BRCA2-deficient cells precipitate telomere shortening upon collapse of stalled replication forks. Here, we report that the dynamic interaction between BRCA2 and telomeric G-quadruplex (G4), the non-canonical four-stranded secondary structure, underlies telomere replication homeostasis. We find that the OB-folds of BRCA2 binds to telomeric G4, which can be an obstacle during replication. We further demonstrate that BRCA2 associates with G-triplex (G3)-derived intermediates, which are likely to form during direct interconversion between parallel and non-parallel G4. Intriguingly, BRCA2 binding to G3 intermediates promoted RAD51 recruitment to the telomere G4. Furthermore, MRE11 resected G4-telomere, which was inhibited by BRCA2. Pathogenic mutations at the OB-folds abrogated the binding with telomere G4, indicating that the way BRCA2 associates with telomere is innate to its tumor suppressor activity. Collectively, we propose that BRCA2 binding to telomeric G4 remodels it and allows RAD51-mediated restart of the G4-driven replication fork stalling, simultaneously preventing MRE11-mediated breakdown of telomere.

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BRCA2-deficient cells undergo telomere shortening upon collapse of stalled replication forks, particularly during lagging-strand telomere synthesis. The molecular mechanism underlying fork collapse remains unclear. Here we find that the BRCA2 C-terminus, which includes an OB-fold, specifically interacts with G-quadruplex (G4) structures generated during lagging-strand telomere replication. We demonstrate that BRCA2 associates with G-triplex (G3)-derived intermediates using electrophoretic mobility shift assay and single-molecule FRET. These G3 intermediates form during direct interconversion between parallel and non-parallel G4 structures. Intriguingly, MRE11 nuclease can resect G4-forming telomere sequences, a function that is inhibited by BRCA2. BRCA2 depletion consistently resulted in increased telomeric damage, which was relieved by MRE11 knockdown. These data suggest that BRCA2 interaction with telomeric G4 prevents MRE11-mediated resection. The specific interaction between BRCA2 and G4 therefore contributes to telomere stability and genome integrity.

The packaging of eukaryotic DNA with histones into chromatin acts to both compact it into the nucleus as well as provide a mechanism for regulation of the genome. Spatial and temporal remodeling of chromatin structure is critical for all DNA-templated processes and dysregulation of these pathways is associated with a number of diseases. The BRG1/BRM associated factor (BAF) complex, is an ATP-dependent remodeling complex that plays an important role in gene regulation, several subunits of which are mutated in human cancers. The Brahma related gene 1 (BRG1) subunit of BAF, which provides the ATPase activity, contains a bromodomain (BD) at its C-terminus. BDs are well-characterized readers of acetylated lysines on histones, and the BRG1-BD has been shown to bind H3K14ac. However, we have recently discovered that in addition to binding acetyl-lysine, the BRG1-BD associates with DNA, a novel function for BDs. In addition, we have demonstrated that an adjacent AT-hook contributes to a multivalent mechanism of association with DNA, increasing affinity and specificity (Morrison et al., Nature Communications, 2017). Here we present our continued studies on this newly recognized composite DNA binding domain. We use systematic evolution of ligands by exponential enrichment (SELEX-seq) to generate a biophysical model of sequence specificity for the domain that we are exploring structurally using NMR spectroscopy. To determine the kinetic and thermodynamic basis of association we used biolayer interferometry (BLI). Here we present a structure model where the BRG1 BD is positioned onto the major groove and the AT-hook is inserted into the minor groove of the SELEX-determined DNA sequence. Together, our results are revealing the molecular details of how this novel DNA binding domain functions to navigate chromatin, and how dysregulation of the ATBD may impact BAF function.

Lysosome is closely linked to autophagy, which plays a vital role in pancreatic adenocarcinoma (PDAC) tumor biology. This study investigated whether lysosome storage dysfuncton(LSD) contributes to PDAC development. Germline putative pathogenic variants (PPVs) in genes involved in lysosome functions were compared between PDAC patients (N=418) and healthy controls (N=845). Furthermore, Galc-knockout mouse pancreas organoids and human PDAC organoids were used to evaluate the consequences of PPV status in PDAC development and establishment. LSD PPVs were enriched in PDAC patients (Log2OR=1.65, P=3.08×10−3). PPV carriers diagnosed with PDAC were younger than non-carriers (61.5 vs. 65.3 years, P=0.031). Hampered autophagolysosome activity with increased autophagy flux and elevated Ki-67 index were observed following GALC downregulation. RNA sequencing of human PDAC organoids revealed upregulation of metabolism related to LSD. Genetically defined lysosome dysfunction is frequently observed in young-age onset PDACs. Lysosome dysfunction might contribute to PDAC development via altered metabolism and impaired autophagolysosome activity.