From single-molecule to synchrotron: New tools for investigating the molecular mechanisms of aberrant DNA diseases.

Abstract

Damage in DNA can interrupt and alter its conformational flexibility—which drives crucial site-specific functions—leading to aberrant DNA diseases. It is not fully understood exactly what role these conformations play in the processes undertaken by DNA, nor how they might be affected by various damage. Here we present an overview and comparison of two novel and emergent techniques to study such conformations; single molecule Förster resonance energy transfer (smFRET) and X-ray scattering interferometry (XSI). We are focusing on the RNaseH2 complex, mutations that are responsible for Aicardi-Goutières syndrome (AGS), to establish how the search space is minimised as it finds and cleaves RNA/DNA hybrids, and whether this is driven by conformational changes. We also introduce O6-methylguanine (O6-MeG), ribonucleotide, nicks and gaps; all prevalent modifications to DNA which without repair would lead to toxic intermediates. It is hypothesised that when the damage is present the increase in flexibility of DNA signals proteins to promote repair. smFRET was used to measure the conformational changes when the different types of damage, were present in a DNA duplex. A change in FRET efficiency is seen for all the types of damage, with the nick and gap damage having a greater impact on the mean and spread of the FRET efficiency. These results provide initial support for the hypothesis that damage alters the overall structure and dynamics of DNA duplexes and that the presence of these lesions and their respective repair mechanisms binds the DNA in varying levels of flexibility.