Application of Difference 3D Electron Microscopy to Identify DSS1 Subunits and Induced Conformational Changes in the Complex with Rad52

Daniel Němeček¹, Jarmila Mlčoušková², Barbora Štefanovie², Lumír Krejčí^2,3

¹Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, CZ

²National Center for Biomolecular Research, Masaryk University, Kamenice 5, Brno, CZ

³Department of Biology, Masaryk University, Kamenice 5, Brno, CZ

The Rad52 protein is a recombination enzyme conserved among eukaryotic organisms that plays an important role in the initial steps of the homology-directed DNA repair by loading Rad51 recombinase on ssDNA as well as by catalyzing annealing of two complementary ssDNA strands [1]. However in human cells, the recombination mediator function is carried out primarily by BRCA2, while Rad52 constitutes a secondary pathway [2]. The BRCA2 protein is stabilized by a small highly acidic protein DSS1 [3]. We discovered that DSS1 is also able to directly interact with human Rad52 and might regulate homologous recombination repair in mammalian cells.

Previous biophysical and structural studies have shown that the full-length human Rad52 protein assembles into a heptameric ring with a funnel-like shape [4]. The conserved N-terminal part of human Rad52 was crystallized as an undecameric ring and provided insight into the interaction of Rad52 with DNA [5, 6]. However, the molecular mechanism and structural basis of the full-length Rad52 function as well as the role of the DSS1 protein remain unknown.

We used electron microscopy and 3-D image processing to determine the structure of the full-length Rad52 heptamer and its complex with DSS1. Initial reference-free class averages of Rad52 appeared as tilted top-side views of rings with a diameter 110-130 Å, consistently with previous studies [4]. Refined 3-D structure of the ring using C7 symmetry showed a funnel-like structure with a central channel wide ~40 Å. The top and bottom rims of the central channel are connected by strong densities that form the body of the channel and likely correspond to the compact fold of the Rad52 N-terminal part. The top of the channel is covered by a weak density.

The 3D reconstruction of the Rad52-DSS1 complex revealed significant conformational changes induced by DSS1. The Rad52 ring is wider and flatter, while the bottom of the central channel is sealed with solid density. The lobes at the ring circumference are larger and likely include additional density of the 8-kDa DSS1 subunits. The DSS1 subunits could this way act as a molecular switch by modulating Rad52 affinity towards DNA.

[1] Liu, J. and Heyer, W.-D. (2011) Proc. Natl. Acad. Sci. USA, 108:441-442.

[2] Feng, Z. et al. (2011) Proc. Natl. Acad. Sci. USA, 108:686-691.

[3] Li, J. et al. (2006) Oncogene, 25:1186-94.

[4] Stasiak, A.Z., et al. (2000) Curr. Biol., 10:337–340.

[5] Kagawa, W., et al. (2002) Mol. Cell, 10:359–371.

[6] Singleton, M.R., et al. (2002) PNAS, 99:13492-13497.