Wasmuth, E. V. | bioRxiv Science

bioRxiv2024

Distinct roles of the two BRCA2 DNA binding domains in DNA damage repair and replication fork preservation

Homologous recombination (HR) is a highly conserved tool for the removal of DNA double-strand breaks (DSBs) and the preservation of stalled and damaged DNA replication forks. Successful completion of HR requires the tumor suppressor BRCA2. Germline mutations in BRCA2 lead to familial breast, ovarian, and other cancers, underscoring the importance of this protein for maintaining genome stability. BRCA2 harbors two distinct DNA binding domains, one that possesses three oligonucleotide/oligosaccharide binding (OB) folds (known as the OB-DBD), and with the other residing in the C-terminal recombinase binding domain (termed the CTRB-DBD) encoded by the last gene exon. Here, we employ a combination of genetic, biochemical, and cellular approaches to delineate contributions of these two DNA binding domains toward HR and the maintenance of stressed DNA replication forks. We show that OB-DBD and CTRB-DBD confer ssDNA and dsDNA binding capabilities to BRCA2, respectively, and that BRCA2 variants mutated in either DNA binding domain are impaired in the ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected for HR, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DNA binding domains in genome maintenance.

biochemistry↗

bioRxiv2024

Single particle cryo-electron microscopy with an enhanced 200 kV cryo-TEM configuration achieves near-atomic resolution

Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom ([A]) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S). First, we evaluated data quality on the standard benchmarking sample, rabbit muscle aldolase, using three of the most frequently used cryo-EM data collection software: SerialEM, Leginon and EPU, and found that - despite sample heterogeneity - all final reconstructions yield same overall resolutions of 2.6 [A] and map quality when using either of the three software. Furthermore, comparison between Glacios-F4-S and a 300 kV cryo-TEM (Titan Krios with Falcon 4) revealed nominal resolution differences in overall reconstructions of a reconstituted human nucleosome core particle, achieving 2.8 and 2.5 [A], respectively. Finally, we performed comparative data analysis on the human RAD51 paralog complex, BCDX2, a four-protein complex of approximately 150 kilodaltons, and found that a small dataset ([≤]1,000 micrographs) was sufficient to generate a 3.3 [A] reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg+2. In summary, this study provides evidence that the Glacios-F4-S operates equally well with all standard data collection software, and is sufficient to obtain high resolution structural information of novel macromolecular complexes, readily acquiring single particle data rivaling that of 300 kV cryo-TEMs.

biophysics↗