Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The replication of the pT181 plasmid is dependent on the plasmid-encoded initiator protein RepC. We have previously shown that RepC protein has sequence-specific endonuclease and topoisomerase-like activities. In this paper we demonstrate that this initiator protein has sequence-specific DNA-binding properties. Based on filter binding of plasmid restriction fragments, RepC protein specifically recognizes only the pT181 origin region. Using DNase I and neocarzinostatin "footprinting" techniques, we show that RepC protein specifically binds to a 32-base-pair sequence within the origin that is part of the initiator cistron. Using dimethyl sulfate as a chemical probe, we have identified the purine residues that interact with the initiator protein. The features of the DNA region that interacts with RepC protein include sequences with the potential to form Z DNA and/or hairpin structures. The specific DNA-protein interaction at the origin may be critical in the initiation of pT181 DNA replication by RepC protein in association with other host initiation proteins.
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PMID:Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication. 346 45

Expression of the DNA topoisomerase IIalpha (topoIIalpha) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIalpha mRNA was increased 1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 degreesC for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIalpha gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) -295 to +85) and of three deletion constructs (nt -197 to +85, -154 to +85, and -74 to +85) was increased approximately 3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt -20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt -74 and -21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3-24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIalpha gene promoter. Finally, in vivo footprint analysis of the topoIIalpha gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIalpha gene by heat shock requires the release of a negative regulatory factor from ICE1.
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PMID:The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIalpha gene by heat shock. 955 15

The bacteriophage Mu mom gene encodes the unique DNA-modification function of the phage. Regulation of the mom gene at the transcriptional level is brought about by the transactivator protein C of the phage. The mom promoter is an activator-dependent weak promoter having poor -10 and -35 elements separated by a 19 bp suboptimal spacer region. These features could constrain RNA polymerase occupancy at the promoter. Here, we have probed into the mechanism by which C protein acts as a transcriptional activator at Pmom. In vivo dimethyl sulfate footprinting studies demonstrate C protein-mediated asymmetric distortion of its specific site at the mom regulatory region. Using a coupled topoisomerase assay, we demonstrate that C protein induces the unwinding of DNA. This C-mediated unwinding seems to be localised to the 3' flanking region of the C binding site located adjacent to and overlapping the -35 element of Pmom. These results suggest that C protein-mediated torsional changes could be reorienting the -10 and -35 elements to a favorable conformation for RNA polymerase occupancy at the mom promoter.
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PMID:Transcriptional activator C protein-mediated unwinding of DNA as a possible mechanism for mom gene activation. 983 13

Liposomal irinotecan (irinotecan liposome injection, nal-IRI), a liposomal formulation of irinotecan, is designed for extended circulation relative to irinotecan and for exploiting discontinuous tumor vasculature for enhanced drug delivery to tumors. Following tumor deposition, nal-IRI is taken up by phagocytic cells followed by irinotecan release and conversion to its active metabolite, SN-38. Sustained inhibition of topoisomerase 1 by extended SN-38 exposure as a result of delivery by nal-IRI is hypothesized to enable superior antitumor activity compared with traditional topoisomerase 1 inhibitors such as conventional irinotecan and topotecan. We evaluated the antitumor activity of nal-IRI compared with irinotecan and topotecan in preclinical models of small-cell lung cancer (SCLC) including in a model pretreated with carboplatin and etoposide, a first-line regimen used in SCLC. Nal-IRI demonstrated antitumor activity in xenograft models of SCLC at clinically relevant dose levels, and resulted in complete or partial responses in DMS-53, DMS-114, and NCI-H1048 cell line-derived models as well as in three patient-derived xenograft models. The antitumor activity of nal-IRI was superior to that of topotecan in all models tested, which generally exhibited limited control of tumor growth and was superior to irinotecan in four out of five models. Further, nal-IRI demonstrated antitumor activity in tumors that progressed following treatment with topotecan or irinotecan, and demonstrated significantly greater antitumor activity than both topotecan and irinotecan in NCI-H1048 tumors that had progressed on previous carboplatin plus etoposide treatment. These results support the clinical development of nal-IRI in patients with SCLC.
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PMID:Extended topoisomerase 1 inhibition through liposomal irinotecan results in improved efficacy over topotecan and irinotecan in models of small-cell lung cancer. 2885 67