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Biophys. J. BioFAST: First Published March 18, 2005. doi:10.1529/biophysj.104.055657
© 2005 by the Biophysical Society.

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NUCLEIC ACIDS

Gapped DNA and Cyclization of Short DNA Fragments

Quan Du 1, Maria Vologodskaia 1, Heiko Kuhn 2, Maxim Frank-Kamenetskii 2 and Alexander Vologodskii 1*

1 New York University
2 Boston University

* To whom correspondence should be addressed. E-mail: alex.vologodskii{at}nyu.edu.

Submitted on November 4, 2004
Revised on December 10, 2004
Accepted on 15 March 2005


  Abstract
We use the cyclization of small DNA molecules, about 200 bp in length, to study conformational properties of DNA fragments with single-stranded gaps. The approach is extremely sensitive to DNA conformational properties and, being complemented by computations, allows a very accurate determination of the fragment's conformational parameters. Sequence-specific nicking endonucleases are used to create the 4-nt-long gap. We determined the bending rigidity of the single-stranded region in the gapped DNA. We found that the gap of 4 nucleotides in length makes all torsional orientations of DNA ends equally probable. Our results also show that the gap has isotropic bending rigidity. This makes it very attractive to use gapped DNA in the cyclization experiments to determine DNA conformational properties, since the gap eliminates oscillations of the cyclization efficiency with the DNA length. As a result, the number of measurements is greatly reduced in the approach and the analysis of the data is greatly simplified. We have verified our approach on DNA fragments containing well-characterized intrinsic bends caused by A-tracts. The obtained experimental results and theoretical analysis demonstrate that gapped-DNA cyclization is an exceedingly sensitive and accurate approach for the determination of DNA bending.

Key Words: DNA bend, DNA conformation, DNA flexibility, Single-stranded DNA, j-factor




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Copyright © 2005 by the Biophysical Society.