Roles of the human Rad51 L1 and L2 loops in DNA binding.
The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.
Matsuo
Yusuke
Y
Graduate School of Science and Engineering, Waseda University, Tokyo, Japan.
Sakane
Isao
I
Takizawa
Yoshimasa
Y
Takahashi
Masayuki
M
Kurumizaka
Hitoshi
H
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D004268:DNA-Binding Proteins; D014443:Tyrosine; D014364:Tryptophan; D051135:Rad51 Recombinase; D000409:Alanine