An atypical BRCT-BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex

Nucleic Acids Res. 2021 Jan 11;49(1):306-321. doi: 10.1093/nar/gkaa1188.

Abstract

The XRCC1-DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT-BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS-MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT-BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Gel
  • Crystallography, X-Ray
  • DNA Ligase ATP / chemistry
  • DNA Ligase ATP / metabolism*
  • DNA Repair*
  • Dimerization
  • Humans
  • Microscopy, Electron
  • Models, Molecular
  • Multiprotein Complexes
  • Mutation
  • Mutation, Missense
  • Negative Staining
  • Point Mutation
  • Protein Conformation
  • Protein Domains
  • Protein Interaction Mapping
  • Recombinant Proteins / metabolism
  • Scattering, Small Angle
  • X-ray Repair Cross Complementing Protein 1 / chemistry
  • X-ray Repair Cross Complementing Protein 1 / genetics
  • X-ray Repair Cross Complementing Protein 1 / metabolism*

Substances

  • Multiprotein Complexes
  • Recombinant Proteins
  • X-ray Repair Cross Complementing Protein 1
  • XRCC1 protein, human
  • DNA Ligase ATP