Novel molecular beacon assays for DNA cleavage reactions

2001 ◽  
Author(s):  
Jianwei J. Li ◽  
Charles Z. Cao ◽  
Ronald Geyer ◽  
Weihong Tan
Keyword(s):  
Dna Cleavage ◽  
Molecular Beacon ◽  
Cleavage Reactions

Molecular Beacon Enables Combination of Highly Processive and Highly Sensitive Rolling Circle Amplification Readouts for Detection of DNA-Modifying Enzymes

Nano LIFE ◽  
2015 ◽  
Vol 05 (02) ◽  
pp. 1541002 ◽  
Author(s):  
Emil L. Kristoffersen ◽  
Maria Gonzalez ◽  
Magnus Stougaard ◽  
Cinzia Tesauro
Keyword(s):  
Real Time ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Drug Response ◽  
Molecular Beacon ◽  
Rolling Circle Amplification ◽  
Dna Topoisomerase ◽  
Rolling Circle ◽  
Topoisomerase Ib ◽  
Highly Sensitive

Here we present an optimized readout format for detection of the circularized products from a DNA-based sensor for measurement of DNA-modifying enzymes including DNA Topoisomerase I. The basic design of the DNA-sensor relies on the use of a substrate that can be circularized by the activity of DNA-modifying enzymes like type IB Topoisomerases and subsequently amplified by a rolling circle amplification (RCA) mechanism. The RCA process can be followed in real-time by the addition of a molecular beacon with a fluorophore/quencher pair. Upon hybridization to the amplified product, the fluorophore/quencher pair is separated, giving rise to a fluorescent signal, measurable in pseudo real-time using a qPCR machine or in a fluorimeter. The RCA products in complex with the molecular beacon can subsequently be moved to microscopic slides and analyzed in a fluorescence microscope. We describe the proof of the principle of this molecular beacon-based method combining the qPCR readout format with the standard Rolling circle Enhanced Enzymatic Assay previously reported. Although the qPCR setup is less sensitive, it allows easy, fast, and high-throughput measurement of enzyme activities. Human Topoisomerase IB (TopIB) is a well-known target for the clinically used anticancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular TopIB activity affecting reversibly the Topoisomerase/DNA cleavage complexes. Therefore, we envisioned that the presented method may find use for the prediction of cellular drug response and for drug screening to discover novel molecules that specifically inhibit TopIB or other DNA-modifying enzymes.

A New Method for Determining the Stereochemistry of DNA Cleavage Reactions:  Application to theSfiI andHpaII Restriction Endonucleases and to the MuA Transposase†

Biochemistry ◽  
1999 ◽  
Vol 38 (14) ◽  
pp. 4640-4648 ◽  
Author(s):  
Kiyoshi Mizuuchi ◽  
Timothy J. Nobbs ◽  
Stephen E. Halford ◽  
Kenji Adzuma ◽  
Jun Qin
Keyword(s):  
Dna Cleavage ◽  
Restriction Endonucleases ◽  
New Method ◽  
Cleavage Reactions

Hydroxylation, Epoxidation, and DNA Cleavage Reactions Mediated by the Biomimetic Mn-TMPyP/O2/Sulfite Oxidation System†

1999 ◽  
Vol 38 (18) ◽  
pp. 4123-4127 ◽  
Author(s):  
Karine Wietzerbin ◽  
James G. Muller ◽  
Rachel A. Jameton ◽  
Geneviève Pratviel ◽  
Jean Bernadou ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Sulfite Oxidation ◽  
Cleavage Reactions ◽  
Oxidation System

DNA cleavage reactions of the dinuclear chemotherapeutic agent copper(II) bis-1,10- phenanthroline terephthalate

2012 ◽  
Vol 50 (01) ◽  
pp. 79-81 ◽  
Author(s):  
Andrew Kellett ◽  
Malachy McCann ◽  
Orla Howe ◽  
Mark O’Connor ◽  
Michael Devereux
Keyword(s):  
Dna Cleavage ◽  
Chemotherapeutic Agent ◽  
Cleavage Reactions

scholarly journals DNA Cleavage Activity of the V(D)J Recombination Protein RAG1 Is Autoregulated

2004 ◽  
Vol 24 (15) ◽  
pp. 6850-6860 ◽  
Author(s):  
Pallabi De ◽  
Mandy M. Peak ◽  
Karla K. Rodgers
Keyword(s):  
Dna Cleavage ◽  
Active Site Residues ◽  
Inhibitory Effects ◽  
Central Domain ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Recombination Protein ◽  
Cleavage Reactions ◽  
Hydrolysis Of ◽  
B And T Lymphocytes

ABSTRACT RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.

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