The architecture of Tetrahymena telomerase holoenzyme.

Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis.

In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed "Inverted Repeat Binding Protein (IRBP) 18" and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.

An RPA-related sequence-specific DNA-binding subunit of telomerase holoenzyme is required for elongation processivity and telomere maintenance.

Telomerase ribonucleoprotein complexes copy an internal RNA template to synthesize DNA repeats. DNA-interacting subunits other than telomerase reverse transcriptase (TERT) and telomerase RNA (TER) have been hypothesized to account for high repeat addition processivity of telomerase holoenzyme compared to the minimal catalytic RNP. Here, we present the identification of three additional subunits of Tetrahymena thermophila telomerase holoenzyme. Each of seven telomerase proteins is required for telomere maintenance and copurifies active RNP. The catalytic core (p65-TER-TERT) is assembled with a three-protein subcomplex (p75-p45-p19) and two peripheral subunits (p82 and p50). Remarkably, only a p82-enriched subset of the total holoenzyme population is capable of high repeat addition processivity, as shown by p82 immunodepletion and add-back. The RPA-like p82 subunit binds sequence specifically to multiple telomeric repeats. These discoveries establish the existence of a telomerase holoenzyme processivity subunit with sequence-specific DNA binding.

Structural basis for Tetrahymena telomerase processivity factor Teb1 binding to single-stranded telomeric-repeat DNA.

Telomerase copies its internal RNA template to synthesize telomeric DNA repeats. Unlike other polymerases, telomerase can retain its single-stranded product through multiple rounds of template dissociation and repositioning to accomplish repeat addition processivity (RAP). Tetrahymena telomerase holoenzyme RAP depends on a subunit, Teb1, with autonomous DNA-binding activity. Sequence homology and domain modeling suggest that Teb1 is a paralog of RPA70C, the largest subunit of the single-stranded DNA-binding factor replication protein (RPA), but unlike RPA, Teb1 binds DNA with high specificity for telomeric repeats. To understand the structural basis and significance of telomeric-repeat DNA recognition by Teb1, we solved crystal structures of three proposed Teb1 DNA-binding domains and defined amino acids of each domain that contribute to DNA interaction. Our studies indicate that two central Teb1 DNA-binding oligonucleotide/oligosaccharide-binding-fold domains, Teb1A and Teb1B, achieve high affinity and selectivity of telomeric-repeat recognition by principles similar to the telomere end-capping protein POT1 (protection of telomeres 1). An additional C-terminal Teb1 oligonucleotide/oligosaccharide-binding-fold domain, Teb1C, has features shared with the RPA70 C-terminal domain including a putative direct DNA-binding surface that is critical for high-RAP activity of reconstituted holoenzyme. The Teb1C zinc ribbon motif does not contribute to DNA binding but is nonetheless required for high-RAP activity, perhaps contributing to Teb1 physical association with the remainder of the holoenzyme. Our results suggest the biological model that high-affinity DNA binding by Teb1AB recruits holoenzyme to telomeres and subsequent Teb1C-DNA association traps product in a sliding-clamp-like manner that does not require high-affinity DNA binding for high stability of enzyme-product association.

Multiple mechanisms for elongation processivity within the reconstituted tetrahymena telomerase holoenzyme.

To maintain telomeres, telomerase evolved a unique biochemical activity: the use of a single-stranded RNA template for the synthesis of single-stranded DNA repeats. High repeat addition processivity (RAP) of the Tetrahymena telomerase holoenzyme requires association of the catalytic core with the telomere adaptor subcomplex (TASC) and an RPA1-related subunit (p82 or Teb1). Here, we used DNA binding and holoenzyme reconstitution assays to investigate the mechanism by which Teb1 and TASC confer high RAP. We show that TASC association with the recombinant telomerase catalytic core increases enzyme activity. Subsequent association of the Teb1 C-terminal domain with TASC confers the capacity for high RAP even though the Teb1 C-terminal domain does not provide a high-affinity DNA interaction site. Efficient RAP also requires suppression of nascent product folding mediated by the central Teb1 DNA-binding domains (DBDs). These sequence-specific high-affinity DBDs of Teb1 can be functionally substituted by the analogous DBDs of Tetrahymena Rpa1 to suppress nascent product folding but only if the Rpa1 high-affinity DBDs are physically tethered into holoenzyme context though the Teb1 C-terminal domain. Overall, our findings reveal multiple mechanisms and multiple surfaces of protein-DNA and protein-protein interaction that give rise to elongation processivity in the synthesis of a single-stranded nucleic acid product.

ALK, ROS1, and NTRK Rearrangements in Metastatic Colorectal Cancer.

Background: ALK, ROS1, and NTRK fusions occur in 0.2% to 2.4% of colorectal cancers. Pioneer cases of metastatic colorectal cancer (mCRC) patients bearing rearrangements who benefited from anti-ALK, ROS, and TrkA-B-C therapies have been reported previously. Here we aimed at characterizing the clinical and molecular landscape of ALK, ROS1, and NTRK rearranged mCRC. Methods: Clinical features and molecular characteristics of 27 mCRC patients bearing ALK, ROS1, and NTRK rearranged tumors were compared with those of a cohort of 319 patients not bearing rearrangements by means of Fisher's exact, χ2 test, or Mann-Whitney test as appropriate. Overall survival curves were estimated with the Kaplan-Meier method and compared using the log-rank test. A Cox proportional hazard model was adopted in the multivariable analysis. Deep molecular and immunophenotypic characterizations of rearranged cases, including those described in The Cancer Genome Atlas database, were performed. All statistical tests were two-sided. Results: Closely recalling the "BRAF history," ALK, ROS1, and NTRK rearrangements more frequently occurred in elderly patients (P = .02) with right-sided tumors (P < .001) and node-spreading (P = .03), RAS wild-type (P < .001), and MSI-high (P < .001) cancers. All patients bearing ALK, ROS1, and NTRK fusions had shorter overall survival (15.6 months, 95% confidence interval [CI] = 0.0 to 20.4 months) than negative patients (33.7 months, 95% CI = 28.3 to 42.1 months), both in the univariate (hazard ratio [HR] = 2.17, 95% CI = 1.03 to 4.57, P < .001) and multivariable models (HR = 2.33, 95% CI = 1.10 to 4.95, P = .02). All four evaluable patients with rearrangements showed primary resistance to anti-epidermal growth factor receptor agents. Frequent association with potentially targetable RNF43 mutations was observed in MSI-high rearranged tumors. Conclusions: ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.

P element excision and repair by non-homologous end joining occurs in both G1 and G2 of the cell cycle.

P element excision generates a DNA double-strand break at the transposon donor site. Genetic studies have demonstrated a strong bias toward repair of P element-induced DNA breaks by homologous recombination with the sister chromatid, suggesting that P element excision occurs after DNA replication, in G2 of the cell cycle. We developed methods to arrest Drosophila tissue culture cells and assay P element excision in either G1- or G2-arrested cells. Dacapo or tribbles transgene expression arrests cells in either G2 or G2, respectively. RNA-mediated gene interference (RNAi) directed against cyclin E or cyclin A arrests cells in G1 or G2, respectively. P element excision occurs efficiently in both G1- and G2-arrested cells, suggesting that cell cycle regulation of P element transposase does not occur in our somatic cell system. DNA double-strand break repair occurs by two predominant mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is thought to be restricted to the post-replicative, G2, phase of the cell cycle, while NHEJ may occur throughout the cell cycle. Our results indicate that NHEJ repair of an extrachromasomal plasmid substrate occurs at least as efficiently in G2-arrested cells as in asynchronous cells or in G1-arrested cells.

YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression.

Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach.

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.

Interplay between Drosophila Bloom's syndrome helicase and Ku autoantigen during nonhomologous end joining repair of P element-induced DNA breaks.

P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. The broken DNA ends generated during transposition can be repaired via the homology-directed synthesis-dependent strand annealing or by nonhomologous end joining (NHEJ). Genetic studies have demonstrated an interaction between the gene (mus309, for mutagen-sensitive) encoding the Drosophila Bloom's syndrome helicase homolog (DmBLM) and the Ku70 gene, which is involved in NHEJ. We have used RNA interference (RNAi) to knock down expression of DmBLM and one or both of the Drosophila Ku subunits, DmKu70 or DmKu80. Our results show that upon reduction of DmKu, an increase in small deletions (1-49 bp) and large deletions (>/=50 bp) flanking the site of P element-induced breaks is observed, and a reduction in large deletions at these sites is found upon reduction of DmBLM. Moreover, double RNAi of DmKu and DmBLM results in an increase in small deletions characteristic of the DmKu RNAi and also partially suppresses the reduction in repair efficiency observed with DmKu RNAi. These results suggest that there are DNA double-strand break recognition and/or processing events involving DmKu and DmBLM that, when eliminated by RNAi, lead to deletions. Finally, these results raise the possibility that, unlike the situation in mammals, where BLM appears to function exclusively in the homologous repair pathway, in Drosophila, DmBLM may be directly involved in, or at least influence the double-strand break recognition that leads to the NHEJ repair pathway.