TY - JOUR
T1 - Gapped DNA and cyclization of short DNA fragments
AU - Du, Quan
AU - Vologodskaia, Maria
AU - Kuhn, Heiko
AU - Frank-Kamenetskii, Maxim
AU - Vologodskii, Alexander
N1 - Funding Information:
This work was supported by National Institutes of Health grant GM54215 to A.V.
PY - 2005/6
Y1 - 2005/6
N2 - We use the cyclization of small DNA molecules, ∼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 nt 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.
AB - We use the cyclization of small DNA molecules, ∼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 nt 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.
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U2 - 10.1529/biophysj.104.055657
DO - 10.1529/biophysj.104.055657
M3 - Article
C2 - 15778443
AN - SCOPUS:22244443662
SN - 0006-3495
VL - 88
SP - 4137
EP - 4145
JO - Biophysical journal
JF - Biophysical journal
IS - 6
ER -