Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding

J Biol Chem. 2005 Jul 15;280(28):26303-11. doi: 10.1074/jbc.M503244200. Epub 2005 May 21.


Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.

Publication types

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

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphate / chemistry
  • Amino Acid Sequence
  • Arginine / chemistry
  • DNA / chemistry
  • DNA Helicases
  • DNA Repair Enzymes
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism*
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Glutamic Acid / chemistry
  • Glutathione Transferase / metabolism
  • Glycine / chemistry*
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Plasmids / metabolism
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Rad51 Recombinase
  • Rec A Recombinases / chemistry
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Sodium Chloride / pharmacology
  • Software
  • Two-Hybrid System Techniques


  • DNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • Glutamic Acid
  • Sodium Chloride
  • Adenosine Triphosphate
  • DNA
  • Arginine
  • Glutathione Transferase
  • RAD51 protein, S cerevisiae
  • Rad51 Recombinase
  • Rec A Recombinases
  • Adenosine Triphosphatases
  • RAD54 protein, S cerevisiae
  • DNA Helicases
  • DNA Repair Enzymes
  • Glycine