Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching
<p>Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexit...
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| Formato: | Tesis |
| Lenguaje: | English |
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2013
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| _version_ | 1826297857552941056 |
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| author | Uphoff, S |
| author2 | Kapanidis, A |
| author_facet | Kapanidis, A Uphoff, S |
| author_sort | Uphoff, S |
| collection | OXFORD |
| description | <p>Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexity of molecular biology demands new experimental concepts that resolve the structural and functional diversity of biomolecules.</p> <p>In this thesis, I describe fluorescence methods that give a direct view on protein-DNA interactions at the single-molecule level. These methods employ photoswitching to control the number of active fluorophores in the sample. Forster Resonance Energy Transfer (FRET) measures the distance between a donor and an acceptor fluorophore to report on biomolecular structure and dynamics in vitro. Because a single distance gives only limited structural information, I developed "switchable FRET" that employs photoswitching to sequentially probe multiple FRET pairs per molecule. Switchable FRET resolved two distances within static and dynamic DNA constructs and protein-DNA complexes. Towards application of switchable FRET, I investigated aspects of the nucleotide selection mechanism of DNA polymerase.</p> <p>I further explored application of single-molecule imaging in the complex environment of the living cell. Photoswitching was used to resolve the precise localisations of individual fluorophores. I constructed a super-resolution fluorescence microscope to image fixed cellular structures and track the movement of individual fluorescent fusion proteins in live bacteria. I applied the method to directly visualise DNA repair processes by DNA polymerase I and ligase, generating a quantitative account of their repair rates, search times, copy numbers, and spatial distribution in the cell. I validated the approach by tracking diffusion of replisome components and their association with the replication fork. Finally, super-resolution microscopy showed dense clusters of SMC (Structural Maintenance of Chromosomes) protein complexes in vivo that have previously been hidden by the limited resolution of conventional microscopy.</p> |
| first_indexed | 2024-03-07T04:38:01Z |
| format | Thesis |
| id | oxford-uuid:d0a52864-6d26-44a4-8fb7-5d12624a04ba |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:38:01Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:d0a52864-6d26-44a4-8fb7-5d12624a04ba2022-03-27T07:51:25ZStudying protein-DNA interactions in vitro and in vivo using single-molecule photoswitchingThesishttp://purl.org/coar/resource_type/c_db06uuid:d0a52864-6d26-44a4-8fb7-5d12624a04baBiophysicsEnglishOxford University Research Archive - Valet2013Uphoff, SKapanidis, A<p>Protein-DNA interactions govern the fundamental cellular processes of DNA replication, transcription, repair, and chromosome organisation. Despite their importance, the detailed molecular mechanisms of protein-DNA interactions and their organisation in the cell remain elusive. The complexity of molecular biology demands new experimental concepts that resolve the structural and functional diversity of biomolecules.</p> <p>In this thesis, I describe fluorescence methods that give a direct view on protein-DNA interactions at the single-molecule level. These methods employ photoswitching to control the number of active fluorophores in the sample. Forster Resonance Energy Transfer (FRET) measures the distance between a donor and an acceptor fluorophore to report on biomolecular structure and dynamics in vitro. Because a single distance gives only limited structural information, I developed "switchable FRET" that employs photoswitching to sequentially probe multiple FRET pairs per molecule. Switchable FRET resolved two distances within static and dynamic DNA constructs and protein-DNA complexes. Towards application of switchable FRET, I investigated aspects of the nucleotide selection mechanism of DNA polymerase.</p> <p>I further explored application of single-molecule imaging in the complex environment of the living cell. Photoswitching was used to resolve the precise localisations of individual fluorophores. I constructed a super-resolution fluorescence microscope to image fixed cellular structures and track the movement of individual fluorescent fusion proteins in live bacteria. I applied the method to directly visualise DNA repair processes by DNA polymerase I and ligase, generating a quantitative account of their repair rates, search times, copy numbers, and spatial distribution in the cell. I validated the approach by tracking diffusion of replisome components and their association with the replication fork. Finally, super-resolution microscopy showed dense clusters of SMC (Structural Maintenance of Chromosomes) protein complexes in vivo that have previously been hidden by the limited resolution of conventional microscopy.</p> |
| spellingShingle | Biophysics Uphoff, S Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching |
| title | Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching |
| title_full | Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching |
| title_fullStr | Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching |
| title_full_unstemmed | Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching |
| title_short | Studying protein-DNA interactions in vitro and in vivo using single-molecule photoswitching |
| title_sort | studying protein dna interactions in vitro and in vivo using single molecule photoswitching |
| topic | Biophysics |
| work_keys_str_mv | AT uphoffs studyingproteindnainteractionsinvitroandinvivousingsinglemoleculephotoswitching |