Investigating cis and trans factors involved in common fragile site breakage and healing using Flex1, a subregion of FRA16D
Kaushal, Simran.
2018
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Abstract: Common Fragile Sites (CFSs) are regions of DNA that display gaps and breaks in metaphase chromosomes under replication stress. While CFSs are not normally expressed, or broken, in individuals, conditions of replication stress result in their breakage. Evidence of such sites has been found in organisms ranging from yeast to humans. Further, CFSs are often sites of rearrangement in cancer ... read morecell lines, therefore understanding their expression is very relevant to human disease. CFSs are late replicating, and conditions of replication stress result in their under-replication at the point of chromatin condensation in the cell cycle. CFSs are enriched in DNA sequences that can form abnormal, or secondary, structures, which could also play a role in their breakage. There are several theories for the cause of CFS breakage, each with varying levels of support, and this is largely due to the difficulty of studying complicated DNA sequences in mammalian cells. Here, we studied Flex1, a roughly 300 bp subregion of CFS FRA16D in S. cerevisiae that has a perfect AT repeat that is highly polymorphic in humans and predicted to form stable secondary structures in vivo. Flex1 also stalls replication in an AT repeat length-dependent manner. Working in yeast allows precise genetic control of DNA sequences, making it an excellent model system to investigate current theories for CFS breakage. We have found that breakage at Flex1 is dependent on structure-specific endonuclease (SSE) complexes Mus81-Mms4, Slx1-Slx4, and Rad1-Rad10, similar to what has been found for human SSEs at FRA16D. Thus, Flex1 serves as a model system for studying breakage and healing at a CFS sequence. The cleavage of Flex1 by Mus81 is dependent on the formation of a secondary structure by the AT repeats. By comparing the effects of different Flex1 sequences on breakage and healing, we have evidence to propose a new theory for CFS expression: CFSs are not only prone to breakage but also impaired in their ability to heal following fragility. We propose that breakage in FRA16D is initiated by SSE cleavage at Flex1, followed by difficulty healing after fragility due to the propensity of adjacent sequences to form hairpins. We also discovered a role for the fork stabilization function of the Mrc1 (hClaspin protein) in protecting against fragility at Flex1, supporting an important link between fork stalling and fragility.
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Biology.
Advisor: Catherine Freudenreich.
Committee: Mitch McVey, Sergei Mirkin, and Sharon Cantor.
Keywords: Genetics, Molecular biology, and Biology.read less - ID:
- fj236f040
- Component ID:
- tufts:28610
- To Cite:
- TARC Citation Guide EndNote
