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. / Graduation date: 2005
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/29967 |
| Date | 14 April 2005 |
| Creators | Hays, Franklin A. |
| Contributors | Ho, Pui Shing |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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