Homologous Recombination and DNA Intermediates Analyzed by Electron Microscopy

Homologous recombination (HR) is a high-fidelity DNA repair pathway that uses a homologous DNA sequence as a template. Recombinase proteins are the central HR players in the three kingdoms of life. RecA/RadA/Rad51 assemble on ssDNA, generated after the processing of double-strand breaks or stalled replication forks into an active and dynamic presynaptic helical nucleofilament. Presynaptic filament formation is regulated by a series of partners of the recombinase, such as scRad52/hBRCA2 mediators or anti-recombinase proteins, to form an active machinery involved in homology search, pair-matching, and invasion within homologous sequences. During homology search, but also during strand invasion, the multiprotein complexes that form the nucleofilament induce the formation of a variety of DNA intermediate states. Here we present specific approaches to study and characterize the different DNA and DNA-protein intermediates formed during homologous recombination. The combination of powerful electron microscopy and sample preparation methods provides a better understanding of these proteins’ molecular activity and their interactions.