X-ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double-strand break repair impacting cell and cancer biology.

Michal Hammel,John A Tainer

doi:10.1002/pro.4133

Abstract

Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non‐homologous end joining (NHEJ) as the primary conserved DNA double‐strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB‐inducing agents, generation of antibody and T‐cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X‐ray scattering (SAXS) results combined with X‐ray crystallography (MX) and cryo‐electron microscopy (cryo‐EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra‐molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo‐EM or MX structures. In the long‐range synaptic complex, X‐ray repair cross‐complementing 4 (XRCC4) plus XRCC4‐like‐factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA‐PKcs (DNA‐dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto‐phosphorylation opens DNA‐PKcs dimer licensing NHEJ via concerted conformational transformations of XLF‐XRCC4, XLF–Ku80, and LigIVBRCT–Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short‐range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation‐of‐function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.

Highlights

Macromolecular flexibility, unstructured linkers, dynamic conformations, and metastable complexes are essential functional aspects of DNA damage response (DDR) regulatory mechanisms
For DNA repair and damage responses ranging from oxidized base repair to DNA double-strand break (DSB) repair (DSBR), we have found that accurate measures of flexibility, conformational change, and dynamic complexes from smallangle X-ray scattering (SAXS) are often important for understanding and dissecting multifunctional mechanisms, as exemplified by the intrinsically disordered tail of Nei Like DNA Glycosylase 1 (NEIL1) acting in efficient oxidized base repair[13,14] and by ATP-driven RAD50 assembly and conformational states acting in the homology-directed repair (HDR) of double-strand break repair (DSBR).[15,16,17,18,19,20]
For Rad[51], which acts in HDR, the functionally flexible polymerization motif lies in the linker region between domains; this made it so challenging to see correct assemblies that a thermophile was employed to define the first intact Rad[51] structure and assembly.[22]

Summary

Introduction

Macromolecular flexibility, unstructured linkers, dynamic conformations, and metastable complexes are essential functional aspects of DNA damage response (DDR) regulatory mechanisms.

Results

Conclusion