FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guaninerich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.The identification of FANCJ mutations in early-onset breast cancer patients (3, 34) and Fanconi anemia (FA) group J patients (22,23,25) implicates FANCJ as a tumor suppressor caretaker that ensures genomic stability. FANCJ interacts with the tumor suppressor BRCA1 (3) and is a DNA helicase that catalytically unwinds duplex DNA in an ATP hydrolysis-dependent manner (2, 14). FA-J cells are hypersensitive to interstrand cross-linking (ICL) agents (1, 25); untreated FA-J cells exhibit diminished BRCA1 foci, and the cells show delayed formation of ionizing radiation-induced BRCA1 foci (31). In response to DNA damage or replicational stress, FANCJ colocalizes with the single-stranded DNA (ssDNA) binding protein replication protein A (RPA), which serves as an auxiliary factor for the unwinding function of FANCJ (15). FANCJ interacts with the mismatch repair complex MutL␣, and this interaction is required for the correction of the ICL response in FA-J cells (32). The activation of FANCJ helicase activity is required for timely progression through S phase of the cell cycle (20); however, the precise functions of the FANCJ helicase in S-phase progression remain to be understood.Although a role for FANCJ in DNA replication has been proposed previously, definitive evidence for a role of the FANCJ helicase in preventing genomic instability is lacking. One source of genomic instability is alternate DNA structures that may impede the replication fork. Guanine-rich nucleic acids have the potential to f...

Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.terpene synthase | bacteria | heterologous expression

Quadruplex ligands are often considered as telomerase inhibitors. Given the fact that some of these molecules are present in the clinical setting, it is important to establish the validity of this assertion. To analyze the effects of these compounds, we used a direct assay with telomerase-enriched extracts. The comparison of potent ligands from various chemical families revealed important differences in terms of effects on telomerase initiation and processivity. Although most quadruplex ligands may lock a quadruplex-prone sequence into a quadruplex structure that inhibits the initiation of elongation by telomerase, the analysis of telomerase-elongation steps revealed that only a few molecules interfered with the processivity of telomerase (i.e., inhibit elongation once one or more repeats have been incorporated). The demonstration that these molecules are actually more effective inhibitors of telomeric DNA amplification than extension by telomerase contributes to the already growing suspicion that quadruplex ligands are not simple telomerase inhibitors but, rather, constitute a different class of biologically active molecules. We also demonstrate that the popular telomeric repeat amplification protocol is completely inappropriate for the determination of telomerase inhibition by quadruplex ligands, even when PCR controls are included. As a consequence, the inhibitory effect of many quadruplex ligands has been overestimated.G-quadruplex ͉ telomere ͉ telomeric repeat amplification protocol assay ͉ direct assay T he unlimited proliferative potential of cancer cells depends on telomere maintenance (1). As a consequence, several classes of telomerase inhibitors have been developed for anticancer purposes. Optimal telomerase activity requires an unfolded single-stranded telomeric overhang (2-4). This overhang may adopt an unusual DNA structure called a G-quadruplex, composed of G-quartets and formed in the presence of cations (5). Therefore, ligands that selectively bind to and stabilize telomeric G-quadruplex structures could act as indirect telomerase inhibitors (Fig. 1A) (6). The number of identified G-quadruplex ligands has grown rapidly over the past few years (for a recent review, see ref. 7); some of these molecules exhibit potent in vivo antitumor activity, and clinical trials started recently.Although various methods are available to measure telomerase activity, the telomeric repeat amplification protocol (TRAP) is most widely used (8). With a few notable exceptions (6, 9-12), TRAP [or a variant called TRAP-G4 (13)] has been the standard method to measure telomerase inhibition by G-quadruplex ligands (reviewed in ref. 7). The original method was improved by the use of an internal control (often called ITAS for internal telomerase assay standard) that coamplifies with the telomerase products and allows the detection of nonspecific Taq polymerase inhibitors. Notably, the ITAS product does not contain any telomeric sequence and therefore is not able to form a G-quadruplex (5,14). We demonstrate here that TRAP is...

An industrial microorganism Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.

Mps One Binder Kinase Activator (MOB)1A/1B are core components of the Hippo pathway that coactivate large tumor suppressor homolog (LATS) kinases. Mob1a/1b double deficiency in mouse liver (LMob1DKO) results in hyperplasia of oval cells and immature cholangiocytes accompanied by inflammatory cell infiltration and fibrosis. More than half of mutant mice die within 3 wk of birth. All survivors eventually develop liver cancers, particularly combined hepatocellular and cholangiocarcinomas (cHC-CCs) and intrahepatic cholangiocellular carcinomas (ICCs), and die by age 60 wk. Because this phenotype is the most severe among mutant mice lacking a Hippo signaling component, MOB1A/1B constitute the critical hub of Hippo signaling in mammalian liver. LMob1DKO liver cells show hyperproliferation, increased cell saturation density, hepatocyte dedifferentiation, enhanced epithelial–mesenchymal transition and cell migration, and elevated transforming growth factor beta(TGF-β)2/3 production. These changes are strongly dependent on Yes-Associated Protein-1 (Yap1) and partially dependent on PDZ-binding motif (Taz) and Tgfbr2, but independent of connective tissue growth factor (Ctgf). In human liver cancers, YAP1 activation is frequent in cHC-CCs and ICCs and correlates with SMAD family member 2 activation. Drug screening revealed that antiparasitic macrocyclic lactones inhibit YAP1 activation in vitro and in vivo. Targeting YAP1/TAZ with these drugs in combination with inhibition of the TGF-β pathway may be effective treatment for cHC-CCs and ICCs.