Structure and Function of Holliday Junctions Complexed with Ions and HU Protein

Abstract

DNA four-way junctions (4WJ) are central intermediates in processes such as replication and recombination. This project studies how different conformations of the junction relate to function, and how proteins interact with the junction to enhance or repress function. Previously, we have examined the 4WJ structure in the presence of different ions, and used Forster Resonance Energy Transfer (FRET) to map the binding site of the protein HU onto the junction. It was found that in the presence of high salt concentrations the junction is stabilized into a stacked form rather than open form. These FRET measurements were used to build four different models for the binding of HU protein to the Holliday Junction, an interaction with a stoichiometry of 2 to 1. This was followed by molecular dynamics simulation to ensure that the models were energetically stable. The three stable models included HU bound to the major groove, minor groove, or face to face. The fourth model, which contained an open junction, though stable, did not agree with the FRET data acquired for the system, and was therefore not considered plausible. To distinguish between these models further FRET measurements are needed to narrow the possible orientations for HU binding the junction. To address the function of the protein bound junction, a new, migrating four way junction is being created and characterized. This junction will be analyzed by FRET in the presence of different ions and possibly HU as well to compare to the characteristics of the stationary junction used in the previous experiments. We will also be examining the free energy of both the mobile and immobile junctions by molecular dynamics to analyze the stability of each construct on their own and when complexed with HU.