Graduate Thesis Or Dissertation

Crystal structures of DNA four-way junctions

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  • DNA four-way junctions (also known as Holliday junctions) are the primary structural intermediate during recombination, an important process responsible for biological evolution and maintenance of genomes. These junctions arise from the assembly of four nucleic acid strands to produce double-helical regions extending from a central point. Although much progress has been made in understanding the general structural and functional properties of four-way junctions, we are only now beginning to elucidate the atomic interactions that stabilize such structures. This thesis deals with the sequence-and drug-dependent formation of Holliday junctions at the atomic level, as determined by single-crystal x-ray diffraction. The first crystal structure of a DNA four-way junction from an inverted repeat sequence is described here. The conformation of the junction corresponds to that of the stacked-X form previously characterized in solution, in which helical arms are coaxially stacked to form two pseudo-continuous duplexes related in a right-handed sense. The junction structure is held rigidly in place by a sequence-dependent network of hydrogen bonds at the point of strand exchange, and the relative geometries of the arms are influenced by contacts further removed from the junction. Comparison of this structure with a nearly identical nucleotide sequence but containing base mismatches details both the effect of non-standard base pairing on the junction and a general structural motif in a consensus ACC-triplet that forms the core of a stable junction. We also describe the first atomic resolution structures of oligonucleotides crosslinked with the photochemotheraputic agent psoralen, a drug used in the clinical treatment of skin disorders and one of the oldest known natural products. We find that the psoralen cross-link can induce the formation of a four-way junction in the crystal by destabilizing the duplex at the six-membered pyrone ring of the drug. These structures also further detail the nature of the junction seen in the uncross-linked sequence. In total, the structures presented in this thesis shed light on a number of important issues regarding the intrinsic stability of four-way junctions, such as branch migration and the sequence-dependent distribution of stacking conformers, as well as raise interesting questions regarding the role of recombination during repair of DNA lesions.
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