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The flexibility of locally melted DNA.

ABSTRACT: Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We have expanded this model to incorporate sequence and temperature dependence of the local melting, and tested it for three sequences at temperatures from 23 degrees C to 42 degrees C. We find that small melted bubbles are significantly more flexible than double-stranded DNA and can alter DNA flexibility at physiological temperatures. However, these bubbles are not flexible enough to explain the recently observed very sharp bends in DNA.

SUBMITTER: Forties RA

PROVIDER: S-EPMC2724272 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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The flexibility of locally melted DNA.

Forties Robert A RA Bundschuh Ralf R Poirier Michael G MG

Nucleic acids research 20090531 14

Protein-bound duplex DNA is often bent or kinked. Yet, quantification of intrinsic DNA bending that might lead to such protein interactions remains enigmatic. DNA cyclization experiments have indicated that DNA may form sharp bends more easily than predicted by the established worm-like chain (WLC) model. One proposed explanation suggests that local melting of a few base pairs introduces flexible hinges. We have expanded this model to incorporate sequence and temperature dependence of the local ...[more]

PMID: 19487242

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Project description:Prokaryotic Argonaute proteins (pAgos) widely participate in hosts to defend against the invasion of nucleic acids. Compared with the CRISPR-Cas system, which requires a specific motif on the target and can only use RNA as guide, pAgos exhibit precise endonuclease activity on any arbitrary target sequence and can use both RNA and DNA as guide, thus rendering great potential for genome editing applications. Hitherto, most in-depth studies on the structure-function relationship of pAgos were conducted on thermophilic ones, functioning at ∼60 to 100°C, whose structures were, however, determined experimentally at much lower temperatures (20-33°C). It remains unclear whether these low-temperature structures can represent the true conformations of the thermophilic pAgos under their physiological conditions. The present work studied three pAgos, PfAgo, TtAgo and CbAgo, whose physiological temperatures differ significantly (95, 75 and 37°C). By conducting thorough experimental and simulation studies, we found that thermophilic pAgos (PfAgo and TtAgo) adopt a loosely-packed structure with a partially-melted surface at the physiological temperatures, largely different from the compact crystalline structures determined at moderate temperatures. In contrast, the mesophilic pAgo (CbAgo) assumes a compact crystalline structure at its optimal function temperature. Such a partially-disrupted structure endows thermophilic pAgos with great flexibility both globally and locally at the catalytic sites, which is crucial for them to achieve high DNA-cleavage activity. To further prove this, we incubated thermophilic pAgos with urea to purposely disrupt their structures, and the resulting cleavage activity was significantly enhanced below the physiological temperature, even at human body temperature. Further testing of many thermophilic Agos present in various thermophilic prokaryotes demonstrated that their structures are generally disrupted under physiological conditions. Therefore, our findings suggest that the highly dynamical structure with a partially-melted surface, distinct from the low-temperature crystalline structure, could be a general strategy assumed by thermophilic pAgos to achieve the high DNA-cleavage activity.

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The flexibility of locally melted DNA.

Publications

The flexibility of locally melted DNA.

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