Sequence-dependent structural features of the DNA double helix have a stron
g influence on the base pair opening dynamics. Here we report a detailed st
udy of the kinetics of base pair breathing in tracts of GC base pairs in DN
A duplexes derived from H-1 NMR measurements of the imino proton exchange r
ates upon titration with the exchange catalyst ammonia. In the limit of inf
inite exchange catalyst concentration, the exchange times of the guanine im
ino protons of the GC tracts extrapolate to much shorter base pair lifetime
s than commonly observed for isolated GC base pairs. The base pair lifetime
s in the GC tracts are below 5 ms for almost all of the base pairs. The unu
sually rapid base pair opening dynamics of GC tracts are in striking contra
st to the behavior of AT tracts, where very long base pair lifetimes are ob
served, The implication of these findings for the structural principles gov
erning spontaneous helix opening as well as the DNA-binding specificity of
the cytosine-5-methyltransferases, where flipping of the cytosine base has
been observed, are discussed.