Linking Structure and Function for the DNA Repair Complex Mre11-Rad50-Nbs1

Abstract

Repair of DNA damage is an essential process in all cells and an important mechanism to avoid cancer development in animals.
The repair of DNA double strand breaks (DSB) requires many component proteins including the Mre11-Rad50-Nbs1 (MRN) complex that serves different functions in this process.
This thesis explores how the organization of the MRN components in the complex contributes to and controls functions such as DNA binding and DNA tethering.
Changes in the structure of the complex upon DNA binding are likely responsible for its role in DNA tethering and damage signaling.
The first chapter reviews the homologous recombination (HR) and non-homologous end joining (NHEJ) pathway of DSB repair and the known roles of Mre11, Rad50 and Nbs1.
The conformational changes induced by ATP binding as well as some standing questions regarding the dynamics and stoichiometry of this protein complex are discussed.
The experimental work described in the rest of the thesis used single molecule imaging by scanning force microscopy (SFM) to visualize nanometer scale conformational changes that accompany different functions. Biochemical analysis and electrophoretic mobility shift assays (EMSAs) were employed to characterize component-specific functions and the importance of protein interfaces and the role of ATP in DNA binding and DNA tethering.
A biochemical analysis of human Rad50 shows that this component can by itself bind DNA although it is defective in ATP-dependent oligomerization.
Mre11, previously thought to be essential for DNA binding and DNA tethering, is not required to maintain these functions.
However, it is clear that proper functions in DNA repair and damage signaling are affected by several specific subunit interactions and that ATP binding triggers changes in interactions with DNA in complex ways, including promoting the necessary tethering activity. The ability to describe changes in protein complex activity and its nanometer resolved structure reveal how complex reactions like DNA repair are accomplished and coordinated within cells.