Measurement of the DNA bend angle induced by the catabolite activator protein using Monte Carlo simulation of cyclization kinetics.

A Monte Carlo simulation method for studying DNA cyclization (or ring-closure) has been extended to the case of protein-induced bending, and its application to experimental data has been demonstrated. Estimates for the geometric parameters describing the DNA bend induced by the catabolite activator protein (CAP or CRP) were obtained which correctly predict experimental DNA cyclization probabilities (J factors), determined for a set of 11 150 to 166 bp DNA restriction fragments bearing A tracts phased against CAP binding sites. We find that simulation of out-of-phase molecules is difficult and time consuming, requiring the geometric parameters to be optimized individually rather than globally. A wedge angle model for DNA bending was found to make reasonable predictions for the free DNA. The bend angle in the CAP-DNA complex is estimated to be 85 to 90 degrees, in agreement with estimates from gel electrophoresis and X-ray co-crystal structures. Since the DNA is found to have a pre-existing bend of 15 degrees, the change in bend angle induced by CAP is 70 to 75 degrees, in a agreement with an estimate from topological measurements. We find evidence for slight (approximately 10 degrees) unwinding by CAP. The persistence length and helical repeat of the unbound portion of the DNA are in accord with literature-cited values, but the best-fit DNA torsional modulus C is found to be 1.7 (+/- 0.2) x 10(-19) erg. cm, versus literature estimates and best-fit values for the free DNA of 2.0 x 10(-19) to 3.4 x 10(-19) erg.com. Simulations using this low value of C predict that cyclization of molecules with out-of-phase bends proceeds via undertwisting or overtwisting of the DNA between the bends, so as to align the bends, rather than through conformations with substantial writhe. We present experiments on the topoisomers formed by cyclization with CAP which support this conclusion, and thereby rationalize the surprising result that cyclization can actually be enhanced by out-of-phase bends if the twist required to align the bends improves the torsional alignment of the ends. The relationship between the present work and previous studies on DNA bending by CAP is discussed, and recommendations are given for the efficient application of the cyclization/simulation approach to DNA bending.