UbcH7 regulates 53BP1 stability and DSB repair

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.Prompt response to double-strand breaks (DSBs) caused by, for example, ionization radiation (IR), requires sequential and coordinated assembly of DNA damage response (DDR) proteins at damage sites (1). Recent research findings reveal key roles of the tumor suppressor p53-binding protein 1 (53BP1) and BRCA1 in the decision making of DSB repair. 53BP1, together with Rif1, suppress BRCA1-dependent homologous recombination (HR), thereby promoting nonhomologous end-joining (NHEJ) in G1 phase (2–6). Conversely, BRCA1 antagonizes 53BP1/Rif1, favoring HR in S and G2 phases (7, 8). In the absence of BRCA1 or with enhanced retention of 53BP1 at DSB sites, cells primarily use the error-prone NHEJ to repair DSBs throughout the cell cycle, which leads to gene rearrangement, cell death, and increased sensitivity to anticancer therapies (9–11). Consistently, BRCA1-null mice are early embryonic lethal (12, 13) and codepletion of TP53BP1 rescued the lethality phenotype of BRCA1-null mice (12–14).Low expression level of 53BP1 was found to be associated with poor clinical outcome in triple negative breast cancer patients with BRCA1 mutation (12, 15), as well as resistance to genotoxins and poly(ADP-ribose) polymerase inhibitors (12, 16, 17). This finding is probably because loss of 53BP1 restored HR and promoted cell survival (12–14). Reduced expression of 53BP1 was also observed in tumors from the brain (18), lymph node (19), and pancreas (20). These data indicate that loss of 53BP1 might be a common mechanism for advanced tumors to evade from radiotherapy or chemotherapy. However, molecular mechanisms controlling the protein level of 53BP1 remain less well understood.Here we show that UbcH7, an E2 enzyme involved in the ubiquitin (Ub) pathway, controls the protein stability of 53BP1, thereby determining the DSB repair choice. Loss of UbcH7 stabilizes 53BP1, forcing cells to choose NHEJ, but not HR, to repair DSBs, which poses a significant threat to cells treated with DNA damage, especially S-phase genotoxins, such as camptothecin (CPT), a topoisomerase 1 (Top1) inhibitor. The ternary CPT-Top1-DNA complex places a roadblock in the path of advancing DNA replication forks, leading to replication fork collapse and generation of one-ended DSBs. Such one-ended DSBs require HR, but not NHEJ, to repair (8). In contrast, repair of one-ended DSBs by NHEJ leads to radial chromosomes and cell death (12–14). Therefore, stabilization of 53BP1 by UbcH7 depletion increased the sensitivity of cancers cells to CPT and other DNA damaging agents. Our data suggest a novel strategy in enhancing the anticancer effect of radiotherapy or chemotherapy through stabilizing or increasing 53BP1.     

BRCA2 regulates DMC1-mediated recombination through the BRC repeats

In somatic cells, BRCA2 is needed for RAD51-mediated homologous recombination. The meiosis-specific DNA strand exchange protein, DMC1, promotes the formation of DNA strand invasion products (joint molecules) between homologous molecules in a fashion similar to RAD51. BRCA2 interacts directly with both human RAD51 and DMC1; in the case of RAD51, this interaction results in stimulation of RAD51-promoted DNA strand exchange. However, for DMC1, little is known regarding the basis and functional consequences of its interaction with BRCA2. Here we report that human DMC1 interacts directly with each of the BRC repeats of BRCA2, albeit most tightly with repeats 1–3 and 6–8. However, BRC1–3 bind with higher affinity to RAD51 than to DMC1, whereas BRC6–8 bind with higher affinity to DMC1, providing potential spatial organization to nascent filament formation. With the exception of BRC4, each BRC repeat stimulates joint molecule formation by DMC1. The basis for this stimulation is an enhancement of DMC1–ssDNA complex formation by the stimulatory BRC repeats. Lastly, we demonstrate that full-length BRCA2 protein stimulates DMC1-mediated DNA strand exchange between RPA–ssDNA complexes and duplex DNA, thus identifying BRCA2 as a mediator of DMC1 recombination function. Collectively, our results suggest unique and specialized functions for the BRC motifs of BRCA2 in promoting homologous recombination in meiotic and mitotic cells.The breast cancer susceptibility protein 2, BRCA2, regulates RAD51-mediated homologous recombination (HR) (1–3). Both RAD51, a DNA strand exchange protein, and its meiotic counterpart, DMC1 (disrupted meiotic cDNA 1 or DNA meiotic recombinase 1), promote HR through the formation of a nucleoprotein filament on ssDNA (4). This filament finds and invades a homologous template, resulting in a DNA strand invasion product called a joint molecule or a displacement-loop (D-loop). The joint molecule provides a primer template for the new DNA synthesis required to repair the DNA double strand break (DSB).The first evidence implicating BRCA2 in meiosis came from studies in Ustilago maydis, where strains lacking the BRCA2 ortholog, Brh2, resulted in absence of meiotic products (5). Shortly thereafter, mouse BRCA2 was inferred to coordinate the activities of RAD51 and DMC1 (6). The first direct interaction between BRCA2 and DMC1 was observed in plants (7) and later in humans (8). In the plant, Arabidopsis thaliana, the interaction between Brca2 and Dmc1 was mapped to the BRC repeats (9), a highly conserved motif comprising a sequence of ∼35 amino acids that is present at least once in all BRCA2-like proteins (10). In humans, BRCA2 contains eight BRC repeats that bind with different affinities to RAD51, and they segregate into two functional classes (11). Within a BRC repeat, two motifs that bind RAD51 have been identified: one comprising the consensus sequence FxxA that mimics the oligomerization interface (12) and contacts the catalytic domain of RAD51; the other binding module comprises the alpha-helical region of the BRC repeat, contains the consensus sequence LFDE, and binds to RAD51 through a different hydrophobic pocket (13).Importantly, loss of Brca2 in plants causes chromosomal aberrations during meiosis (14). In humans, GST-pull down assays using peptide fragments of BRCA2 mapped a unique DMC1 interacting site to residues 2386–2411 (8). However, in mouse, mutation of a key residue (Phe-2406) within this site, which had been shown to disrupt the interaction of BRCA2 with DMC1 by peptide array analysis, had no effect in meiosis (15), suggesting that another site or sites in BRCA2 provide the functions needed during meiosis in this organism (6). A direct physical interaction was indeed established for purified full-length human BRCA2 and DMC1 (2), but the functional relevance of this interaction was not elaborated.We have previously shown that the BRC repeats of BRCA2 modulate the DNA binding selectivity of RAD51 to stimulate the assembly on ssDNA by inhibiting its ATP hydrolysis and preventing its association with dsDNA (2, 11, 16); as a result, BRCA2 catalyzes the recombination activity of RAD51 (17).A comprehensive analysis of aligned sequences of RAD51 orthologs and human RAD51 paralogues suggested that most eukaryotic RAD51 proteins, including DMC1, could interact with the BRC repeats, at least in principle (10). Here we investigate whether and how BRCA2 modulates DMC1-mediated recombination.     

Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance.The breast cancer 1, early onset (BRCA1) gene is commonly mutated in hereditary breast and ovarian cancers. The BRCA1 protein has multiple domains that mediate protein interactions; BRCA1 gene mutations may produce truncated proteins that lose the ability to interact with associated proteins. Additionally, mutations in the BRCA C-terminal (BRCT) domain of BRCA1 create protein folding defects that result in protease-mediated degradation (1–3).Cells that contain dysfunctional BRCA1 proteins are hypersensitive to DNA damaging agents (4). In particular, BRCA1-deficient cell lines are exquisitely sensitive to poly(ADP-ribose) polymerase (PARP) inhibition (5). Despite initial responses of BRCA1-mutant cancers to PARP inhibitor treatment (6), acquired resistance universally develops. Resistance may result from secondary mutations in the BRCA1 gene that restore the reading frame and produce a functional BRCA1 protein (7, 8). In Brca1-mutated mouse mammary tumors, activation of p-glycoprotein or loss of p53 binding protein 1 (53BP1) expression resulting from truncating TP53BP1 mutations confers PARP inhibitor resistance (9). Loss of 53BP1 in BRCA1-deficient cells provides the C-terminal binding protein interacting protein (CtIP) with unrestricted access to DNA breaks, facilitating DNA end resection, an early step in homologous recombination (HR) (9–11).Following BRCA1-CtIP–mediated activation of DNA end resection, eventual BRCA2-mediated assembly of the RAD51 recombinase in nucleoprotein filaments is a critical step in HR. A role for BRCA1 in RAD51 loading and the mechanisms by which it participates have not been fully clarified. Of note, in PARP inhibitor-resistant BRCA1- and 53BP1-deficient tumors and derived cell lines, RAD51 γ-irradiation–induced foci were detected, although at a lower level than in BRCA1- and 53BP1-proficient cells (9). Previous studies demonstrated that RAD51 foci were partially reduced in BRCA1- or partner and localizer of BRCA2 (PALB2)-deficient cells reconstituted with BRCA1 or PALB2 constructs carrying mutations that disrupt the BRCA1–PALB2 interaction (12, 13), suggesting that BRCA1 may enlist PALB2, which in turn organizes the recruitment of BRCA2 and RAD51.To date, the described mechanisms of PARP inhibitor resistance occur in only a fraction of the BRCA1 mutant patient population or in PARP inhibitor-resistant Brca1-mutated mouse mammary tumors (8, 10). Here, we used a human breast cancer cell line that contains a BRCT domain BRCA1 mutation to identify additional mechanisms of acquired PARP inhibitor resistance, and demonstrate that stabilization of the mutant BRCA1 protein is critical for the restoration of RAD51 focus formation.     

Structural insights into the histone H1-nucleosome complex

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.Eukaryotic genomic DNA is packaged into chromatin through association with positively charged histones to form the nucleosome, the structural unit of chromatin (1–3). The nucleosome core consists of an octamer of histones with two copies of H2A, H2B, H3, and H4, around which ∼146 bp of DNA winds in ∼1.65 left-handed superhelical turns (4). At this level of the DNA packaging, chromatin resembles a beads-on-a-string structure, with the nucleosome core as the beads and the linker DNA between them as the strings (5). At the next level of DNA packaging, H1 histones bind to the linker DNA and the nucleosome to further condense the chromatin structure (6, 7). H1-mediated chromatin condensation plays important roles in cellular functions such as mitotic chromosome architecture and segregation (8), muscle differentiation (9), and regulation of gene expression (10, 11).Linker H1 histones typically are ∼200 amino acid residues in length, with a short N-terminal region, followed by a ∼70–80-amino acid structured globular domain (gH1) and a ∼100-amino acid unstructured C-terminal domain that is highly enriched in Lys residues. H1 stabilizes the nucleosome and facilitates folding of nucleosome arrays into higher-order structures (12–15). gH1 alone confers the same protection from micrococcal nuclease digestion to the nucleosome as the full-length H1 does (16). The N-terminal region of H1 is not important for nucleosome binding (16, 17), whereas the C terminus is required for H1 binding to chromatin in vivo (18, 19) and for the formation of a stem structure of linker DNA in vitro (17, 20, 21).The globular domain structures of avian H5 (22) and budding yeast Hho1 (23), which are both H1 homologs, have been determined at atomic resolution and show similar structures. In addition, numerous studies have indicated that gH1/gH5 binds around the dyad region of the nucleosome (14, 24), leading to many conflicting structural models for how the globular domain of H1/H5 binds to the nucleosome (SI Appendix, Fig. S1) (24–26). These models are divided into two major classes, symmetric and asymmetric, on the basis of the location of gH1/gH5 in the nucleosome. In the symmetric class, gH1/gH5 binds to the nucleosomal DNA at the dyad and interacts with both linker DNAs (16, 17, 27, 28). In the asymmetric class, gH1/gH5 binds to the nucleosomal DNA in the vicinity of the dyad axis and to 10 bp (27, 29–32) or 20 bp (19, 29, 33, 34) of one linker DNA, or is located inside the DNA gyres, where it interacts with histone H2A (35). In addition, Zhou and colleagues also characterized the orientation of gH5 in the gH5-nucleosome complex (29). The use of nonuniquely positioned nucleosomes and indirect methods may have contributed to the differences in these models (SI Appendix, Fig. S1).Multidimensional nuclear magnetic resonance (NMR), and in particular methyl-based NMR, provides a direct approach to the structural characterization of macromolecular complexes (36, 37). We have previously assigned chemical shifts of the methyl groups of the side chains of residues Ile, Leu, and Val in the core histones (38) and the backbone amides in the disordered histone tails (39), which provide the fingerprints for investigating the interactions between H1 and the nucleosome. Here, we used NMR, along with several other methods, to determine the location and orientation of the globular domain of a stable mutant of Drosophila H1 on a well-positioned nucleosome.     

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-β signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.Heterotopic ossification (HO) is defined as bone formation in soft tissue where bone normally does not exist. It can be the result of surgical operations, trauma, or genetic conditions, one of which is fibrodysplasia ossificans progressiva (FOP). FOP is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification (1–6). The responsive mutation for classic FOP is 617G > A (R206H) in the intracellular glycine- and serine-rich (GS) domain (7) of ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP) (8–10). ACVR1 mutations in atypical FOP patients have been found also in other amino acids of the GS domain or protein kinase domain (11, 12). Regardless of the mutation site, mutated ACVR1 (FOP-ACVR1) has been shown to activate BMP signaling without exogenous BMP ligands (constitutive activity) and transmit much stronger BMP signaling after ligand stimulation (hyperactivity) (12–25).To reveal the molecular nature of how FOP-ACVR1 activates BMP signaling, cells overexpressing FOP-ACVR1 (12–20), mouse embryonic fibroblasts derived from Alk2^R206H/+ mice (21, 22), and cells from FOP patients, such as stem cells from human exfoliated deciduous teeth (23), FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) (24, 25) and induced mesenchymal stromal cells (iMSCs) from FOP-iPSCs (FOP-iMSCs) (26) have been used as models. Among these cells, Alk2^R206H/+ mouse embryonic fibroblasts and FOP-iMSCs are preferred because of their accessibility and expression level of FOP-ACVR1 using an endogenous promoter. In these cells, however, the constitutive activity and hyperactivity is not strong (within twofold normal levels) (22, 26). In addition, despite the essential role of BMP signaling in development (27–31), the pre- and postnatal development and growth of FOP patients are almost normal, and HO is induced in FOP patients after physical trauma and inflammatory response postnatally, not at birth (1–6). These observations led us to hypothesize that FOP-ACVR1 abnormally responds to noncanonical BMP ligands induced by trauma or inflammation.Here we show that FOP-ACVR1 transduced BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling (10, 32–34) and contributes to inflammatory responses (35, 36). Our in vitro and in vivo data indicate that activation of TGF-β and aberrant BMP signaling by Activin-A in FOP-cells is one cause of HO in FOP. These results suggest a possible application of anti–Activin-A reagents as a new therapeutic tool for FOP.     

From the Cover: Population-based screening for breast and ovarian cancer risk due to BRCA1 and BRCA2

In the Ashkenazi Jewish (AJ) population of Israel, 11% of breast cancer and 40% of ovarian cancer are due to three inherited founder mutations in the cancer predisposition genes BRCA1 and BRCA2. For carriers of these mutations, risk-reducing salpingo-oophorectomy significantly reduces morbidity and mortality. Population screening for these mutations among AJ women may be justifiable if accurate estimates of cancer risk for mutation carriers can be obtained. We therefore undertook to determine risks of breast and ovarian cancer for BRCA1 and BRCA2 mutation carriers ascertained irrespective of personal or family history of cancer. Families harboring mutations in BRCA1 or BRCA2 were ascertained by identifying mutation carriers among healthy AJ males recruited from health screening centers and outpatient clinics. Female relatives of the carriers were then enrolled and genotyped. Among the female relatives with BRCA1 or BRCA2 mutations, cumulative risk of developing either breast or ovarian cancer by age 60 and 80, respectively, were 0.60 (± 0.07) and 0.83 (± 0.07) for BRCA1 carriers and 0.33 (± 0.09) and 0.76 (± 0.13) for BRCA2 carriers. Risks were higher in recent vs. earlier birth cohorts (P = 0.006). High cancer risks in BRCA1 or BRCA2 mutation carriers identified through healthy males provide an evidence base for initiating a general screening program in the AJ population. General screening would identify many carriers who are not evaluated by genetic testing based on family history criteria. Such a program could serve as a model to investigate implementation and outcomes of population screening for genetic predisposition to cancer in other populations.Inherited mutations in BRCA1 and BRCA2 predispose to high risks of breast and ovarian cancer. Among female mutation carriers, presymptomatic surgical measures significantly reduce morbidity and mortality (1, 2). In particular, risk-reducing salpingo-oophorectomy (i.e., the removal of ovaries and fallopian tubes from a woman without ovarian cancer) reduces risk both of breast cancer and of ovarian cancer, as well as overall mortality (1). However, for many mutation carriers identified following their first cancer diagnosis, genetic testing was not previously indicated because family history did not suggest inherited cancer predisposition (3–5, 6). From a prevention perspective, it is a missed opportunity to identify a woman as a BRCA1 or BRCA2 mutation carrier only after she develops cancer.Among Ashkenazi (European) Jews (AJ), three mutations, BRCA1 185delAG, BRCA1 5382insC, and BRCA2 6174delT, account for the great majority of inherited cancer risk due to BRCA1 and BRCA2 (7). In the AJ population, 2.5% of persons carry one of these three mutations (8), and the mutations account for 11% of breast cancer (3) and 40% of ovarian cancer (9, 10). These observations suggest that genetic testing in the AJ population for these mutations fulfills WHO criteria for population screening (11, 12): The disease is an important public health burden to the target population; prevalence and attributable risk of disease due to the mutations are known; and effective interventions exist. However, one necessary piece of information remains unknown: What is the disease risk to mutation carriers ascertained from the general population, rather than carriers identified based on family history (13)?Previous studies assessing cancer risks due to mutations in BRCA1 and BRCA2 ascertained carriers through high-incidence families (14), through a single index case with breast or ovarian cancer (3, 15) or through both affected and unaffected carriers (16). In a 1997 study of AJ volunteers, most index cases had no previous cancer diagnosis, but the percentage of index cases with a family history of breast cancer was approximately double that of unselected AJs (17). In principle, these strategies could have yielded risk estimates different from those of carriers ascertained from the local host population, if cancer risk in BRCA1 or BRCA2 carriers were influenced by familial factors other than the BRCA1 or BRCA2 mutation, such as modifier genes or shared environment (18). In addition, in almost all of these studies, risk estimates were based on imputing carrier status, rather than on direct genetic testing of BRCA1 and BRCA2. This year, the Recommendation Statement on BRCA Testing from the US Preventive Services Task Force recommended against population screening for BRCA1 and BRCA2 mutations, because cancer risk to mutation carriers in the general population was not yet known (19). To address this gap, in this study we assessed breast and ovarian cancer risks in confirmed carriers of BRCA1 and BRCA2 mutations ascertained from the general population. The study was undertaken in the AJ population, because screening for only three founder mutations is sufficient to capture nearly all inherited cancer risk in this population due to BRCA1 and BRCA2 (7).     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Quantitative analysis of vesicle recycling at the calyx of Held synapse

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).Synaptic vesicle recycling ensures synaptic transmission during sustained neuronal activity (1–3). Despite its crucial role, the cycle is poorly understood. In contrast to vesicle exocytosis and endocytosis, which can be directly assayed by presynaptic capacitance measurements and postsynaptic current recordings, vesicle recycling is usually investigated by fluorescence imaging and electron microscopy (EM) with limited signal or temporal resolution (4–7). Likely owing to technical difficulties, the basic properties of vesicle recycling, such as the size of the recycling pool (RP) (3, 6, 8–11), the kinetics of vesicle recycling (6, 8–12), and how the RP supports synaptic transmission (1, 13–15) remain to be elucidated. Classically, presynaptic vesicles can be functionally divided into three populations: the readily releasable pool (RRP), the reserve pool, and the resting pool (3, 16, 17). The RRP is defined as being composed of docked and immediately releasable vesicles (17), which are usually depleted by high-frequency stimulation, prolonged presynaptic depolarization, or the application of hypertonic solution (18–21). The reserve pool functions as a reservoir and serves to maintain vesicle refilling into the RRP (2, 3). These two pools together are commonly referred to as the RP. The resting pool serves as a depot of vesicles for backup use (16, 22). However, it has been debated for a decade whether nerve terminals use the majority (∼100%, from electrophysiology) or only a small fraction (5–40%, from fluorescence imaging and EM) of vesicles in recycling, and whether the RP size undergoes dynamic changes during varied neuronal activity (6, 7, 23–28).The use of vesicles in recycling is a critical determinant of synaptic transmission (1, 13–15). However, it has never been rigorously determined how fast recently recaptured vesicles are organized to recycle and whether vesicles in the RP are homogeneously ready for use (25). Two forms of vesicle retrieval, “kiss-and-run” and full collapse, have been reported for many years. It is still ambiguous whether the rapidly recaptured vesicles in the kiss-and-run mode can be rapidly reused (29–31).Here, we addressed the above issues by developing a new approach to quantify the basic properties of vesicle recycling with unparalleled precision. Different from previous studies in cultured cell systems, the present work combined electrophysiological measurements and EM observations at the calyx of Held synapse in acute brain slices, quantitatively analyzed synaptic vesicle recycling, and kinetically dissected the recycling vesicle pool. We propose a realistic kinetic model and provide new insights into the mechanism that ensures rate-limited but sustainable synaptic transmission.     

Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development

Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.The auxin binding protein 1 (ABP1) was first isolated from maize plants based on its ability to bind auxin (1). The crystal structure of ABP1 demonstrated clearly that ABP1 has an auxin-binding pocket and, indeed, binds auxin (2). However, the elucidation of the physiological functions of ABP1 has been challenging because the first reported abp1 T-DNA insertion mutant in Arabidopsis was not viable (3). Nevertheless, ABP1 has been recognized as an essential gene for plant development and as a key component in auxin signaling (4–9). Because viable abp1 null mutants in Arabidopsis were previously unavailable, alternative approaches have been used to disrupt ABP1 function in Arabidopsis to determine the physiological roles of the protein. Cellular immunization approaches were used to generate ABP1 knockdown plants (10, 11). Inducible overexpression of the single chain fragment variable regions (scFv12) of the anti-ABP1 monoclonal antibody mAb12 both in cell lines and in Arabidopsis plants presumably neutralizes the endogenous ABP1 activities (10, 11). Two such antibody lines, SS12S and SS12K, have been widely used in many ABP1-related studies (4, 6, 9–11). The results obtained from the characterization of the antibody lines suggest that ABP1 regulates cell division, cell expansion, meristem activities, and root development (4, 6, 10, 12, 13). Transgenic plants that overexpress ABP1 antisense RNA were also used to elucidate the physiological functions of ABP1 (4, 10). Moreover, missense point mutation alleles of abp1 have also been generated through the Arabidopsis TILLING project. One such TILLING mutant, named abp1-5, harbors a mutation (His94 >Tyr) in the auxin-binding pocket and has been widely used in many ABP1-related studies (4, 8, 9). Previous studies based on the antisense lines, antibody lines, and Arabidopsis mutant alleles have led to the conclusion that ABP1 is essential for embryogenesis, root development, and many other developmental processes. However, the interpretation of results generated by using the ABP1 antisense and antibody lines are not straightforward and off-target effects have not been completely ruled out. We believe that characterization of abp1 null plants is urgently needed to unambiguously define the roles of ABP1 in auxin signaling and in plant development.In the past several years, studies of the presumed ABP1-mediated auxin signal transduction pathway were carried out in several laboratories. It has been hypothesized that ABP1 is an auxin receptor mediating fast, nongenomic effects of auxin (4–6, 8, 9), whereas the TIR1 family of F-box protein/auxin receptors are responsible for auxin-mediated gene regulation (14, 15). One of the proposed functions of ABP1 is to regulate subcellular distribution of PIN auxin efflux carriers (6, 9, 13). Furthermore, a recent report suggests that a cell surface complex consisting of ABP1 and transmembrane receptor-like kinases functions as an auxin receptor at the plasma membrane by activating the Rho-like guanosine triphosphatases (GTPases) (ROPs) in an auxin-dependent manner (8). ROPs have been reported to play a role in regulating cytoskeleton organization and PIN protein endocytosis (5, 6). However, it is important to unequivocally determine the biological processes that require ABP1 before extensive efforts are directed toward elucidating any ABP1-mediated signaling pathways.In this paper, we generate and characterize new abp1 null mutants in Arabidopsis. We are interested in elucidating the molecular mechanisms by which auxin regulates flower development because our previously identified auxin biosynthetic mutants display dramatic floral defects (16–18). Because ABP1 was reported as an essential gene and ABP1 binds auxin (2, 3), we decided to determine whether ABP1 plays a role in flower development. We used our recently developed ribozyme-based CRISPR gene editing technology (19) to specifically inactivate ABP1 during flower development. Unexpectedly, we recovered a viable abp1 mutant (abp1-c1, c stands for alleles generated by using CRISPR) that contains a 5-bp deletion in the first exon of ABP1. We also isolated a T-DNA abp1 allele (abp1-TD1) that harbors a T-DNA insertion in the first exon of ABP1. We show that both abp1-c1 and abp1-TD1 are null mutants. Surprisingly, the mutants were indistinguishable from wild-type (WT) plants at all of the developmental stages we analyzed. Our data clearly demonstrate that ABP1 is not an essential gene and that ABP1 does not play a major role in auxin signaling and Arabidopsis development under normal growth conditions.     

Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system

Chronic infection with hepatitis C virus (HCV) is an important cause of end stage liver disease worldwide. In the United States, most HCV-related disease is associated with genotype 1 infection, which remains difficult to treat. Drug and vaccine development was hampered by inability to culture patient isolates representing HCV genotypes 1–7 and subtypes; only a recombinant 2a genome (strain JFH1) spontaneously replicated in vitro. Recently, we identified three mutations F1464L/A1672S/D2979G (LSG) in the nonstructural (NS) proteins, essential for development of full-length HCV 2a (J6) and 2b (J8) culture systems in Huh7.5 cells. Here, we developed a highly efficient genotype 1a (strain TN) full-length culture system. We initially found that the LSG substitutions conferred viability to an intergenotypic recombinant composed of TN 5′ untranslated region (5′UTR)-NS5A and JFH1 NS5B-3′UTR; recovered viruses acquired two adaptive mutations located in NS3 and NS4B. Introduction of these changes into a replication-deficient TN full-length genome, harboring LSG, permitted efficient HCV production. Additional identified NS4B and NS5B mutations fully adapted the TN full-length virus. Thus, a TN genome with 8 changes (designated TN cell-culture derived, TNcc) replicated efficiently and released infectious particles of ∼5 log₁₀ focus-forming units per mL; passaged TNcc did not require additional changes. IFN-α and directly acting antivirals targeting the HCV protease, NS5A, and NS5B, each inhibited full-length TN infection dose-dependently. Given the unique importance of genotype 1 for pathogenesis, this infectious 1a culture system represents an important advance in HCV research. The approach used and the mutations identified might permit culture development for other HCV isolates, thus facilitating vaccine development and personalized treatment.Hepatitis C virus (HCV) chronically infects an estimated 130–170 million people worldwide. The infection increases the risk of developing liver cirrhosis and liver cancer and results in more than 350,000 deaths annually. No HCV vaccine is available. Current standard treatment is based on IFN-α/ribavirin, which, however, has low efficacy against the most prevalent HCV variants (1). Incorporation of directly acting antivirals (DAAs) in treatment regimens improves sustained viral response rate, but a favorable outcome is challenged by fast emergence of drug resistance and differential responses of the different HCV genotypes (2). Thus, HCV infection continues to be a huge health and economic burden to the world population, and improved in vitro experimental systems would be important to permit additional studies of new antivirals and associated resistance patterns.HCV is a small enveloped virus belonging to the genus Hepacivirus in the family Flaviviridae. The HCV genome is a positive-sense single-strand RNA (∼9.6 kb), consisting of a single ORF flanked by 5′ and 3′ untranslated regions (UTRs). The ORF encodes virus structural proteins (core, E1, and E2), p7, and six nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (1). HCV isolates are classified into seven major variants (genotypes 1–7) and numerous subtypes (a, b, etc.) differing by ∼30% and ∼20%, respectively, in nucleotide and amino acid sequences (3). Genotype 1 is the most prevalent in the world. In the United States, Japan, China, and southern Europe over 70% of HCV patients are infected with genotype 1 (4–7). In northern Europe, genotype 1 accounts for ∼50% of HCV infections (8). Genotype 1 infection is more resistant to IFN-based treatment than infection with other genotypes (1).In vitro HCV culture systems are needed to study treatment regimens and associated viral resistance. However, drug and vaccine development have been greatly hampered by inability to culture patient isolates in vitro. Although a number of HCV full-length clones were shown to be infectious in chimpanzees (ref. 9 and cited references therein), only JFH1 (genotype 2a) spontaneously replicated in human hepatoma cells (Huh7 and its derivatives) and released infectious virus particles (10, 11). Efficient growth of JFH1 required culture adaptive mutations (12). Recently, we reported efficient J6cc (2a) and J8cc (2b) full-length culture systems (13); subsequently Date et al. reported on a full-length system for 2a strain JFH2 (14). A single full-length genotype 1a genome, H77-S, carrying mutations identified in the subgenomic replicon of the same strain, has been reported to release relatively small amounts of virus particles (15). The Con1 (1b) full-length culture system was reported, but a very low level of replication has limited its utility (16). Thus, efficient HCV full-length culture systems remained limited to genotype 2 isolates. The clinical importance of the marked genetic differences between HCV genotype isolates poses a critical need for development of robust full-length culture systems for HCV genotype 1 isolates.The unique replication capacity of JFH1 has permitted the development of JFH1-based HCV recombinants (17); we and others have reported different inter- and intragenotypic recombinants including core-NS2 (17–24), 5′UTR-NS2 (25), NS3 protease/NS4A (26, 27), NS5A (28), and core-NS3 protease plus NS4A-NS5A (29) of various genotypes. These JFH1-based culture systems have been used for genotype-specific studies of HCV genes (19, 20, 22, 25–28, 30), for testing of HCV DAAs (26–28) and neutralizing antibodies (19, 20, 22, 30), and for studying aspects of virus–host interaction (25, 31, 32). Through studies of J6 (2a) recombinants with the entire or partial NS5B and 3′UTR from JFH1, we recently identified adaptive mutations F1464L in NS3 and A1672S in NS4A, designated LS (13). Combination of LS with selected mutations in NS5B and the 3′UTR allowed replication of the full-length J6 genome, which led to the identification of an additional unique mutation D2979G in NS5B, designated G (13). The LSG substitutions permitted the development of robust HCV full-length culture systems for J6 and for the prototype genotype 2b J8 strain (13). In this study, we used LSG as a critical component and a unique approach to develop a highly efficient full-length genotype 1a (strain TN) infectious culture system, named TN cell-culture derived (TNcc). The TNcc replicated efficiently in culture, released viral particles of ∼10⁵ focus-forming units (FFU)/mL and did not require additional mutations after viral passage. We demonstrated that full-length TN responded dose dependently to IFN-α, as well as HCV DAAs already used in the clinic and being tested in clinical trials.     

Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.Poly(ADP-ribose) polymerase (PARP) inhibition provides a promising therapeutic strategy for targeting homologous recombination (HR)-deficient tumors, such as breast cancer 1 (BRCA1)-mutated cancers (1). Indeed, clinical phase I and phase II trials have shown potent anticancer activity of small molecule inhibitors of PARP, such as olaparib, in patients with BRCA1-associated breast cancer (2, 3). However, it remains to be established whether different breast cancer subtypes in BRCA1 mutation carriers respond equally to PARP inhibition. Reduced sensitivity of breast cancers to anticancer drugs has frequently been associated with an epithelial-to-mesenchymal transition (EMT) (4–7). Metaplastic breast carcinomas (MBCs) are a subset of triple-negative breast cancers (TNBCs) characterized by a claudin-low and EMT-like phenotype (8) and a poor prognosis compared with other TNBCs (9). More than 60% of MBCs have BRCA1 promoter methylation, raising the question whether these tumors can be effectively targeted by using PARP inhibitors (10). To address this issue in an experimentally controlled setting, we set out to generate a genetically engineered mouse model (GEMM) of BRCA1-deficient MBC by inducing EMT via MET overexpression in a previously established GEMM of BRCA1-mutated breast cancer. We report that EMT is associated with olaparib resistance and can be effectively bypassed by administration of AZD2461, a PARP inhibitor with low affinity for the P-glycoprotein (Pgp) drug efflux transporter.