Identification and purification of two distinct complexes containing the five RAD51 paralogs

Genes Dev. 2001 Dec 15;15(24):3296-307. doi: 10.1101/gad.947001.

Abstract

Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Baculoviridae / genetics
  • Chromatography, Gel
  • DNA Repair / genetics
  • DNA Repair / physiology*
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / isolation & purification*
  • DNA-Binding Proteins / metabolism
  • Humans
  • Male
  • Microscopy, Electron
  • Precipitin Tests
  • Protein Binding
  • Protein Isoforms / isolation & purification
  • Protein Isoforms / metabolism
  • Rad51 Recombinase
  • Recombinant Proteins / metabolism
  • Recombination, Genetic
  • Testis / chemistry*
  • Testis / cytology

Substances

  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Protein Isoforms
  • Recombinant Proteins
  • X-ray repair cross complementing protein 3
  • XRCC2 protein, human
  • RAD51 protein, human
  • Rad51 Recombinase
  • Adenosine Triphosphatases