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Analysis of the mechanism of recruitment of the helicase Sen1 at the replisome and its role in the maintenance of genomic stability
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Lones, Emma (2020) Analysis of the mechanism of recruitment of the helicase Sen1 at the replisome and its role in the maintenance of genomic stability. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3520012
Abstract
During the S phase of the cell cycle, the processes of RNA transcription and DNA replication occur on the same template DNA; thus, can interfere with one another. In addition, co-transcriptional DNA:RNA hybrids (R loops) can impede processive DNA replication. When these obstacles are not removed, clashes with replication forks can result in their stalling, affecting genome stability.
The highly conserved DNA:RNA helicase Sen1, required for transcription termination and R loops resolution, travels with replisomes via an interaction with Ctf4 and Mrc1. I have analysed the role of Sen1 at replication forks in S. cerevisiae using a novel separation of function mutant, sen1-3; which no longer binds replisomes, but is fully competent in its transcription termination function. sen1-3 is synthetic lethal combined with deletion of the RNase H enzymes, which digest R loops; and displays synthetic defects combined with the RNA metabolism mutant hpr1D, which are suppressed by RNase H overexpression. This suggests one function of Sen1 at forks may be to remove R loops that they encounter. I have also shown sen1-3 is synthetic defective with the S phase checkpoint mutants mrc1D, ctf18D and rad53D, however the growth defects and increased recombination are not sensitive to RNase H. This indicates Sen1 may also remove other toxic obstacles encountered by replisomes.
Additionally, I have explored further the biochemistry of Sen1 association at forks. While full length Sen1 binding to its replisome partners is restricted to S phase, the Nterminal domain can interact with Mrc1 and Ctf4 throughout the cell cycle. I have identified that the interaction appears to be regulated by S phase cells extracts but does not depend on phosphorylation. I have also observed that the C-terminal of Sen1 interacts with DNA polymerase epsilon outside of the context of the replisome, independently from the cell cycle.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology |
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Library of Congress Subject Headings (LCSH): | DNA helicases, Cell cycle, Genetic transcription, DNA replication | ||||
Official Date: | September 2020 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | De Piccoli, Giacomo ; Gambus, Aga | ||||
Sponsors: | Midlands Integrative Biosciences Training Partnership ; Biotechnology and Biological Sciences Research Council (Great Britain) | ||||
Format of File: | |||||
Extent: | various pagings : illustrations, charts | ||||
Language: | eng |
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