Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

Significance of Knotted Structures for Function of Proteins and Nucleic Acids

Poster Session II

40 – POS Board 12 Detecting DNA Knotting by Strong Confinement and Action of Loop-inducing Proteins Maedeh Roushan, Zubair Azad, Parminder Kaur, Jianguo Lin, Hong Wang, Robert Riehn . North Carolina State University, Raleigh, NC, USA. DNA performs a carefully choreographed ballet during the cell cycle. The organization is driven by the specific binding of proteins to form tertiary DNA-protein-DNA complexes. The search process that precedes the formation must overcome the challenge of very low effective mobility of genomic-sized DNA pieces in the dense cellular environment, and potentially leads to complex topologies such as knots and loops. We have developed nanofluidic devices that gently elongate single DNA molecules through a lateral force without tethering the molecule or blocking the molecule ends. Nanochannel cross- sections are 100x100 nm2, and channels are hundreds of microns long. Because DNA is elongated through confinement, loops and knots with a length down to 2 kb can be directly observed in real time. Channels are made of fused silica, enabling single-molecule observation of both DNA and proteins. Because the effective concentration of DNA inside channels exceeds 1 mg/ml with the channel at the point of DNA-DNA contact, protein-mediated capture cross- sections are very high. We have investigated a range of proteins that have been proposed as part of the telomeric T-loop complex, as well as DNA ligases. We find that a subset of these induce knots that are free to travel along the elongated DNA molecule, but cannot leave the DNA strand at its end. We further find that we can distinguish these knots from simple loops, and that the diffusivity of knots and loops can be modified through transient protein bridges. Finally we are able to distinguish proteins with dense and sparse binding patterns.

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