TY - JOUR
T1 - Homologous Recombination and DNA Intermediates Analyzed by Electron Microscopy
AU - Basto, Clara
AU - Moreira-Tavares, Eliana
AU - Muhammad, Ali Akbar
AU - Baconnais, Sonia
AU - Mazón, Gerard
AU - Le Cam, Eric
AU - Dupaigne, Pauline
N1 - Publisher Copyright:
© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2025/1/1
Y1 - 2025/1/1
N2 - 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.
AB - 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.
KW - Displacement-Loop
KW - DNA intermediates
KW - DNA-proteins complexes
KW - Homologous recombination
KW - Recombination intermediates
KW - Strand-exchange
KW - Synaptic complexes
KW - Transmission Electron microscopy
UR - http://www.scopus.com/inward/record.url?scp=85213517987&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-4280-1_12
DO - 10.1007/978-1-0716-4280-1_12
M3 - Article
C2 - 39704947
AN - SCOPUS:85213517987
SN - 1064-3745
VL - 2881
SP - 239
EP - 257
JO - Methods in molecular biology (Clifton, N.J.)
JF - Methods in molecular biology (Clifton, N.J.)
ER -