Understanding DNA Looping Through Cre-Recombination Kinetics
- 1.8k Downloads
The interior of a cell is a crowded and fluctuating environment where DNA and other biomolecules are both highly constrained and subject to many mechanical forces. The extensive compaction of DNA in living cells is a challenge to many critical biological functions. An evolutionary solution to this challenge may be the juxtaposition of cis-acting elements such that multimeric protein complexes simultaneously interact with two or more protein-binding sites. This mode of biological activity involves the formation of looped DNA structures, which, by themselves, are thermodynamically unfavorable. Our knowledge about the roles of DNA bending, twisting, and their respective energetics in DNA looping has come mainly from analyses of ligase-dependent DNA cyclization experiments, which are quantitatively described by the Jacobson–Stockmayer, or J, factor. In this chapter, we discuss a novel quantitative approach to measuring the probability of DNA loop formation in solution using ensemble Förster resonance energy transfer (FRET) measurements of intramolecular and intermolecular Cre-recombination kinetics. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure.
KeywordsExtract Rate Constant Intermolecular Reaction Transcription Factor Interaction Intramolecular Recombination Repeat loxP Site
We thank Andreas Hanke and Stefan Giovan for calculations of J theor. This work was supported by a grant from the NIH/NSF Joint Program in Mathematical Biology (DMS-0800929 from the National Science Foundation) to SDL.
- 2.T. Förster, Z. Naturforsch. A. 4(7) (1949)Google Scholar
- 17.R. Zhou, T. Ha, Methods Mol. Biol. 922, 85–100 (2012)Google Scholar
- 37.Y. Chen, U. Narendra, E.L. Iype, M.M. Cox, A.P. Rice, Mol. Cell 6, 885–897 (2000)Google Scholar
- 43.D.M. Crothers, J. Drak, J.D. Kahn, S.D. Levene, Methods Enzymol. 212, 3–29 (1992)Google Scholar
- 52.V.A. Bloomfield, D.M. Crothers, I.J. Tinoco, Nucleic acids: structures, properties and functions (University Science Books, Herndon, 2000)Google Scholar