Search results for "Endonucleases"
showing 10 items of 27 documents
A Comparison of Techniques to Evaluate the Effectiveness of Genome Editing
2018
Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use.
PARP inhibition enhances tumor cell-intrinsic immunity in ERCC1-deficient non-small cell lung cancer.
2018
The cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway detects cytosolic DNA to activate innate immune responses. Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as those caused by BRCA1 mutations or ERCC1 defects. Using isogenic cell lines and patient-derived samples, we showed that ERCC1-defective non-small cell lung cancer (NSCLC) cells exhibit an enhanced type I IFN transcriptomic signature and that low ERCC1 expression correlates with increased lymphocytic infiltration. We demonstrated that clinical PARPi, including olaparib and rucaparib, have cell-autonomous immunomodulatory properties in ERCC1-defecti…
A detailed experimental study of a DNA computer with two endonucleases
2017
Abstract Great advances in biotechnology have allowed the construction of a computer from DNA. One of the proposed solutions is a biomolecular finite automaton, a simple two-state DNA computer without memory, which was presented by Ehud Shapiro’s group at the Weizmann Institute of Science. The main problem with this computer, in which biomolecules carry out logical operations, is its complexity – increasing the number of states of biomolecular automata. In this study, we constructed (in laboratory conditions) a six-state DNA computer that uses two endonucleases (e.g. AcuI and BbvI) and a ligase. We have presented a detailed experimental verification of its feasibility. We described the effe…
Ribonucleotide Reductase Messenger RNA Expression and Survival in Gemcitabine/Cisplatin-Treated Advanced Non-Small Cell Lung Cancer Patients
2004
Abstract Purpose: No chemotherapy regimen, including the widely used combination of gemcitabine/cisplatin, confers significantly improved survival over any other in metastatic non-small cell lung cancer (NSCLC); however, the selection of patients according to key genetic characteristics can help to tailor chemotherapy. Ribonucleotide reductase subunit M1 (RRM1) is involved in DNA synthesis and repair and in gemcitabine metabolism, and the excision repair cross-complementing group 1 (ERCC1) gene has been related to cisplatin activity. Experimental Design: Patients were part of a large randomized trial carried out from September 1998 to July 2000, comparing gemcitabine/cisplatin versus gemcit…
SNPs and taxane toxicity in breast cancer patients
2014
Aim: In order to identify genetic variants associated with taxanes toxicity, a panel with 47 SNPs in 20 genes involved in taxane pathways was designed. Patients & methods: Genomic DNA of 113 breast cancer patients was analyzed (70 taking docetaxel, 43 taking paclitaxel). Results: Two SNPs associated with docetaxel toxicity were identified: CYP3A4*1B with infusion-related reactions; and ERCC1 Gln504Lys with mucositis (p ≤ 0.01). Regarding paclitaxel toxicity: CYP2C8 HapC and CYP2C8 rs1934951 were associated with anemia; and ERCC1 Gln504Lys with neuropathy (p ≤ 0.01). Conclusion: Genes involved in DNA repair mechanisms and reactive oxygen species levels influence taxane toxicity in cance…
Covalent DNA adducts formed by benzo[c]chrysene in mouse epidermis and by benzo[c]chrysene fjord-region diol epoxides reacted with DNA and polynucleo…
1997
The metabolic activation in mouse skin of benzo[c]chrysene (B[c]C), a weakly carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar and crude oil, was investigated. Male Parkes mice were treated topically with 0.5 mumol of B[c]C, and DNA was isolated from the treated areas of skin at various times after treatment and analyzed by 32P-postlabeling. Seven adduct spots were detected, at a maximum level of 0.89 fmol of adducts/microgram of DNA. Four B[c]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 mumol of the optically pure putative proximate carcinogens (+)- and (-)-trans-benzo[c]chrysene-9,10-dihydrodiols [(+)- and (-)-B[c]C-diols] led to th…
Molecular modes of action of cantharidin in tumor cells
2005
Cancer chemotherapy is often limited by patient's toxicity and tumor drug resistance indicating that new drug development and modification of existing drugs is critical for improving the therapeutic response. Traditional Chinese medicine is a rich source of potential anticancer agents. In particular, cantharidin (CAN), the active principle ingredient from the blister beetle, Mylabris, has anti-tumor activity, but the cytotoxic mechanism is unknown. In leukemia cells, cantharidin induces apoptosis by a p53-dependent mechanism. Cantharidin causes both DNA single- and double-strand breaks. Colony-forming assays with knockout and transfectant cells lines showed that DNA polymerase beta, but not…
c-Fos is required for excision repair of UV-light induced DNA lesions by triggering the re-synthesis of XPF
2006
Cells deficient in c-Fos are hypersensitive to ultraviolet (UV-C) light. Here we demonstrate that mouse embryonic fibroblasts lacking c-Fos (fos-/-) are defective in the repair of UV-C induced DNA lesions. They show a decreased rate of sealing of repair-mediated DNA strand breaks and are unable to remove cyclobutane pyrimidine dimers from DNA. A search for genes responsible for the DNA repair defect revealed that upon UV-C treatment the level of xpf and xpg mRNA declined but, in contrast to the wild type (wt), did not recover in fos-/- cells. The observed decline in xpf and xpg mRNA is due to impaired re-synthesis, as shown by experiments using actinomycin D. Block of xpf transcription resu…
DNA damage by peroxynitrite characterized with DNA repair enzymes.
1996
The DNA damage induced by peroxynitrite in isolated bacteriophage PM2 DNA was characterized by means of several repair enzymes with defined substrate specificities. Similar results were obtained with peroxynitrite itself and with 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite, nitric oxide and superoxide. A high number of base modifications sensitive to Fpg protein which, according to HPLC analysis, were mostly 8-hydroxyguanine residues, and half as many single-strand breaks were observed, while the numbers of oxidized pyrimidines (sensitive to endonuclease III) and of sites of base loss (sensitive to exonuclease III or T4 endonuclease V) were relativ…
Fen1 is induced p53 dependently and involved in the recovery from UV-light-induced replication inhibition.
2005
Mouse embryonic fibroblasts (MEFs) that lack p53 are hypersensitive to the cytotoxic and genotoxic effect of ultraviolet (UV-C) light. They also display a defect in the recovery from UV-C-induced DNA replication inhibition. An enzyme involved in processing stalled DNA replication forks is flap endonuclease 1 (Fen1). Gene expression profiling of UV-C-irradiated MEFs revealed fen1 to be upregulated, which was confirmed by RT-PCR and Western blot experiments. Increased Fen1 levels upon UV-C exposure are due to transcriptional activation, as revealed by inhibitor studies. Fen1 induction was dose- and time-dependent; it occurred on protein level already 3 h after irradiation. Induction of Fen1 b…