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Dissecting protein-induced DNA looping dynamics in real time.

ABSTRACT: Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA-protein interactions. In addition, it permits many single-molecule experiments to be performed in parallel. Using as a model system the tetrameric Type II restriction enzyme SfiI, that binds two copies of its recognition site, we show here that we can determine the DNA-protein association and dissociation steps as well as the actual process of protein-induced loop capture and release on a single DNA molecule. The result of these experiments is a quantitative reaction scheme for DNA looping by SfiI that is rigorously compared to detailed biochemical studies of SfiI looping dynamics. We also present novel methods for data analysis and compare and discuss these with existing methods. The general applicability of the introduced techniques will further enhance tethered particle motion as a tool to follow DNA-protein dynamics in real time.

SUBMITTER: Laurens N

PROVIDER: S-EPMC2760800 | biostudies-other | 2009 Sep

REPOSITORIES: biostudies-other

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Dissecting protein-induced DNA looping dynamics in real time.

Laurens Niels N Bellamy Stuart R W SR Harms August F AF Kovacheva Yana S YS Halford Stephen E SE Wuite Gijs J L GJ

Nucleic acids research 20090708 16

Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA-protein interactions. In addition, it permits many single-molecule experiments to be per ...[more]

PMID: 19586932

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Project description:Reoviruses are non-enveloped viruses with wide host range, can cause serious infections in animals, plants and microorganism, e.g., aquareovirus, which is capable of causing serious haemorrhagic in aquatic animals. To date, the entry process of aquareovirus infection remains obscure. Real-time single-virus tracking are effective tools for exploring the details in viral infection process, which are crucial for understanding the pathogenic mechanism. Here, we used quantum dots-based single particle tracking technology combined with biochemical assays and ultrastructural observation to reveal unobservable infection steps and map dynamic interactions between a reovirus, Scophthalmus maximus reovirus (SMReV), and its host cell in real time. The results showed that the single membrane-bound reovirus particle can enter into the cell within several seconds through nascent clathrin-caoted pits, and most of the particles could internalize into cytoplasm within 30 min post-infection. The specific inhibitors analysis also showed that entry of SMREV depended on clathrin-mediated endocytosis rather than cavolin-mediated endocytosis. The motion analysis of internalized single particle indicated that the reovirus initially experienced slow and directed motion in the actin-enriched cell periphery, while it underwent relatively faster and directed movement toward the cell interior, suggesting that transport of SMReV was dependent on the cytoskeleton. Further, dual-labeling of virus and cytoskeleton and inhibitor analysis both demonstrated that transport of internalized SMReV was firstly dependent on actin filaments at the cell periphery, and then on microtubules toward the cell interior. Then visualization of SMReV trafficking in the endosomes revealed that the internalized reovirus particles were sorted from early endosomes to late endosomes, then part of them were delivered to lysosome. This study for the first time revealed the entry pathway, intracellular dynamic and the infection fate of fish reovirus in host cell in real time and in situ, which provided new insights into the infection mechanism of non-enveloped viruses.

Dataset Information

Dissecting protein-induced DNA looping dynamics in real time.

Publications

Dissecting protein-induced DNA looping dynamics in real time.

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