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Please use this identifier to cite or link to this item: http://hdl.handle.net/1807/17746

Title: In Vivo Analysis of Cruciform Extrusion and Resolution of DNA Palindromes in Eukaryotes
Authors: Cote, Atina
Advisor: Lewis, Susanna
Department: Molecular and Medical Genetics
Issue Date: 23-Sep-2009
Abstract: 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.
URI: http://hdl.handle.net/1807/17746
Appears in Collections:Doctoral
Department of Molecular Genetics - Doctoral theses

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