Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIα

Kevin H. Thornton, Viswanathan V Krishnan, Mary G. West, Jennifer Popham, Melissa Ramirez, Michael P. Thelen, Monique Cosman

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region. These regions, known as BRCT domains, have been found to mediate important protein-protein interactions. We produced the BRCT domain of DNA ligase IIIα (L3[86]) for biophysical and structural characterization. A glutathione S-transferase (GST) fusion with the L3[86] domain (residues 837-922 of ligase IIIα) was expressed in Escherichia coli and purified by glutathione affinity chromatography. The GST fusion protein was removed by thrombin digestion and further purification steps. Using this method, 15N-labeled and 13C/15N-double-labeled L3[86] proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy. To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied. Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation. However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3[86] or X1BRCTb proteins alone. This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIα activity.

Original languageEnglish (US)
Pages (from-to)401-411
Number of pages11
JournalProtein Expression and Purification
Volume21
Issue number3
DOIs
StatePublished - 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIα'. Together they form a unique fingerprint.

  • Cite this