A continuous assay for DNA cleavage: The application of “break lights” to enediynes, iron-dependent agents, and nucleases

Biggins, John B.; Prudent, James R.; Marshall, David J.; Ruppen, Mark E.; Thorson, Jon S.

doi:10.1073/pnas.240460997

Cited by 110 publications

(82 citation statements)

References 37 publications

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“…Supporting online information online includes experimental procedures for the detection of DNA cleavage activity of TLM A, K-1, and K-2 by break-light assay (39) and Tables S1 for plasmids, Tables S2 for bacterial strains, and Table S3 for oligonucleotides as PCR primers used in this study.…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of tlmK Unveiling Unstable Carbinolamide Intermediates in the Tallysomycin Biosynthetic Pathway

Wang

Tao

Wendt-Pienkoski

et al. 2009

Journal of Biological Chemistry

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Tallysomycins (TLMs) belong to the bleomycin family of anticancer antibiotics. TLMs differ from bleomycins primarily by the presence of a 4-amino-4,6-dideoxy-L-talose sugar attached to C-41 as part of a glycosylcarbinolamide. We previously proposed, on the basis of bioinformatics analysis of the tlm biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158, that the tlmK gene is responsible for the attachment of this sugar moiety. We now report that inactivation of tlmK in S. hindustanus abolished TLM A and TLM B production, the resultant ⌬tlmK mutant instead accumulated five new metabolites, and introduction of a functional copy of tlmK to the ⌬tlmK mutant restored TLM A and TLM B production. Two major metabolites, TLM K-1 and TLM K-2, together with three minor metabolites, TLM K-3, TLM K-4, and TLM K-5, were isolated from the ⌬tlmK mutant, and their structures were elucidated. These findings provide experimental evidence supporting the previous functional assignment of tlmK to encode a glycosyltransferase and unveil two carbinolamide pseudoaglycones as key intermediates in the TLM biosynthetic pathway. TlmK stabilizes the carbinolamide intermediates by glycosylating their hemiaminal hydroxyl groups, thereby protecting them from hydrolysis during TLM biosynthesis. In the absence of TlmK, the carbinolamide intermediates fragment to produce an amide TLM K-1 and aldehyde intermediates, which undergo further oxidative fragmentation to afford carboxylic acids TLM K-2, TLM K-3, TLM K-4, and TLM K-5. Tallysomycins (TLMs)3 belong to the bleomycin (BLM) family of glycopeptide antitumor antibiotics (1, 2) (Fig. 1). The BLMs are currently used clinically under the trade name Blenoxane in combination with a number of other agents for the treatment of Hodgkin lymphoma, carcinomas of the skin, head, and neck, and testicular cancers. Early development of drug resistance and dose-dependent pulmonary toxicity are major limitations of BLMs in chemotherapy (3, 4). Structural modifications to this family of natural products are necessary for improvement in efficacy and reduction of toxicity. Although numerous BLM analogs have been synthesized (5, 6), total synthesis remains of limited practical value because of the complex scaffold of this entire family of natural products. Recent cloning and characterization of the BLM, TLM, and zorbamycin biosynthetic gene clusters from Streptomyces verticillus ATCC15003 (7), Streptoalloteichus hindustanus E465-94 (ATCC 31158) (8), and Streptomyces flavoviridis ATCC21892 (9) opened the possibility toward the production of novel BLM analogs via genetic metabolic engineering approaches.TLMs are structurally related to BLMs but differ from BLMs in three ways; (i) the presence of two hydroxyl groups within the aminoethylbithiazole moiety, one of which is conjugated to a 4-amino-4,6-dideoxy-L-talose sugar as part of a glycosylcarbinolamide, (ii) the presence of two series of C-terminal amine moieties with (A series) or without (B series) a ␤-lysine moiety in the subterminal pos...

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Section: Discussionmentioning

confidence: 99%

Functional Characterization of tlmK Unveiling Unstable Carbinolamide Intermediates in the Tallysomycin Biosynthetic Pathway

Wang

Tao

Wendt-Pienkoski

et al. 2009

Journal of Biological Chemistry

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“…When the probe undergoes a spontaneous conformational change forcing the stem apart, fluorescence occurs. Recently, molecular beacons have been widely used in the investigation of DNA-protein interactions including DNA cleavage and ligation reactions because of their high sensitivity, simplicity, and real-time monitoring [22][23][24][25] . In this study, we describe a new high-throughput assay that translates the IN 3'-processing reaction into a fluorescent signal in real-time using the principle of molecular beacons.…”

Section: +mentioning

confidence: 99%

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

Liu

et al. 2007

Acta Pharmacologica Sinica

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Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening. Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluorescence properties of molecular beacons, the substrate is designed to form a stemloop structure labeled with a fluorophore at the 5' end and a quencher at the 3' end. IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening. Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn 2+ . The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC 50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay. Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

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“…For example, fluorescence-based assays with "molecular break light" probes and "molecular beacons" were reported for monitoring endonucleases. [16][17][18][19][20][21][22] The use of complexes of DNA and cationic conjugated polymer as probes was recently described. [23] Colorimetric methods based on enzyme-responsive gold nanoparticle (AuNP) [24][25][26][27] and DNAzyme [28] systems have also been developed for detecting endonucleases.…”

Section: Introductionmentioning

confidence: 99%

“…These were at least 100 times more sensitive than those of previously reported methods based on gel electrophoresis and chromatography, UV spectroscopy, and the general fluorescence methods. [16][17][18][19][24][25][26][27][28] These results indicated that the proposed method could be used for quantifying multiple endonucleases simultaneously with high sensitivity and specificity.…”

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confidence: 99%

Multiplex Detection of Endonucleases by Using a Multicolor Gold Nanobeacon

Huang

Zhao

Liang

et al. 2011

Chemistry A European J

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A highly sensitive and selective assay based on a novel enzyme-responsive multicolor gold nanobeacon has been developed for the multiplex detection of endonucleases, a group of very important nucleases. The nanobeacon takes advantage of the high specificity of DNA cleavage reactions combined with the unique fluorescence-quenching property of gold nanoparticles (AuNPs). To prepare the nanobeacon, three hairpin DNA reporters, each labeled at the 5' terminus with a fluorescent dye (i.e., fluorescein amidite (FAM), carboxy-X-rhodamine (ROX), cyanine dye (Cy5)), that respond to one of three different endonucleases are co-assembled at the surface of AuNPs (15 nm). This assembly brings the dyes into very close proximity with the AuNP, which leads to significant quenching of the fluorescence due to the nanosurface energy-transfer (NSET) effect. When the nanobeacon is exposed to the targeted endonucleases, specific DNA cleavage occurs and pieces of DNA fragments are released from the AuNP surface along with the fluorescent dye, which results in the fluorescence recovery that provides the basis for a quantitative measurement of endonuclease activity. Three endonucleases, namely HaeIII, EcoRI, and EcoRV, were studied as the proof-of-concept analytes. These endonucleases in homogeneous mixture solutions were simultaneously quantified by the proposed assay with high sensitivity and specificity. The limits of detection obtained were in the range of 5.0×10(-4) U mL(-1) to 1.0×10(-3) U mL(-1) of endonuclease; these limits are at least 100 times more sensitive than the previously reported endonuclease assays. Endonuclease inhibitors impair the DNA cleavage, so it is anticipated that the present method has great potential for screening inhibitors of endonucleases. To demonstrate this application, the inhibitory effects of certain anticancer drugs on HaeIII, EcoRI, and EcoRV activities were studied. The present protocol proved to be sensitive, reliable, and easy to carry out.

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A continuous assay for DNA cleavage: The application of “break lights” to enediynes, iron-dependent agents, and nucleases

Cited by 110 publications

References 37 publications

Functional Characterization of tlmK Unveiling Unstable Carbinolamide Intermediates in the Tallysomycin Biosynthetic Pathway

Functional Characterization of tlmK Unveiling Unstable Carbinolamide Intermediates in the Tallysomycin Biosynthetic Pathway

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

Multiplex Detection of Endonucleases by Using a Multicolor Gold Nanobeacon

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