Book of Abstracts: Albany 2003
June 17-21 2003
Site-Resolved Energetics in a DNA Unwinding Element
The process of DNA replication requires denaturation of a specific region in the origin of replication, which in E.coli contains three A+T rich 13mers (1). Since DnaB helicase cannot initiate the unwinding of the DNA, formation of an open complex is the key step in the initiation of replication at oriC. Stable DNA unwinding is induced by DNA supercoiling at these sites, as shown by hypersensitivity to single-strand specific nuclease and by two-dimensional gel electrophoresis (2). These results indicate that the reduced helical stability at these sites, called DNA unwinding elements (DUE), is responsible for the formation of the open complex. In the present work we use NMR spectroscopy and proton exchange to characterize the dynamics and the stability of Watson-Crick base-pairs in the DNA double helix formed by 5?-GCGATCTATTTATTTGC-3? and its complementary strand. The DNA contains the leftmost 13mer (in bold) from the origin of replication in E.coli. The imino proton resonances of the DNA 17mer and their assignments are shown in the figure below. We have measured the exchange rates of the imino protons as a function of the concentration of exchange catalyst over the temperature range from 10 to 35 °C. The results are used to determine the opening rates and equilibrium constants for opening of single base pairs as a function of temperature. The relation between the overall reduced stability of the DUE and the local stability at the level of individual base-pairs will be discussed.
This work is supported by a grant from the National Institutes of Health.