Book of Abstracts: Albany 2005
Kinking of Nicked DNA
Two types of interaction are well known to contribute to DNA stability: stacking between adjacent base pairs and pairing between complementary bases. Here we determine their relative contributions into the stability of the double helix by studying DNA molecules with sequence-specific solitary nicks.
Our major hypothesis is that nicked DNA exists in solution in the form of equilibrium between the closed state (the stacking between the two base pairs flanking the nick site is preserved) and the open state (stacking in the nick site is completely lost). Our model is presented schematically in Figure 1 (1). The stacked conformation of the nicked DNA is very close to the conformation of an intact DNA duplex. The loss of stacking between the base pairs flanking the nick induces a kink in DNA duplex. Fast equilibration between the two states leads to a differential retardation of the nicked DNA molecules during PAGE depending on the identity of the nicked dinucleotide. In fact, retardation of the nicked molecule is given by the weighted average over the two conformations adopted by the nicked stack. We estimate the occupancies of the two states by measuring PAGE mobilities of nicked DNA with respect to the mobilities of intact molecules (closed state) and gapped molecules (open state).
Figure 1: Schematic representation of the stacked-to-unstacked conformational transition of the DNA fragment at the nick site described by the energy difference ΔGST.
From Boltzmann distribution, stacking free energy parameters, ΔGST, describing the stacked-unstacked equilibrium for all 32 nicked dinucleotide stacks are determined. Only 10 of them are essential and they govern the stacking interactions between adjacent base pairs in intact DNA double helix. From these data and from a classical Marmur-Doty dependence of DNA melting temperature on G·C content (2, 3), the contribution of base-pairing into duplex stability is estimated. The obtained energy parameters of the duplex DNA indicate that base-stacking rather than base-pairing is primarily responsible for stabilizing the DNA double helix.
References and Footnotes
Maxim D. Frank-Kamenetskii*
Center for Advanced Biotechnology