Conformational flexibility and oligomerization of BRCA2 regions induced by RAD51 interaction.

BRCA2 is a key breast cancer associated protein that is predicted to have interspersed regions of intrinsic disorder. Intrinsic disorder coupled with large size likely allows BRCA2 to sample a broad range of conformational space. We expect that the resulting dynamic arrangements of BRCA2 domains are a functionally important aspect of its role in homologous recombination DNA repair. To determine the architectural organization and the associated conformational landscape of BRCA2, we used scanning force microscopy based single molecule analyses to map the flexible regions of the protein and characterize which regions influence oligomerization. We show that the N- and the C-terminal regions are the main flexible regions. Both of these regions also influence BRCA2 oligomerization and interaction with RAD51. In the central Brc repeat region, Brc 1-4 and Brc 5-8 contribute synergistically to BRCA2 interaction with RAD51. We also analysed several single amino acid changes that are potentially clinically relevant and found one, the variant of F1524V, which disrupts key interactions and alters the conformational landscape of the protein. We describe the overall conformation spectrum of BRCA2, which suggests that dynamic structural transitions are key features of its biological function, maintaining genomic stability.

Imaging of DNA and Protein by SFM and Combined SFM-TIRF Microscopy.

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

The bacterial condensin MukB compacts DNA by sequestering supercoils and stabilizing topologically isolated loops.

MukB is a structural maintenance of chromosome-like protein required for DNA condensation. The complete condensin is a large tripartite complex of MukB, the kleisin, MukF, and an accessory protein, MukE. As found previously, MukB DNA condensation is a stepwise process. We have defined these steps topologically. They proceed first via the formation of negative supercoils that are sequestered by the protein followed by hinge-hinge interactions between MukB dimers that stabilize topologically isolated loops in the DNA. MukB itself is sufficient to mediate both of these topological alterations; neither ATP nor MukEF is required. We show that the MukB hinge region binds DNA and that this region of the protein is involved in sequestration of supercoils. Cells carrying mutations in the MukB hinge that reduce DNA condensation in vitro exhibit nucleoid decondensation in vivo.

Architectural plasticity of human BRCA2-RAD51 complexes in DNA break repair.

The tumor suppressor BRCA2 is a large multifunctional protein mutated in 50-60% of familial breast cancers. BRCA2 interacts with many partners and includes multiple regions with potentially disordered structure. In homology directed DNA repair BRCA2 delivers RAD51 to DNA resulting in removal of RPA and assembly of a RAD51 nucleoprotein filament. Dynamic rearrangements of BRCA2 likely drive this molecular hand-off initiating DNA strand exchange. We show human BRCA2 forms oligomers which can have an extended shape. Scanning force microscopy and quantitative single molecule fluorescence define the variety of BRCA2 complexes, reveal dramatic rearrangements upon RAD51 binding and the loading of RAD51 patches on single strand DNA. At sites of repair in cell nuclei, super-resolution microscopy shows BRCA2 and RAD51 arranged in largely separate locations. We identified dynamic structural transitions in BRCA2 complexes suggested to facilitate loading of RAD51 onto RPA coated single strand DNA and subsequent release of BRCA2.

The Mre11-Nbs1 Interface Is Essential for Viability and Tumor Suppression.

The Mre11 complex (Mre11, Rad50, and Nbs1) is integral to both DNA repair and ataxia telangiectasia mutated (ATM)-dependent DNA damage signaling. All three Mre11 complex components are essential for viability at the cellular and organismal levels. To delineate essential and non-essential Mre11 complex functions that are mediated by Nbs1, we used TALEN-based genome editing to derive Nbs1 mutant mice (Nbs1mid mice), which harbor mutations in the Mre11 interaction domain of Nbs1. Nbs1mid alleles that abolished interaction were incompatible with viability. Conversely, a 108-amino-acid Nbs1 fragment comprising the Mre11 interface was sufficient to rescue viability and ATM activation in cultured cells and support differentiation of hematopoietic cells in vivo. These data indicate that the essential role of Nbs1 is via its interaction with Mre11 and that most of the Nbs1 protein is dispensable for Mre11 complex functions and suggest that Mre11 and Rad50 directly activate ATM.

Enhanced chromatin dynamics by FACT promotes transcriptional restart after UV-induced DNA damage.

Chromatin remodeling is tightly linked to all DNA-transacting activities. To study chromatin remodeling during DNA repair, we established quantitative fluorescence imaging methods to measure the exchange of histones in chromatin in living cells. We show that particularly H2A and H2B are evicted and replaced at an accelerated pace at sites of UV-induced DNA damage. This accelerated exchange of H2A/H2B is facilitated by SPT16, one of the two subunits of the histone chaperone FACT (facilitates chromatin transcription) but largely independent of its partner SSRP1. Interestingly, SPT16 is targeted to sites of UV light-induced DNA damage-arrested transcription and is required for efficient restart of RNA synthesis upon damage removal. Together, our data uncover an important role for chromatin dynamics at the crossroads of transcription and the UV-induced DNA damage response.