Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Telomerase is a ribonucleoprotein enzyme that adds telomeric repeats to chromosomes, maintaining telomere length and stabilizing chromosome ends. In vitro, telomerase from the ciliate Tetrahymena elongates single-stranded, guanosine-rich DNA primers by adding repeats of the Tetrahymena telomeric sequence, dT2G4. We have identified two activities of Tetrahymena telomerase in addition to the previously described processive elongation reaction: a 3'-5' nucleolytic cleavage of primer or product DNA and a nonprocessive mode of elongation. The nucleolytic cleavage activity removed residues not conforming to the telomeric repeat sequence from a primer 3' end, eliminating mismatch between DNA primer and RNA template sequences. Template-matched residues were also cleaved from primer or product DNA. Specific primer lengths, sequences, and concentrations stimulated cleavage and processive or nonprocessive elongation differentially. These newly identified activities suggest that telomerase may catalyze a range of telomere synthesis and repair functions and suggest mechanistic similarities between telomerase and RNA polymerase enzymes. On the basis of our results, we propose a model for telomerase primer binding, cleavage, and elongation.
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PMID:Tetrahymena telomerase catalyzes nucleolytic cleavage and nonprocessive elongation. 833 Jul 40

Estrogen-like chemicals are unique compared to nonestrogenic xenobiotics, because in addition to their chemical properties, the estrogenic property of these compounds allows them to act like sex hormones. Whether weak or strong, the estrogenic response of a chemical, if not overcome, will add extra estrogenic burden to the system. At elevated doses, natural estrogens and environmental estrogen-like chemicals are known to produce adverse effects. The source of extra or elevated concentration of estrogen could be either endogenous or exogenous. The potential of exposure for humans and animals to environmental estrogen-like chemicals is high. Only a limited number of estrogen-like compounds, such as diethylstilbestrol (DES), bisphenol A, nonylphenol, polychlorinated biphenyls (PCBs), and dichlorodiphenyltrichloroethane (DDT), have been used to assess the biochemical and molecular changes at the cellular level. Among them, DES is the most extensively studied estrogen-like chemical, and therefore this article is focused mainly on DES-related observations. In addition to estrogenic effects, environmental estrogen-like chemicals produce multiple and multitype genetic and/or nongenetic hits. Exposure of Syrian hamsters to stilbene estrogen (DES) produces several changes in the nuclei of target organ for carcinogenesis (kidney): (1) Products of nuclear redox reactions of DES modify transcription regulating proteins and DNA; (2) transcription is inhibited; (3) tyrosine phosphorylation of nuclear proteins, including RNA polymerase II, p53, and nuclear insulin-like growth factor-1 receptor, is altered; and (4) DNA repair gene DNA polymerase beta transcripts are decreased and mutated. Exposure of Noble rats to DES also produces several changes in the mammary gland: proliferative activity is drastically altered; the cell cycle of mammary epithelial cells is perturbed; telomeric length is attenuated; etc. It appears that some other estrogenic compounds, such as bisphenol A and nonylphenol, may also follow a similar pattern of effects to DES, because we have recently shown that these compounds alter cell cycle kinetics, produce telomeric associations, and produce chromosomal aberrations. Like DES, bisphenol A after metabolic activation is capable of binding to DNA. However, it should be noted that a particular or multitype hit(s) will depend upon the nature of the environmental estrogen-like chemical. The role of individual attack leading to a particular change is not clear at this stage. Consequences of these multitypes of attack on the nuclei of cells could be (1) nuclear toxicity/cell death; (2) repair of all the hits and then acting as normal cells; or (3) sustaining most of the hits and acting as unstable cells. Proliferation of the last type of cell is expected to result in transformed cells.
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PMID:Biochemical and molecular changes at the cellular level in response to exposure to environmental estrogen-like chemicals. 901 29

The EWS gene is fused in Ewing sarcoma-like tumors by a chromosomal translocation to one of the four ETS-family genes: FLI1, ERG, ETV1, and E1AF. The orientation of EWS and FLI1 on chromosomes 22 and 11, respectively, is 5' centromeric and 3' telomeric, whereas that of ERG on chromosome 21 is the reverse. Although 10% of Ewing-family tumors express the EWS-ERG fusion transcript, there have been no reports on tumors with t(21;22)(q22;q12) identified by banding cytogenetics. We found the karyotype 50, XY, +8, +8, +12, +mar in all metaphase cells from a tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis performed on the tumor and direct sequencing of the products identified the EWS-ERG fusion transcript. Subsequent two-color fluorescence in situ hybridization (FISH) analysis with EWS and ERG clones showed the fused signals on the der(21) chromosome, but no ERG signals on the chromosome 22 homologs. Thus, our RT-PCR and FISH analyses indicated that the chromosome 22 fragment containing the 5' portion of EWS had been inverted and inserted into chromosome 21 and had fused to the 3' portion of ERG. This subtle chromosome aberration could not be identified by routine cytogenetics. A chromosomal inversion/insertion has also been described in acute leukemia with the MLL-AF10 fusion gene, and this may be a common pathway for producing fusion of reverse-oriented genes in leukemias and solid tumors.
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PMID:EWS-ERG fusion transcript produced by chromosomal insertion in a Ewing sarcoma. 907 76

Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W. Jiang, K. Middleton, H.-J. Yoon, C. Fouquet, and J. Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolar-cytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperature-sensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5delta null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.
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PMID:The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3. 931 78

It has been previously shown that genes transcribed by RNA polymerase II (RNAP II) are subject to position effect variegation when located near yeast telomeres. This telomere position effect requires a number of gene products that are also required for silencing at the HML and HMR loci. Here, we show that a null mutation of the DNA repair gene RAD6 reduces silencing of the HM loci and lowers the mating efficiency of MATa strains. Likewise, rad6-delta reduces silencing of the telomere-located RNAP II-transcribed genes URA3 and ADE2. We also show that the RNAP III-transcribed tyrosyl tRNA gene, SUP4-o, is subject to position effect variegation when located near a telomere and that this silencing requires the RAD6 and SIR genes. Neither of the two known Rad6 binding factors, Rad18 and Ubr1, is required for telomeric silencing. Since Ubrl is the recognition component of the N-end rule-dependent protein degradation pathway, this suggests that N-end rule-dependent protein degradation is not involved in telomeric silencing. Telomeric silencing requires the amino terminus of Rad6. Two rad6 point mutations, rad6(C88A) and rad6(C88S), which are defective in ubiquitin-conjugating activity fail to complement the silencing defect, indicating that the ubiquitin-conjugating activity of RAD6 is essential for full telomeric silencing.
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PMID:The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae. 934 33

Mechanistic and regulatory aspects of meiotic chromosome pairing and segregation have received increasing attention in recent years. This review is concerned with the role of chromosomal sites and chromosome organization in pairing and sperm development in Drosophila. Two major topics are reviewed. The first concerns the distribution and identification of meiotic pairing sites in male Drosophila. Cytogenetic data show that pairing sites are distributed widely in the euchromatin of autosomes but are absent from centromeric heterochromatin. The reverse distribution holds for the X, where the major pairing site is located in the central region of the centric heterochromatin, co-mapping with the rDNA locus. Recent transgenic studies have demonstrated that this pairing site consists mainly of a 240-bp repeated sequence in the intergenic spacers of the rDNA repeats. These spacer repeats contain RNA polymerase I promoters, which must be functional for the repeats to have pairing activity, suggesting a mechanistic connection between pairing and transcription. The general idea that pairing sites coincide with transcribed sequences is discussed. The second major topic involves the effects of sex chromosome rearrangements on spermiogenesis. A variety of rearrangements involving the sex chromosomes, including heterochromatic deletions and translocations with autosomes, have been shown to lead either to meiotic drive or to sterility. Recent evidence strongly implicates the X chromosome pairing site in the etiology of these effects. These findings are discussed in terms of a novel model that interprets the spermiogenic disruptions associated with sex chromosome rearrangements as resulting from disabling of spermatids due to triggering of a checkpoint concerned with monitoring chromosome alignment at meiotic metaphase.
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PMID:Pairing sites and the role of chromosome pairing in meiosis and spermatogenesis in male Drosophila. 935 84

Telomerase, a specialized cellular reverse transcriptase, compensates for chromosome shortening during the proliferation of most eucaryotic cells and contributes to cellular immortalization. The mechanism used by the single-celled protozoan malaria parasite Plasmodium falciparum to complete the replication of its linear chromosomes is currently unknown. In this study, telomerase activity has for the first time been identified in cell extracts of P. falciparum. The de novo synthesis of highly variable telomere repeats to the 3' end of DNA oligonucleotide primers by plasmodial telomerase is demonstrated. Permutated telomeric DNA primers are extended by the addition of the next correct base. In addition to elongating preexisting telomere sequences, P. falciparum telomerase can also add telomere repeats onto nontelomeric 3' ends. The sequence GGGTT was the predominant initial DNA sequence added to the nontelomeric 3' ends in vitro. Poly(C) at the 3' end of the oligonucleotide significantly alters the precision of the new telomerase added repeats. The efficiency of nontelomeric primer elongation was dependent on the presence of a G-rich cassette upstream of the 3' terminus. Oligonucleotide primers based on natural P. falciparum chromosome breakpoints are efficiently used as telomerase substrates. These results imply that P. falciparum telomerase contributes to chromosome maintenance and to de novo telomere formation on broken chromosomes. Reverse transcriptase inhibitors such as dideoxy GTP efficiently inhibit P. falciparum telomerase activity in vitro. These data point to malaria telomerase as a new target for the development of drugs that could induce parasite cell senescence.
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PMID:Plasmodium falciparum telomerase: de novo telomere addition to telomeric and nontelomeric sequences and role in chromosome healing. 944 88

The telomeric sequence repeats at the ends of eukaryotic chromosomes are maintained by the ribonucleoprotein enzyme telomerase. Telomeric DNA primers are bound by telomerase both at the active site, which includes base-pairing with the RNA template, and at a second anchor site. The stabilities of Euplotes aediculatus primer-telomerase complexes were determined by measuring their dissociation rates (koff), using an assay involving photo-cross-linking at the anchor site. The primer length was varied, and mismatched substitutions were introduced in a systematic manner. We observed that koff does not scale with primer length as expected for accumulated primer-template base-pairing. This suggests that telomerase maintains a more-or-less constant number of base pairs, similar to the transcription bubble maintained by RNA polymerase. An upper limit was estimated by comparing the experimental koff for the primer-telomerase complex to that of a model DNA-RNA duplex. All the binding energy could be attributed to 10 or 11 base pairs; alternatively, there could be <10 base pairs, with the remaining energy contributed by other parts of telomerase. Most primers exhibited biphasic dissociation kinetics, with variations in both the amount in each phase and the rate for each phase. Since the cross-links monitored in the dissociation assay were all formed with the 5' region of the primer, the two phases may arise from different base-pairing registers with the RNA template, possibly representing pre- and post-translocation complexes. A shift from slow phase to fast phase dissociation was observed in the presence of dGTP, which may implicate dGTP as a positive effector of translocation.
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PMID:Euplotes telomerase: evidence for limited base-pairing during primer elongation and dGTP as an effector of translocation. 954 47

Simple sequence repeat telomeric DNA is maintained by a specialized reverse transcriptase, telomerase. The integral RNA subunit of telomerase contains a template region that determines the sequence added to chromosome ends. Aside from providing the template, little is known about the role of the telomerase RNA. In addition, no hypotheses have been suggested to account for the striking evolutionary divergence in size and sequence between telomerase RNAs of ciliates, yeasts, and mammals. We show that the two- to threefold increase in size of the mammalian telomerase RNAs relative to ciliate telomerase RNAs is due to the presence of an extra domain resembling a box H/ACA small nucleolar RNA (snoRNA). The human telomerase RNA (hTR) H/ACA domain is essential in vivo for hTR accumulation, hTR 3' end processing, and telomerase activity. By substituting the U64 box H/ACA snoRNA for the hTR H/ACA domain, we demonstrate that a heterologous snoRNA can function to promote chimeric RNA accumulation and 3' end processing but not telomerase activity. In addition, we show that maturation of full-length hTR and its assembly into active telomerase occur from an mRNA promoter-driven RNA polymerase II transcript but not from a U6 snRNA promoter-driven RNA polymerase III transcript. Finally, we show that a small percentage of hTR is associated with nucleoli. These results have implications for the biogenesis and structure of hTR and the human telomerase ribonucleoprotein complex. They also expand the structural and functional diversity of the box H/ACA snoRNA motif.
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PMID:A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end. 985 80

Telomerase is a ribonucleoprotein complex with reverse-transcriptase activity responsible for telomere reconstitution. High telomerase activity was found in cancer cells, but not in differentiated homologous nonmalignant tissues. We demonstrated previously that the disappearance of telomerase activity is a reliable marker of tumor cell killing in human cancer cell lines. We have investigated the possibility of evaluating chemosensitivity of neoplastic cells of different origin [ovary, lung, breast, gastrointestinal, skin (melanoma)] obtained from cancer patients, by measuring residual telomerase activity after drug treatment in vitro. Using the classical telomeric repeat amplification protocol ("TRAP") assay based on polymerase chain reaction, we examined telomerase activity of untreated or drug-treated tumor cell suspensions, derived from the processing of surgical specimens. Feasibility and reproducibility of the assay were evaluated according to various parameters, including drug concentration, time of in vitro culture, and type of tumor. The results indicated that the assay is highly sensitive and reproducible, and can be performed using surgical specimens in a reasonable percentage of cases, ranging from 40% (breast cancer) to 100% (ovarian cancer). Moreover, the assay provides comparable results using a wide range of tumor cells, and the presence of normal cells does not interfere with the results. Prolonged tumor cell culture is not required because the assay can be completed within 24 to 72 hours after sample collection. In conclusion, the present investigation provides the technical bases for future studies to evaluate whether this assay would be able to predict patient's response to antitumor agents.
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PMID:Suppression of telomerase activity as an indicator of drug-induced cytotoxicity against cancer cells: in vitro studies with fresh human tumor samples. 1046 37


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