dna repair 5 ( 2 0 0 6 ) 718–730 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/dnarepair Defining the salt effect on human RAD51 activities Kang-Sup Shim ∗ , Christoph Schmutte, Kristine Yoder, Richard Fishel ∗∗ Department of Molecular Virology, Immunology, and Medical Genetics, Human Cancer Genetics, The Ohio State University College of Medicine and Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43102, United States article info Article history: Received 13 January 2006 Received in revised form 9 March 2006 Accepted 10 March 2006 Published on line 27 April 2006 Keywords: hRAD51 Salt DNA binding strand exchange Abbreviations: ATPase, ATP hydrolysis activity; bp, base pair; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; X174, bacteriophage X174; ATP, adenosine triphosphate; ATPS, adenosine-5-O-thio triphosphate; ADP, adenosine diphosphate; DTT, dithiothreitol; hRAD51, human RAD51; SSB, single strand binding protein; HEPES, N-[2-hydroxyethyl]piperazine-N ′ -[2- ethanesulfonic acid]; IAB, IAsys biosensor; Mg 2+ , magnesium ion; NPF, nucleoprotein filament; nt, nucleotide; RFI/RFII, replicative form I/II abstract Previous work by Sung and colleagues identified unusual salt requirements for hRAD51 strand exchange compared to RecA [S. Sigurdsson, K. Trujillo, B. Song, S. Stratton, P. Sung, Basis for avid homologous DNA strand exchange by human Rad51 and RPA, J. Biol. Chem. 276 (2001) 8798–8806]. Later studies showed that this salt [(NH 4 ) 2 SO 4 ] appeared to enhance the ability of hRAD51 to distinguish ssDNA from dsDNA [Y. Liu, A.Z. Stasiak, J.Y. Masson, M.J. McIlwraith, A. Stasiak, S.C. West, Conformational changes modulate the activity of human RAD51 protein, J. Mol. Biol. 337 (2004) 817–827]. The mechanism of this salt effect remains enigmatic. Here, we detail the properties of several neutral salts on hRAD51 activities. We found that the cation identity correlated with the stimulatory effect of these neutral salts on hRAD51 ATPase and strand exchange activities. The salt effect appears to be related to the size of the cation, which may be largely mimicked with the cesium ion. These results are consistent with the hypothesis that stimulating cations induce an important conforma- tion and/or transition state in hRAD51. In the presence of an optimal ammonium-based salt (NaNH 4 HPO 4 ), hRAD51 mediated strand exchange was successfully performed using a sim- plified protocol. We confirmed and extend the observation that efficient strand exchange correlated with preferential binding of ssDNA over dsDNA. In addition we observed an induced stability of the hRAD51–DNA complex in the presence of ATP that becomes unstable following ATP hydrolysis (the ADP form or nucleotide free form). These salt-induced char- acteristics of hRAD51 increasingly resemble RecA-mediated recombinase activities, which should help in dissecting the mechanism of these proteins in homologous recombination. © 2006 Elsevier B.V. All rights reserved. ∗ Corresponding author. ∗∗ Corresponding author. Tel.: +1 614 292 2484. E-mail addresses: shim.55@osu.edu (K.-S. Shim), rfishel@osu.edu (R. Fishel). 1568-7864/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2006.03.006