| Autor: |
Niels Laurens, Stephen E. Halford, Yana S. Kovacheva, Gijs J.L. Wuite, August F. Harms, Stuart R.W. Bellamy, Lucy E. Catto, Christian Pernstich |
| Jazyk: |
angličtina |
| Předmět: |
|
| Zdroj: |
Biophysical Journal. (3):366a |
| ISSN: |
0006-3495 |
| DOI: |
10.1016/j.bpj.2008.12.1971 |
| Popis: |
Protein induced DNA looping is a key regulatory mechanism involved in important processes such as gene regulation, DNA-transcription and -replication. The relation between the induced loop topology and DNA-protein dynamics is essential for understanding these processes. Bending and twisting rigidities of DNA are shown to have a profound influence on the formation and stability of these loops. We used FokI, a restriction enzyme that binds two asymmetrical recognition sites enhancing its specificity, as our model system. Controlling the orientation of both binding sites enabled us to explore the impact of the physical properties of DNA by inducing different loop topologies and measuring the resulting changes in DNA-protein dynamics.The looping behavior is quantified using a tethered particle assay. With this assay we obtained the kinetics of protein induced loop formation with a single measurement by tracking up to 50 DNA tethers in parallel. The dwell times are extracted and compared using both a running average method and a hidden Markov analysis.We used DNA substrates with a range of different spacing's between the two asymmetric recognition sequences. In addition we varied the orientation of these recognition sites and sampled how binding and loop formation is influenced by these different topologies. We show that both, the separation and orientation of the two recognition sites have a profound influence on the formation and stability of these looped DNA-protein structures. The results are understood and modeled in terms of the helical pitch and the bending energy involved in protein induced loop formation. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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