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

Journal Article · · Nucleic Acids Research
DOI:https://doi.org/10.1093/nar/gkaa1188· OSTI ID:1766538
 [1];  [2];  [3];  [4];  [1];  [5];  [3];  [4];  [6];  [2]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Univ. of New Mexico, Albuquerque, NM (United States)
  3. Univ. of Montreal, QC (Canada)
  4. Univ. of Cincinnati, OH (United States)
  5. Washington Univ., St. Louis, MO (United States)
  6. Univ. of Texas, Houston, TX (United States)
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.
Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
Cancer Prevention Research Institute of Texas (CPRIT); National Institutes of Health (NIH); Natural Sciences and Engineering Research Council of Canada (NSERC); USDOE Office of Science (SC); V Foundation
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1766538
Journal Information:
Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 1 Vol. 49; ISSN 0305-1048
Publisher:
Oxford University PressCopyright Statement
Country of Publication:
United States
Language:
English

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