Single-molecule views on homologous recombination Andrea Candelli 1 , Mauro Modesti 2,3,4,5 , Erwin J. G. Peterman 1 and Gijs J. L. Wuite 1 * 1 LaserLaB and Department of Physics and Astronomy, VU University Amsterdam, Amsterdam, The Netherlands 2 Centre de Recherche en Cance ´rologie de Marseille, CNRS, UMR7258, Marseille F-13009, France 3 Inserm U1068, Marseille F-13009, France 4 Institut Paoli-Calmettes, Marseille F-13009, France 5 Aix-Marseille Universite ´, Marseille F-13284, France Abstract. All organisms need homologous recombination (HR) to repair DNA double-strand breaks. Defects in recombination are linked to genetic instability and to elevated risks in developing cancers. The central catalyst of HR is a nucleoprotein filament, consisting of recombinase proteins (human RAD51 or bacterial RecA) bound around single-stranded DNA. Over the last two decades, single-molecule techniques have provided substantial new insights into the dynamics of homologous recombination. Here, we survey important recent developments in this field of research and provide an outlook on future developments. 1. Introduction 323 1.1. Double-stranded DNA (dsDNA) breaks and homologous recombination (HR) 323 1.2. Structure and conformations of human RAD51 and E. coli RecA 325 2. Single-molecule techniques for the study of DNA–protein interactions 326 3. Single-molecule experiments 329 3.1. Assembly of the NPF 329 3.2. Conformational transitions of the NPF 334 3.3. Searching for a needle in the haystack 336 3.4. Strand-exchange at the single-molecule level 339 3.5. RAD51 disassembly from DNA 342 4. Conclusions and outlook 344 5. Acknowledgement 345 6. References 345 1. Introduction 1.1 Double-stranded DNA (dsDNA) breaks and homologous recombination (HR) Ultimately, the biological essence of life consists in the accurate transmission of genetic infor- mation from one generation to the next. Preserving genome integrity against DNA damages is therefore crucial for living organisms. Among the different types of DNA damage encountered, * Author for Correspondence : G. J. L. Wuite, LaserLaB and Department of Physics and Astronomy, VU University Amsterdam, Amsterdam, The Netherlands. gwuite@nat.vu.nl Quarterly Reviews of Biophysics 46, 4 (2013), pp. 323–348. f Cambridge University Press 2013 323 doi:10.1017/S0033583513000073 Printed in the United States of America