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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Among the polymerases, DNA polymerase alpha-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase alpha-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase alpha-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase alpha-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase alpha-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)(5)-3'/5'-(TAACCC)(5)T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were approximately 40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase alpha uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.
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PMID:In vitro expansion of mammalian telomere repeats by DNA polymerase alpha-primase. 1093 27

The length variability of four human interstitial telomeric sequences (ITs) is described. Three of the ITs contain short telomeric stretches ranging between 53 and 84 bp and are localized in 21q22, 2q31, and 7q36; the fourth IT derives from the subtelomeric domain of chromosome 6p and contains a tract of a few hundred basepairs of exact and degenerate repeats. Using primers flanking the repeats, we amplified the genomic DNA from unrelated individuals and from family members, and we found that all the loci are polymorphic. At the 21q22 IT locus, two equally frequent alleles were found, while the number of alleles at the 2q31, 7q36, and 6pter IT loci was 8, 6, and 4, respectively. Sequence analysis revealed that in the three loci containing short ITs the alleles differ from one another for multiples of the hexanucleotide; it is likely that the mechanism leading to the polymorphism is DNA polymerase slippage. These loci were also unstable in gastric tumor cells characterized by microsatellite instability. At the 6pter IT locus, the four alleles range in length from about 500 to about 700 bp; this variability is probably due to unequal exchange or gene conversion. Our data indicate that stretches of exact internal telomeric repeats can be highly unstable, like microsatellites with shorter units, and that they can be useful polymorphic markers for linkage analysis, for forensic applications, and for the detection of genetic instability in tumors.
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PMID:Instability of interstitial telomeric sequences in the human genome. 1096 8

Telomerase is a specialized DNA polymerase that adds telomeric sequences onto chromosome ends. The functional telomerase complex contains a telomerase reverse transcriptase (TERT) and also a telomerase RNA (TR). Although it is well established that the human telomerase reverse transcriptase (hTERT) can function well in different human cell lines, it has not been shown whether it is compatible with telomerase template RNA from other species. Here we report that the expressed hTERT is functionally compatible with rabbit telomerase template RNA (rTR) as demonstrated by TRAP assay. The direct interaction between hTERT and rTR is further confirmed by immunoprecipitation-linked RT-PCR in which rTR is detected from the complex immunoprecipitated by an anti-hTERT antibody. The hTERT expressed in rabbit lens epithelial cells demonstrates two major functions: modulation of expression of other genes and attenuation of apoptosis. Thus, telomerase has a variety of functions besides telomere synthesis, and the template RNA is functionally conserved between human and rabbit.
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PMID:hTERT can function with rabbit telomerase RNA: regulation of gene expression and attenuation of apoptosis. 1109 41

Since the introduction of PRimed IN Situ labeling (PRINS) as a rapid and extremely sensitive alternative method to conventional fluorescence in situ hybridization (FISH), its application in clinical cytogenetics has been limited to the detection of highly repeated sequences, such as centromeric and telomeric regions. In the original PRINS method, unlabeled oligonucleotide probes are annealed to their repeated complementary target sequences in fixed human metaphase chromosomes on a slide. The probes serve as primers for subsequent in situ chain elongation with Taq DNA polymerase and labeled nucleotides. In contrast to conventional PCR, cyclic in situ amplification of the chromosomal target DNA with paired primers remained both difficult and strictly limited to highly repeated sequences, since the maintenance of constant reaction conditions on the slide during temperature and pressure shifts presents a major problem. We developed a new system for in situ PCR that allows the amplification of target sequences analogous to PCR in the test tube. We applied this method successfully for the detection of highly repeated sequences, for the detection of low copy repeats, and in one case, for the detection of a single-copy DNA sequence. The significance of this development for further in situ PCR applications will be discussed.
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PMID:A new technique for cyclic in situ amplification and a case report about amplification of a single copy gene sequence in human metaphase chromosomes through PCR-PRINS. 1118 May 96

Telomerase is a telomere-specific DNA polymerase consisting of protein and RNA components, which is activated in germline cells and the majority of cancers and serves to counter the consequences of telomere shortening. The protein component, hTERT, is believed to be the catalytic subunit of human telomerase and its expression at the mRNA level correlates well with telomerase activity in vitro. Current techniques for assaying telomerase activity detect only the mean activity in a sample and are unable to isolate specific cell sub-populations. This report describes the development and validation of a cellular, immunofluorescence-based flow cytometry assay that allows detection of intranuclear hTERT while maintaining identifiable cell population characteristics. The assay was shown to be both sensitive to changes in telomerase expression and was semi-quantitative. In both cell line differentiation experiments and in primary cells, a good correlation existed between hTERT expression measured by flow cytometry and telomerase activity detected by the telomeric repeat amplification protocol (TRAP). The method developed offers a quick, simple and reproducible cellular-based assay for hTERT expression. This assay will provide a useful, new tool for future investigations, facilitating the analysis of hTERT expression in mixed cell populations.
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PMID:Detection of hTERT protein by flow cytometry. 1118 8

Cdc13 is a single-strand telomeric DNA-binding protein that positively regulates yeast telomere replication by recruiting telomerase to chromosome termini through a site on Cdc13 that is eliminated by the cdc13-2 mutation. Here we show that Cdc13 has a separate role in negative regulation of telomere replication, based on analysis of a new mutation, cdc13-5. Loss of this second regulatory activity results in extensive elongation of the G strand of the telomere by telomerase, accompanied by a reduced ability to coordinate synthesis of the C strand. Both the cdc13-5 mutation and DNA polymerase alpha mutations (which also exhibit elongated telomeres) are suppressed by increased expression of the Cdc13-interacting protein Stn1, indicating that Stn1 coordinates action of the lagging strand replication complex with the regulatory activity of CDC13. However, the association between Cdc13 and Stn1 is abolished by cdc13-2, the same mutation that eliminates the interaction between Cdc13 and telomerase. We propose that Cdc13 participates in two regulatory steps-first positive, then negative-as a result of successive binding of telomerase and the negative regulator Stn1 to overlapping sites on Cdc13. Thus, Cdc13 coordinates synthesis of both strands of the telomere by first recruiting telomerase and subsequently limiting G-strand synthesis by telomerase in response to C-strand replication.
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PMID:Cdc13 both positively and negatively regulates telomere replication. 1123 Jan 49

Human TTAGGG repeat-binding factor 1 (TRF1) is involved in the regulation of telomere length in vivo, but the mechanism of regulation remains largely undefined. We have developed an in vitro system for assessing the effect of TRF1 on DNA synthesis using purified proteins and synthetic DNA substrates. Results reveal that TRF1, when bound to telomeric duplex DNA, inhibits DNA synthesis catalyzed by DNA polymerase alpha/primase (pol alpha). Inhibition required that TRF1 be bound to duplex telomeric DNA as no effect of TRF1 was observed on nontelomeric, random DNA substrates. Inhibition was shown to be dependent on TRF1 concentration and the length of the telomeric duplex region of the DNA substrate. When bound in cis to telomeric duplex DNA, TRF1 was also capable of inhibiting pol alpha-catalyzed DNA synthesis on nontelomeric DNA sequences from positions both upstream and downstream of the extending polymerase. Inhibition of DNA synthesis was shown to be specific for TRF1 but not necessarily for the DNA polymerase used in the extension reaction. In a series of control experiments, we assessed T7 DNA polymerase-catalyzed synthesis on a DNA template containing tandem gal4 operators. In these experiments, the addition of the purified Gal4-DNA binding domain (Gal4-DBD) protein has no effect on the ability of T7 polymerase to copy the DNA template. Interestingly, TRF1 inhibition was observed on telomeric DNA substrates using T7 DNA polymerase. These results suggest that TRF1, when bound to duplex telomeric DNA, serves to block extension by DNA polymerases. These results are discussed with respect to the role of TRF1 in telomere length regulation.
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PMID:TRF1 inhibits telomere C-strand DNA synthesis in vitro. 1132 63

It is known that in mice the centromeric heterochromatin remains compact during the whole cell cycle and at interphase is referred to as "chromocentres". In the current study, by the use of antibodies against prekinetochores and DNA polymerase (a PCNA antigen), we showed that in murine L929 cells chromocentres remain spatially associated with prekinetochores during the entire interphase, including the late S-period, when DNA chromocentres replicate. Augmentation of prekinetochore fluorescence increases concomitantly with the heterochromation replication, but the prekinetochore duplication occurs only in G2 period. A conclusion has been made that murine interphase cells can be used for biochemical fractionation of chromocentres associated with prekinetochore proteins.
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PMID:[Stability of spatial interactions between chromocenters and pre-kinetochores in the interphase murine cells]. 1139 13

More than 80% of human malignancies express telomerase activity, while normal somatic tissues in general lack it. During each normal cell division, there is a constant loss of DNA sequences at chromosomal ends, which is due to the 'end-replication problem' of conventional DNA polymerase. Critical shortening of telomeres induces cell cycle arrest and eventually cell death. Telomerase, a ribonucleoprotein complex with a RNA (TR) and a catalytic subunit (TERT) as core components, is able to add reitineratedly telomeric repeat sequences to the very ends of chromosomes. It was suggested that activation of telomerase in tumor cells has a major impact on their continuous growth. Indeed, transfection of TERT constructs into various normal human cell types led to telomere elongation or stabilization and, most importantly, cellular immortalization. Conversely, inhibition of telomerase in tumor cell lines induced growth arrest, at least in first experimental settings. Such initial success implies that drug-mediated abrogation of telomerase action might be an ideal adjuvant treatment for cancer patients. There are, however, legitimate concerns about the generalization of such an approach.
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PMID:Targeted inhibition of telomerase in human cancer: will it be a double-edged sword? 1144 Dec 76

The Saccharomyces cerevisiae Ku complex, while important for nonhomologous DNA end joining, is also necessary for maintaining wild-type telomere length and a normal chromosomal DNA end structure. Yeast cells lacking Ku can grow at 23 degrees C but are unable to do so at elevated temperatures due to an activation of DNA damage checkpoints. To gain insights into the mechanisms affected by temperature in such strains, we isolated and characterized a new allele of the YKU70 gene, yku70-30(ts). By several criteria, the Yku70-30p protein is functional at 23 degrees C and nonfunctional at 37 degrees C. The analyses of telomeric repeat maintenance as well as the terminal DNA end structure in strains harboring this allele alone or in strains with a combination of other mutations affecting telomere maintenance show that the altered DNA end structure in yeast cells lacking Ku is not generated in a telomerase-dependent fashion. Moreover, the single-stranded G-rich DNA on such telomeres is not detected by DNA damage checkpoints to arrest cell growth, provided that there are sufficient double-stranded telomeric repeats present. The results also demonstrate that mutations in genes negatively affecting G-strand synthesis (e.g., RIF1) or C-strand synthesis (e.g., the DNA polymerase alpha gene) allow for the maintenance of longer telomeric repeat tracts in cells lacking Ku. Finally, extending telomeric repeat tracts in such cells at least temporarily suppresses checkpoint activation and growth defects at higher temperatures. Thus, we hypothesize that an aspect of the coordinated synthesis of double-stranded telomeric repeats is sensitive to elevated temperatures.
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PMID:Maintenance of double-stranded telomeric repeats as the critical determinant for cell viability in yeast cells lacking Ku. 1188 5


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