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Nucleic Acids Research 2006 34(10):2864-2877; doi:10.1093/nar/gkl382
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Published online 24 May 2006

Nucleic Acids Research, 2006, Vol. 34, No. 10 2864-2877
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article

DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force

Gregory J. Gemmen, Rachel Millin and Douglas E. Smith*

Department of Physics Mail Code 0379 University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA

*To whom correspondence should be addressed. Tel: +1 858 534 5241; Email: des{at}physics.ucsd.edu

Received February 22, 2006. Revised April 4, 2006. Accepted May 2, 2006.

Proteins interacting at multiple sites on DNA via looping play an important role in many fundamental biochemical processes. Restriction endonucleases that must bind at two recognition sites for efficient activity are a useful model system for studying such interactions. Here we used single DNA manipulation to study sixteen known or suspected two-site endonucleases. In eleven cases (BpmI, BsgI, BspMI, Cfr10I, Eco57I, EcoRII, FokI, HpaII, NarI, Sau3AI and SgrAI) we found that substitution of Ca2+ for Mg2+ blocked cleavage and enabled us to observe stable DNA looping. Forced disruption of these loops allowed us to measure the frequency of looping and probability distributions for loop size and unbinding force for each enzyme. In four cases we observed bimodal unbinding force distributions, indicating conformational heterogeneity and/or complex binding energy landscapes. Measured unlooping events ranged in size from 7 to 7500 bp and the most probable size ranged from less than 75 bp to nearly 500 bp, depending on the enzyme. In most cases the size distributions were in much closer agreement with theoretical models that postulate sharp DNA kinking than with classical models of DNA elasticity. Our findings indicate that DNA looping is highly variable depending on the specific protein and does not depend solely on the mechanical properties of DNA.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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