are structural intermediates involved in a growing number of biological processes including DNA repair, genetic recombination, and viral integration. Although previous studies have focused on understanding the conformational variability and sequence-dependent formation of Holliday junctions in solution there have been relatively few insights into junction structure at the atomic level. Recent crystallographic studies have demonstrated that the more compact stacked-X junction form has an antiparallel alignment of DNA strands and standard Watson-Crick base pairs across the central crossover region. Junction formation within this crystallographic system was seen to be dependent on a common trinucleotide sequence motif ("ACC-triplet" at the 6th, 7th and 8th positions of the decanucleotide sequence d(CCnnnN₆N₇N₈GG)) containing a series of stabilizing direct and solvent-mediated hydrogen bonding interactions. This thesis addresses questions concerning the nucleotide sequence-dependent formation and conformational variability of DNA Holliday junctions as determined by single crystal x-ray diffraction. We have used the modified bases 2,6-diaminopurine and inosine to demonstrate that minor groove interactions adjacent to the trinucleotide junction core are not major contributors to overall conformation. In addition, incorporation of guanine into the sixth position of this core does not have a significant effect on junction geometry. Meanwhile, incorporation of 5-bromouracil into the eighth position perturbs the geometry in terms of the interduplex angle as well as the defined conformational variables, J[subscript roll] and J[subscript slide]. These novel junction structures demonstrate that the nucleotide sequence within the central core generates a position specific relationship between molecular interactions at the junction crossover and overall structural geometry. A systematic crystallographic screen of the trinucleotide core region is presented here as an unbiased, comprehensive, search for sequences that stabilize junctions. As the result of this screen, we can extend the core sequence motif to 'N₆Y₇C₈' where N₆ is an adenine, guanine, or cytosine nucleotide and Y₇ is either a cytosine or thymine (if N₆ = adenine) nucleotide. Using these novel junction structures, we demonstrate that base sequence within the central core has a significant effect on the overall geometry of the junction. Thus, this central region of the structure may serve as a linchpin for determining the local and global conformation and overall variability of the four-stranded DNA Holliday junction. These observations raise some interesting questions regarding the importance of this core region in biological processes such as genetic recombination.
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