Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction

Nucleic Acids Res. 2013 Oct;41(19):9020-32. doi: 10.1093/nar/gkt691. Epub 2013 Aug 8.

Abstract

RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model's depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51(E42A), RAD51(E59A), RAD51(E237A), RAD51(E59A/E237A) and RAD51(E42A/E59A/E237A) maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction.

Publication types

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

MeSH terms

  • BRCA2 Protein / chemistry*
  • BRCA2 Protein / metabolism
  • Models, Molecular
  • Mutation
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Interaction Domains and Motifs
  • Rad51 Recombinase / chemistry*
  • Rad51 Recombinase / genetics
  • Rad51 Recombinase / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Structural Homology, Protein

Substances

  • BRCA2 Protein
  • Peptides
  • Saccharomyces cerevisiae Proteins
  • RAD51 protein, S cerevisiae
  • RAD51 protein, human
  • Rad51 Recombinase