title: Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding.
abstract: 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.
authors.affiliation: Section of Microbiology, University of California, Davis, California 95616-8665, USA.
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Zhang
authors.forename: Kyung-Im
authors.initials: KI
authors.lastname: Lee
authors.forename: Jachen A
authors.initials: JA
authors.lastname: Solinger
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kiianitsa
authors.forename: Wolf-Dietrich
authors.initials: WD
authors.lastname: Heyer
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D005998:Glycine / Q000737:chemistry*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase; D011938:Rec A Recombinases / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D012984:Software; D020798:Two-Hybrid System Techniques
chemical_list: D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine; D005982:Glutathione Transferase; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D005998:Glycine