Biology subjects

Jensen, R. B.

Publications and source records attributed to Jensen, R. B..

5 recordsLinked to original sources

bioRxiv2024

RAD51 Paralogs and RAD51 Paralog Complexes BCDX2 and CX3 Interact with BRCA2

Homologous recombination (HR) is an important mechanism for repairing DNA double-strand breaks (DSBs) and preserving genome integrity. Pathogenic mutations in the HR proteins BRCA2 and the RAD51 paralogs predispose individuals to breast, ovarian, pancreatic, and prostate cancer. The RAD51 paralogs: RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 form two complexes RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3). Similar to BRCA2, loss of RAD51 paralog functions in mammalian cells lead to chromosomal abnormalities, growth defects, disrupted RAD51 foci formation, and PARP inhibitor sensitivity. Despite significant e]ort over the past three decades, the specific molecular functions of the human RAD51 paralogs have remained elusive due to technical challenges such as low protein expression in human cell lines and instability of the purified proteins. Recent studies have determined the molecular structures of the BCDX2 and CX3 complexes dramatically enhancing our understanding of these challenging proteins. Using multiple approaches, we demonstrate that the RAD51 paralogs interact with BRCA2 at two distinct interaction hubs located in the BRC repeats and the DNA binding domain. We confirm, using a yeast 3-hybrid approach, that human RAD51 paralogs interact directly with BRC repeats one and two (BRC1-2) of BRCA2. Because of the dynamic nature of the RAD51B C-terminal domain (CTD), identified in the recently solved cryo-EM structures, we focused on elucidating the interaction with RAD51B. We determined that BRCA2 interacts with the CTD of RAD51B and not the N-terminal domain (NTD) that is involved in stacking interactions with RAD51C and RAD51D. Furthermore, the interaction with RAD51B is dependent upon an FxxA motif located on a surface exposed region of the CTD. Our study has identified novel interactions between the RAD51 paralogs and BRCA2 and further demonstrated that a previously unrecognized FxxA motif located within a mobile element of RAD51B is critical for the interaction.

molecular biology↗

bioRxiv2024

Cell cycle regulation of polymerase theta mediated end-joining underlies its sensitivity to loss of BLM-DNA2

Polymerase theta-mediated end-joining (TMEJ) is a salvage DNA repair pathway that anneals and ligates microhomologies flanking a DNA double-strand break (DSB) downstream of aborted homologous recombination to ensure DSBs are resolved before chromosome segregation. Here, we use a sensitive deep-sequencing approach to identify DSB repair outcomes at an endogenous locus. We observe that TMEJ is impaired by depletion or inhibition of DNA2, a component of the DSB resection machinery. Mechanistically we find that DNA2 deficiency influences TMEJ efficiency by slowing S-phase progression, thereby preventing cells from reaching mitotic commitment at the G2/M transition, when we find that TMEJ is optimally licensed to occur. Further interrogation of the cell cycle regulation of TMEJ reinforces a strict requirement for the mitotic kinase PLK1 in enabling TMEJ at the G2/M transition. We surprisingly find CDK1 to be largely dispensable for TMEJ, but important for a small subset of repair events likely occurring in mitosis. These findings have important implications for modulation of TMEJ in response to cell cycle kinase inhibitors that are in active development as anti-tumor agents.

molecular biology↗

bioRxiv2023

PARG inhibition induces nuclear aggregation of PARylated PARP1

PARG inhibitors are currently under clinical development for the treatment of DNA repair-deficient cancers, however, their precise mechanism of action is still unclear. Here we report that PARG inhibition causes increased nuclear PARylated PARP1 that limits PARP1 chromatin binding in response to DNA damage. This PARylated PARP1 accumulates as aggregates at sites distinct from the site of DNA damage, leading to the mis-localization of PARP1. Additionally, these aggregates are formed through PAR chains as abrogating PARP1 catalytic activity prevents their formation. Finally, these PARP1 nuclear aggregates persist long-term and are associated with cleaved cytoplasmic PARP1, a cell death hallmark, which ultimately leads to a non-apoptotic form of cell death. Overall, our data uncovers a novel mechanism of PARG inhibitor cytotoxicity, which will inform ongoing clinical studies.

cancer biology↗

bioRxiv2022

BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individuals lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, Replication Protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins--of which BRCA2 is the defining member in humans --alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to--and the assembly of RAD51 filaments on--a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short pre-assembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation. SignificanceDespite decades of genetic and cell biological studies, mechanistic biochemical analyses of human BRCA2 function in recombinational DNA repair have only been possible since the purification of full-length BRCA2. These mechanistic studies crucially inform with respect to the molecular function of BRCA2 in genome maintenance. Here, we use single-molecule methods to visualize the assembly of RAD51 on individual RPA-coated ssDNA molecules and to see how this process is regulated by the tumor suppressor protein, BRCA2. We show that BRCA2 serves as a chaperone to nucleate RAD51 and deliver it to RPA-coated ssDNA. This work advances understanding of the molecular functions of BRCA2 and, consequently, the molecular etiology of breast cancer in an important way.

biochemistry↗

bioRxiv2021

Cancer missense mutations in the BRC repeats of BRCA2 protein disrupt RAD51 binding and activity leading to chemotherapeutic sensitivity

Pathogenic mutations in the BRCA2 tumor suppressor gene predispose to breast, ovarian, pancreatic, prostate, and other cancers. BRCA2 maintains genome stability through homology-directed repair (HDR) of DNA double-strand breaks (DSBs) and replication fork protection. Nonsense or frameshift mutations leading to truncation of the BRCA2 protein are typically considered pathogenic, however, missense mutations resulting in single amino acid substitutions can be challenging to functionally interpret. The majority of missense mutations in BRCA2 have been classified as Variants of Uncertain Significance (VUS) with unknown functional consequences. In this study, we identified three BRCA2 VUS located within the BRC repeat region to determine their impact on canonical HDR and fork protection functions. We provide evidence that S1221P and T1980I, which map to conserved residues in the BRC2 and BRC7 repeats, compromise the cellular response to chemotherapeutics and ionizing radiation, and display deficits in fork protection. We further demonstrate biochemically that S1221P and T1980I disrupt RAD51 binding and diminish the ability of BRCA2 to stabilize RAD51-ssDNA complexes. The third variant, T1346I, located within the spacer region between BRC2 and BRC3 repeats, is fully functional. We conclude that T1346I is a benign allele whereas S1221P and T1980I are hypomorphic disrupting the ability of BRCA2 to fully engage and stabilize RAD51 nucleoprotein filaments. Our results underscore the importance of correctly classifying BRCA2 VUS as pathogenic variants can impact both future cancer risk and guide therapy selection during cancer treatment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/461749v2_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1e5e3c6org.highwire.dtl.DTLVardef@1cd0e60org.highwire.dtl.DTLVardef@16adf4corg.highwire.dtl.DTLVardef@16a0089_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗