Regulation of eukaryotic DNA replication: genome architecture and polymerase switching
<p>Eukaryotic DNA replication is tightly regulated, requiring the coordinated action of many different cellular processes and proteins to facilitate duplication of the entire genomic template.</p> <p>This work investigates two different aspects of this coordination: the timing re...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2021
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| _version_ | 1826302520686804992 |
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| author | Oldach, P |
| author2 | Muller, C |
| author_facet | Muller, C Oldach, P |
| author_sort | Oldach, P |
| collection | OXFORD |
| description | <p>Eukaryotic DNA replication is tightly regulated, requiring the coordinated action of many different cellular processes and proteins to facilitate duplication of the entire genomic template.</p>
<p>This work investigates two different aspects of this coordination: the timing regulation that leads some genomic loci to replicate early in S phase and some late, and the enzymatic regulation that allows nascent strand synthesis to be carried out in an efficient and error-minimizing way by one of a family of distinct DNA polymerases.</p>
<p>First, the role of 3D genome architecture in human cell replication timing is analyzed with the use of a system for inducible cohesin degradation. Loss of cohesin has been shown to drive loss of topologically associating domain (TAD) structure across the genome. Thus, high throughput sequencing and microscopy were used to assess replication timing differences between wildtype and cohesin- ablated S phase conditions. Neither acute S phase cohesin ablation nor sustained ablation from G1 through S phase resulted in genome-wide changes to replication timing domains (RD). Furthermore, specific genomic loci hypothesized to show a replication timing response to cohesin loss – such as TAD boundaries, CTCF binding sites, and genes that showed differential expression in the absence of cohesin – showed no significant changes. Thus, it was concluded that cohesin is necessary for neither the establishment nor execution of replication timing domains.</p>
<p>Second, the potential for switching during ongoing leading strand synthesis by polymerase epsilon is investigated in budding yeast. A system was developed to incorporate (1) an inducible and tagged polymerase epsilon Pol2 subunit in addition to the endogenous Pol2 subunit, (2) a long replicon free of licensed replication origins, (3) robust cell cycle synchronization, and (4) a quantitative ChIP-Seq method for detection of the induced and endogenous Pol2 subunits on chromatin. Preliminary experimentation utilizing this system suggest an absence of rapid and widespread polymerase epsilon switching during ongoing replication. This opens the door for future experiments assessing polymerase switching in response to replication stress and obstacles such as UV-induced DNA damage.</p> |
| first_indexed | 2024-03-07T05:48:48Z |
| format | Thesis |
| id | oxford-uuid:e820f21f-e989-4fb9-ac23-7a6f6481cf52 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:48:48Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | oxford-uuid:e820f21f-e989-4fb9-ac23-7a6f6481cf522022-03-27T10:44:26ZRegulation of eukaryotic DNA replication: genome architecture and polymerase switchingThesishttp://purl.org/coar/resource_type/c_db06uuid:e820f21f-e989-4fb9-ac23-7a6f6481cf52DNA replicationEnglishHyrax Deposit2021Oldach, PMuller, CRaff, JNorbury, C <p>Eukaryotic DNA replication is tightly regulated, requiring the coordinated action of many different cellular processes and proteins to facilitate duplication of the entire genomic template.</p> <p>This work investigates two different aspects of this coordination: the timing regulation that leads some genomic loci to replicate early in S phase and some late, and the enzymatic regulation that allows nascent strand synthesis to be carried out in an efficient and error-minimizing way by one of a family of distinct DNA polymerases.</p> <p>First, the role of 3D genome architecture in human cell replication timing is analyzed with the use of a system for inducible cohesin degradation. Loss of cohesin has been shown to drive loss of topologically associating domain (TAD) structure across the genome. Thus, high throughput sequencing and microscopy were used to assess replication timing differences between wildtype and cohesin- ablated S phase conditions. Neither acute S phase cohesin ablation nor sustained ablation from G1 through S phase resulted in genome-wide changes to replication timing domains (RD). Furthermore, specific genomic loci hypothesized to show a replication timing response to cohesin loss – such as TAD boundaries, CTCF binding sites, and genes that showed differential expression in the absence of cohesin – showed no significant changes. Thus, it was concluded that cohesin is necessary for neither the establishment nor execution of replication timing domains.</p> <p>Second, the potential for switching during ongoing leading strand synthesis by polymerase epsilon is investigated in budding yeast. A system was developed to incorporate (1) an inducible and tagged polymerase epsilon Pol2 subunit in addition to the endogenous Pol2 subunit, (2) a long replicon free of licensed replication origins, (3) robust cell cycle synchronization, and (4) a quantitative ChIP-Seq method for detection of the induced and endogenous Pol2 subunits on chromatin. Preliminary experimentation utilizing this system suggest an absence of rapid and widespread polymerase epsilon switching during ongoing replication. This opens the door for future experiments assessing polymerase switching in response to replication stress and obstacles such as UV-induced DNA damage.</p> |
| spellingShingle | DNA replication Oldach, P Regulation of eukaryotic DNA replication: genome architecture and polymerase switching |
| title | Regulation of eukaryotic DNA replication: genome architecture and polymerase switching |
| title_full | Regulation of eukaryotic DNA replication: genome architecture and polymerase switching |
| title_fullStr | Regulation of eukaryotic DNA replication: genome architecture and polymerase switching |
| title_full_unstemmed | Regulation of eukaryotic DNA replication: genome architecture and polymerase switching |
| title_short | Regulation of eukaryotic DNA replication: genome architecture and polymerase switching |
| title_sort | regulation of eukaryotic dna replication genome architecture and polymerase switching |
| topic | DNA replication |
| work_keys_str_mv | AT oldachp regulationofeukaryoticdnareplicationgenomearchitectureandpolymeraseswitching |