What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

Abstract

DNA damage outcomes depend upon the efficiency and fidelity of DNA damage responses (DDRs) for different cells and damage. As such, DDRs represent tightly regulated prototypical systems for linking nanoscale biomolecular structure and assembly to the biology of genomic regulation and cell signaling. However, the dynamic and multifunctional nature of DDR assemblies can render elusive the correlation between the structures of DDR factors and specific biological disruptions to the DDR when these structures are altered. In this chapter, we discuss concepts and strategies for combining structural, biophysical, and imaging techniques to investigate DDR recognition and regulation, and thus bridge sequence-level structural biochemistry to quantitative biological outcomes visualized in cells. We focus on representative DDR responses from PARP/PARG/AIF damage signaling in DNA single-strand break repair and nonhomologous end joining complexes in double-strand break repair. Methods with exemplary experimental results are considered with a focus on strategies for probing flexibility, conformational changes, and assembly processes that shape a predictive understanding of DDR mechanisms in a cellular context. Integration of structural and imaging measurements promises to provide foundational knowledge to rationally control and optimize DNA damage outcomes for synthetic lethality and for immune activation with resulting insights for biology and cancer interventions.

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

“…More broadly, current and emerging MRN results are aiding in addressing grand challenges of the postgenomic era for biochemistry and cancer biology: ( a ) an accurate prediction of pathology for damage response sequence variants, ( b ) an actionable mechanistic understanding of oncogenic replication and repair stress for biology and medicine, and ( c ) an advanced assessment of functional biochemical complexes and networks controlling outcomes from molecules to cells (235). Existing insights are already changing our concepts of damage detection, signaling, and repair with implications for strategies of controlling MRN activities for cancer, T cell aging, immune responses, and viral infections.…”

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

“…However, despite important structural and biophysical studies on NHEJ complexes 28 , the absence of high resolution structures of Ku-KBMs complexes limits our understanding of the roles and specificity of the different molecular interactions in the recruitment of NHEJ factors to DSBs. Mapping KBM-binding sites in structures is also needed to clarify potential competition of all the Ku interacting factors on limited positions (Supplementary figure 1).…”

Small angle X‐ray scattering and cross‐linking for data assisted protein structure prediction in CASP 12 with prospects for improved accuracy

“…Consider that single amino acid mutations, small ligands, and pH can drastically alter protein conformation. SAXS can accurately show the conformation and assembly state that can vary with ligand binding, as seen for proteins such as abscisic acid receptor and apoptosis inducing factor . SAXS can similarly distinguish differences in aggregation or assembly resulting from single residue changes, as found for superoxide dismutase mutations that correlate to Amyotrophic Lateral Sclerosis prognosis, for macromolecular interactions controlling pathogenesis, for nanomachines orchestrating genetic integrity, and for design of mega‐protein assemblies .…”