CHARACTERIZATION OF THE ATF CREB SITE AND ITS COMPLEX WITH GCN4/

We have studied DNA minicircles containing the ATF/CREB binding site f or GCN4 by using a combination of cyclization kinetics experiments and Monte Carlo simulations, Cyclization rates were determined with and w ithout GCN4 for DNA constructs containing the ATF/CREB site separated from a phased A-tract multimer bend by a variable length phasing adapt or. The cyclization results show that GCN4 binding does not significan tly change the conformation of the ATF/CREB site, which is intrinsical ly slightly bent toward the major groove. Monte Carlo simulations quan titate the ATF/CREB site structure as an 8 degrees bend toward the maj or groove in a coordinate frame near the center of the site. The ATF/C REB site is underwound by 53 degrees relative to the related AP-I site DNA. The effect of GCN4 binding can be modeled either as a decrease i n the local flexibility, corresponding to an estimated 60% increase in the persistence length for the 10-bp binding site, or possibly as a s mall decrease (1 degrees) in intrinsic bend angle. Our results agree w ith recent electrophoretic and crystallographic studies and demonstrat e that cgclization and simulation can characterize subtle changes in D NA structure and flexibility.