Zabolotnaya, Ekaterina and Mela, Ioanna and Williamson, Mark and Bray, Sian and Yau, Siu Kei and Papatziamou, Dimitra and Edwardson, J. Michael and Robinson, Nick and Henderson, Robert (2020) Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy. Proceedings of the National Academy of Sciences of the United States of America, 117 (26). pp. 14936-14947. ISSN 0027-8424
Full text not available from this repository.Abstract
Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1–2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.