Identification of the Subunit – Subunit Interface of Xenopus Rad51.1 Protein: Similarity to RecA Tassadite Selmane 1 , Jean-Michel Camadro 2 , Se´bastien Conilleau 1 Fabrice Fleury 1 , Vinh Tran 1 , Chantal Pre ´vost 3 and Masayuki Takahashi 1 * 1 FRE 2230 Unite ´ de Recherche sur la Biocatalyse, Centre National de la Recherche Scientifique and University of Nantes, 2 rue de la Houssiniere 44322 Nantes cedex 3, France 2 Laboratoire d’Inge ´ nie ´ rie des Prote ´ ines et Contro ˆ le Me ´ tabolique, Institut Jacques-Monod, UMR 7592 Centre National de la Recherche Scientifique and Universite ´ s Paris 6 and 7, F-75251 Paris cedex 05, France 3 UPR 9080 Laboratoire de Biochimie The ´ orique, Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique 75005 Paris, France Rad51,like its prokaryotic homolog RecA, forms a helicalfilamentfor homologous DNA recombination and recombinationalDNA repair. Comparison of the three-dimensional structures ofhuman Rad51 and Escherichia coli RecA indicated that the tyrosine residue at position 191 in human Rad51 lies atthe centre ofa putative subunit – subunit contact interface.We inserted a tryptophan residue as a fluorescent probe at the corresponding position in Xenopus Rad51.1 and found that its fluorescence depended upon the protein concentration, indicating that the residue is truly in the subunit – subunitinterface.We also found that 3 M urea, which promoted the dissociation ofRad51 filamentwithout complete unfolding of the protein, exposed the tryptophan residue to solvent. The fluorescence was notmodified by binding to DNA and only slightly modified by ATP,indicating thatthe same site is used for formation of the active ATP-Rad51-DNA filament. The slight changes in fluorescence caused by ATP and ADP suggest that the subunit – subunit contactis altered,leading to the elongation of the filament by these nucleotides, as with the RecA filament. Thus, Rad51 forms filaments by subunit –subunit contact much like RecA does. q 2003 Elsevier Ltd. All rights reserved. Keywords: fluorescence; filament formation; homologous recombination; protein assembly; Rad51 protein *Corresponding author Introduction The eukaryote protein Rad51 is the homolog of the Escherichia coli protein RecA and is involved in homologousrecombination and recombinational DNA repair. 1 – 3 Its over-production increases the resistanceof cells to DNA-damaging chemicals and X-ray irradiation. 4,5 Rad51 also interacts with tumor suppressorssuch as p53, BRCA1 and BRCA2. 6 – 9 The protein may thus be important for preventing formation ofcancers.A variant has been found in patients having bilateral breast cancer. 10 Its capacity to repair damaged DNA also influences the resistance of cancer cells to chemo- therapy and radiotherapy. 11 Inhibiting its production with antisense RNA or ribozyme increases the efficiency of these anti-cancer treatments. 12,13 Rad51 is phosphorylated and activated by cAbl and Arg tyrosine kinases, 14 – 16 which are also involved in the resistance of cancer cells to chemotherapy, 17 further indicating its importance in the resistance of cancer cells. Lastly, Rad51 influences the immortality of cancer cells. Immortalized cells contain three to four times more of the protein than do normalcells, 18 and cancer cells contain more Rad51 than do normal cells. 19 Inhibiting Rad51 synthesis alone prolongs the survivalof mice bearing a cancer. 12 Thus the protein could be a targetfor the treatmentand diagnosis of cancer. 0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. Present address: T. Selmane, National Institutes of Health, Genetics and Biochemistry Branch, Bethesda, MD 20892-0538, USA. E-mail address of the corresponding author: masa@chimbio.univ-nantes.fr Abbreviations used: XlRad51.1, Xenopus laevis protein 51.1; CD, circular dichroism. doi:10.1016/j.jmb.2003.11.045 J. Mol. Biol. (2004) 335, 895–904