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 Förster Resonance Energy Transfer (FRET) to map the binding site of the protein HU onto the junction. 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. Molecular dynamics simulations ensured 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. 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 4WJ is being created and characterized. This 4WJ will be analyzed by FRET in the presence of ions and proteins to compare to the characteristics of the stationary junction. The free energy of both the mobile and immobile junctions will be analyzed by molecular dynamics to predict stability of each construct alone and when complexed with HU.This research is supported by an the NSF (MCB‐0316625; MCB‐0843656 to I.M.), the NIH (GM‐076490 to D.L.B.), and by NIGMS Training grant T32GM08271.