DNA palindromes are implicated in several examples of gross chromosomal aberrations in the human genome, however, the molecular mechanism(s) that govern palindrome instability are largely under-investigated. Because of their propensity for intrastrand base pairing, it is suspected that the acquisition of a secondary structure, such
as a hairpin or cruciform, instigates the rearrangement process. A significant hurdle in defining palindrome-provoked instability lies in the fact that reliable methods for
examining in vivo cruciform extrusion remain underdeveloped. A challenge is to provide
straightforward evidence for cruciform extrusion in eukaryotic cells. Here, I present a plasmid system for use in Saccharomyces cerevisiae that enables for the detection of cruciforms in vivo. Cruciform extrusion, of either an in vitro-prepared palindrome or a near-palindrome from the human genome, is monitored by scoring for the product of cruciform resolution, being a dually hairpin-capped linear DNA molecule. These results not only provide evidence for the occurrence of cruciform extrusion in eukaryotic
chromatin, they also identify a novel source of endogenous double strand break formation.
A screen for candidate genes that are required for resolution revealed that the
Mus81 Endonuclease, a candidate Holliday junction resolvase, provides the majority of
cruciform resolution activity in mitotic cells, validating the notion that cellular HJ
resolvases can misrecognize a cruciform for a Holliday junction. A second screen
identified a requirement for the Sgs1-Top3-Rmi1 complex in the prevention of double
strand break formation, including cruciform resolution, of DNA palindromes. These
results uncover a new role for the RecQ helicase in prevention of palindrome-provoked
instability, possibly through the intrusion of cruciform structures. Together, this work
contributes significantly to our understanding of cruciform metabolism in eukaryotes and supports suggestions that cruciform extrusion instigates instability in the human genome.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/17746 |
| Date | 23 September 2009 |
| Creators | Cote, Atina |
| Contributors | Lewis, Susanna |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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