Probing transient protein-mediated DNA linkages using nanoconfinement.
We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNApolymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches.
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Maedeh
M
Roushan
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Parminder
P
Kaur
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Alena
A
Karpusenko
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Preston J
PJ
Countryman
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Carlos P
CP
Ortiz
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Shuang
S
Fang Lim
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Hong
H
Wang
Department of Physics, NC State University , Raleigh, North Carolina 27695, USA.
Robert
R
Riehn
DNA; atomic force microscopy; biochemistry; biological techniques; biomechanics; catalysis; channel flow; enzymes; molecular biophysics; molecular configurations; nanobiotechnology; nanofluidics; polymers