期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 104, 期 31, 页码 12646-12650出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0610673104
关键词
RecA; single molecule; vesicle encapsulation
资金
- NIGMS NIH HHS [GM 074526, R01 GM065367, R21 GM074526] Funding Source: Medline
Vesicle encapsulation offers a biologically relevant environment for many soluble proteins and nucleic acids and an optimal immobilization medium for single-molecule fluorescence assays. Furthermore, the confinement of biomolecules within small volumes opens up new avenues to unique experimental configurations. Nevertheless, the vesicles' impermeability, even toward ions and other small molecules such as ATP, hinders more general applications. We therefore developed methods to induce pores into vesicles. Porous vesicles were then used to modulate the interaction between Escherichia coli RecA proteins and ssDNA by changing the extravesicular nucleotides. Repetitive binding and dissociation of the same RecA filament on the DNA was observed with a rebinding rate two orders of magnitude greater than in the absence of confinement, suggesting a previously unreported nucleation pathway for RecA filament. This method provides a biofriendly and simple alternative to surface tethering that is ideal for the study of transient and weakly interacting biological complexes.
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