The tumor suppressor complex BRCA1-BARD1 functions in DNA double-strand break repair by homologous recombination. Therein, BRCA1-BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumor suppressor complex BRCA2-PALB2 and the recombinase RAD51. By examining purified BRCA1-BARD1 and mutants, we show that BRCA1 and BARD1 both bind DNA and interact with RAD51, and that BRCA1-BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1-BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. Evidence is provided that BRCA1 and BARD1 are both indispensable for RAD51 stimulation. Importantly, BRCA1-BARD1 mutants weakened for RAD51 interaction are compromised for DNA joint formation and for the mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1-BARD1 in homologous recombination, a novel attribute of the tumor suppressor complex that could be targeted in cancer therapy.

The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55–Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2–F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55–Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

The TGFβ and Ras-MAPK pathways play critical roles in cell development and cell cycle regulation, as well as in tumor formation and metastasis. In the absence of cellular transformation, these pathways operate in opposition to one another, where TGFβ maintains an undifferentiated cell state and suppresses proliferation, while Ras-MAPK pathways promote proliferation, survival and differentiation. However, in colorectal and pancreatic cancers, the opposing pathways' mechanisms are simultaneously activated in order to promote cancer progression and metastasis. Here, we highlight the roles of the TGFβ and Ras-MAPK pathways in normal and malignant states, and provide an explanation for how the concomitant activation of these pathways drives tumor biology. Finally, we survey potential therapeutic targets in these pathways.

Highlights d FANCD2 colocalizes with co-transcriptional R-loops in human cells d Human FANCI-FANCD2 robustly binds ssRNA, but not RNA:DNA hybrids d Human FANCI-FANCD2 binds R-loops via the displaced ssDNA strand and ssRNA tail d ssRNA and R-loop can stimulate robust FANCI-FANCD2 monoubiquitination

Telomerase is essential for continuous cellular proliferation. Substantial insights have come from studies of budding yeast telomerase, which consists of a catalytic core in association with two regulatory proteins, ever shorter telomeres 1 and 3 (Est1 and Est3). We report here a high-resolution structure of the Est3 telomerase subunit determined using a recently developed strategy that combines minimal NMR experimental data with Rosetta de novo structure prediction algorithms. Est3 adopts an overall protein fold which is structurally similar to that adopted by the shelterin component TPP1. However, the characteristics of the surface of the experimentally determined Est3 structure are substantially different from those predicted by prior homology-based models of Est3. Structure-guided mutagenesis of the complete surface of the Est3 protein reveals two adjacent patches on a noncanonical face of the protein that differentially mediate telomere function. Mapping these two patches on the Est3 structure defines a set of shared features between Est3 and HsTPP1, suggesting an analogous multifunctional surface on TPP1.RASREC Rosetta | OB-fold protein T elomerase is a telomere-dedicated DNA polymerase that is responsible for telomere-length maintenance in most eukaryotes. In cells that lack telomerase, gradual erosion due to incomplete replication of duplex telomeric DNA leads to an eventual block to cellular proliferation. Ectopic expression of telomerase in human cells is sufficient to confer cellular immortality (1) and it is up-regulated in over 90% of tumor biopsies (2). Conversely, reduced telomerase activity is responsible for the age-dependent effects on organs that rely on continual replenishment throughout a normal human life span and can lead to bone marrow failure, pulmonary fibrosis, or aplastic anemia (3). Hence, an increased understanding of the roles of telomerase and its accessory proteins in telomere length homeostasis has the potential to impact several different aspects of human health.The yeast telomerase holoenzyme is composed of three proteins [the catalytic ever shorter telomere 2 (Est2) subunit, along with the Est1 and Est3 regulatory proteins], which together form a complex with the TLC1 RNA (4, 5). In vivo, telomerase is highly regulated, in that only a subset of telomeres are elongated in each cell cycle (6); however, the mechanism that restricts telomerase to a limited number of substrates is still poorly understood. This deficit stems at least in part from the fact that the surface of yeast telomerase represents a largely unexplored territory. Even though there are numerous interaction surfaces on the three Est proteins with the potential to regulate important interactions, the only well-characterized regulatory step involving Est proteins is the recruitment of telomerase to the telomere through the direct interaction of Est1 and the end-binding protein Cdc13 (7), which was originally uncovered using a labor-intensive genetic approach (8).High-resolution structural information provides a str...