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:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1-3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and 1-beta-D-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.
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PMID:Collateral sensitivity to gemcitabine (2',2'-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines. 1133 Oct 76

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase beta. Second, on the basis of sequence homology between topo V and polymerase beta, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5' 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3'-hydroxyl and 5'-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.
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PMID:A type IB topoisomerase with DNA repair activities. 1135 38

Helix-hairpin-helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)(2) domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH(2) terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes.
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PMID:Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases. 1236 75

We designed a method by which to generate antibiotic-resistant strains of Streptococcus pneumoniae at frequencies 4 orders of magnitude greater than the spontaneous mutation rate. The method is based on the natural ability of this organism to be genetically transformed with PCR products carrying sequences homologous to its chromosome. The genes encoding the targets of ciprofloxacin (parC, encoding the ParC subunit of DNA topoisomerase IV), rifampin (rpoB, encoding the beta subunit of RNA polymerase), and streptomycin (rpsL, encoding the S12 ribosomal protein) from susceptible laboratory strain R6 were amplified by PCR and used to transform the same strain. Resistant mutants were obtained with a frequency of 10(-4) to 10(-5), depending on the fidelity of the DNA polymerase used for PCR amplifications. Ciprofloxacin-resistant mutants, for which the MICs were four-to eightfold higher than that for R6, carried a single mutation of a residue in the quinolone resistance-determining region: S79 (change to A, F, or Y) or D83 (change to N or V). Rifampin-resistant strains, for which the MICs were at least 133-fold higher than that for R6, contained a single mutation within cluster I of rpoB: S482 (change to P), Q486 (change to L), D489 (change to V), or H499 (change to L or Y). Streptomycin-resistant mutants, for which the MICs were at least 64-fold higher than that for R6, carried a mutation at either K56 (change to I, R, or T) or K101 (change to E). PCR products obtained from the mutants were able to transform R6 to resistance with high efficiency (>10(4)). This method could be used to efficiently obtain resistant mutants for any drug whose target is known.
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PMID:High-efficiency generation of antibiotic-resistant strains of Streptococcus pneumoniae by PCR and transformation. 1265 55

A subset (gamma(2)) of late herpes simplex virus 1 genes depends on viral DNA synthesis for its expression. For optimal expression, a small number of these genes, exemplified by U(S)11, also requires two viral proteins, the alpha protein infected cell protein (ICP) 22 and the protein kinase U(L)13. Earlier we showed that U(L)13 and ICP22 mediate the stabilization of cdc2 and the replacement of its cellular partner, cyclin B, with the viral DNA polymerase processivity factor U(L)42. Here we report that cdc2 and its new partner, U(L)42, bind a phosphorylated form of topoisomerase II alpha. The posttranslational modification of topoisomerase II alpha and its interaction with cdc2-U(L)42 proteins depend on ICP22 in infected cells. Although topoisomerase II is required for viral DNA synthesis, ICP22 is not, indicating a second function for topoisomerase II alpha. The intricate manner in which the virus recruits topoisomerase II alpha for post-DNA synthesis expression of viral genes suggests that topoisomerase II alpha also is required for untangling concatemeric DNA progeny for optimal transcription of late genes.
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PMID:Herpes simplex virus 1 activates cdc2 to recruit topoisomerase II alpha for post-DNA synthesis expression of late genes. 1266 17

The adenovirus (Ad) genome is a linear double-stranded (ds) molecule containing about 36 kilobase pairs. At each end of the genome an approximately 100 base pair (bp) inverted terminal repeat (ITR) is found, the exact length depending on the serotype. To the 5'-end of each ITR, a 55-kDa terminal protein (TP) is covalently coupled. The Ad DNA replication system was one of the first replication systems that could be reconstituted in vitro (Challberg and Kelly 1979). The system requires three virally encoded proteins: precursor TP (pTP), DNA polymerase (Pol) and the DNA binding protein (DBP). In addition, three stimulating human cellular proteins have been identified. These are the transcription factors NFI (Nagata et al. 1982) and Oct-1 (Pruijn et al. 1986) and the type I topoisomerase NFII (Nagata et al. 1983). Ad DNA replication uses a protein primer for replication initiation. The transition from initiation to elongation is marked by a jumping back mechanism (King and van der Vliet 1994), followed by elongation. In order to elongate DBP is required. In this review we discuss the roles of DBP during initiation and elongation and we relate biochemical data on the jumping back mechanism used by Ad Pol to the recently solved crystal structure of a Pol alpha-like replication complex (Franklin et al. 2001). We comment on the conditions and possible functions of jumping back and propose a model to describe the jumping back mechanism.
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PMID:Adenovirus DNA replication: protein priming, jumping back and the role of the DNA binding protein DBP. 1274 51

The response of different tumours to radiation varies. This variation has been attributed to, among others, varying DNA repair capabilities The response of three tumour lines, differing in their sensitivities to radiation, namely, murine fibrosarcoma, lymphosarcoma and ascites, was studied by following the activities of enzymes known to be involved in DNA repair. The activities of poly (ADP-ribose) polymerase (PARP), DNA polymerase b and DNA ligase in fibrosarcoma, lymphosarcoma and ascites recorded varying degrees of increase following gamma irradiation (2 Gy). The increase was more pronounced in fibrosarcoma, which recorded a maximum 2 h after irradiation for b polymerase, and at 4 h for ligase and PARP, thereafter declining to near normal levels after 24 h. In contrast, the activity of DNA Topoisomerase I declined, corresponding to an increase in the PARP activity. The maximum increase in the activity of beta polymerase, ligase and PARP from lymphosarcoma and ascites was observed 2 h after irradiation with a corresponding decrease in Topoisomerase I activity. Search for the target enzymes and proteins for modification by PARP in gamma -irradiated fibrosarcoma tumour cells revealed that nuclei, and not chromatin, were preferentially modified by PARP. Among the nuclear proteins, histones were found to be ribosylated. The enzyme topoisomerase was ribosylated by PARP in vitro, and this modification was found to inhibit topoisomerase activity. We speculate that a possible role of PARP is to coordinate the activities of other enzymes in DNA repair by selectively inhibiting certain enzymes by the ribosylation process.
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PMID:Response of DNA repair enzymes in murine fibrosarcoma, lymphosarcoma and ascites cells following gamma irradiation. 1464 26

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.
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PMID:Role of protein-protein interactions during herpes simplex virus type 1 recombination-dependent replication. 1502 9

Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polymerase (E9), an essential and stoichiometric component of the processive polymerase (A20), a single-strand DNA-binding protein (I3), a type I topoisomerase (H6), the uracil DNA glycosylase (D4), a nucleic acid-independent nucleoside triphosphatase (D5), a serine/threonine protein kinase (B1), and a Holliday Junction resolvase (A22). All of these factors work in concert to faithfully duplicate the viral genome. Although a replication origin has not been defined for the poxviruses, cis-acting sequences found within the telomeric 200 bp have been implicated as necessary and sufficient for minichromosome replication. Replication occurs within cytoplasmic foci from approx 3 to 12 h postinfection. This chapter includes several methodologies to assay and quantitate replication in vivo, visualize replication foci microscopically, and test the integrity of central replication enzymes in vitro.
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PMID:Methods for analysis of poxvirus DNA replication. 1511 16

Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.
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PMID:Mismatch repair and DNA damage signalling. 1527 97


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