Vaccinia Topoisomerase
Vaccinia Topoisomerase - Related Videos
02:02
DNA Topoisomerases
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2020
Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA. Types and Mechanism of action Topoisomerases are divided into two main types. Type I...
09:16
A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors
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Cited by 13 •
2016
Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.
10:48
Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression
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Cited by 5 •
2014
We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.
09:27
Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins
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Cited by 9 •
2014
A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently tagged proteins simultaneously using the method of transient dominant selection is described here.
02:52
Expansion of Mutant Vaccinia Virus in Mammalian Cells
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2026
Source: Yuan, M., et al. A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors. J. Vis. Exp. (2016)This video demonstrates the expansion of plaque-derived recombinant virus in adherent mammalian cells. The virus carries a fluorescent reporter in place of a virulent gene, enabling infection tracking. Infected cells are harvested for PCR verification of the genetic modification.
FAQs
Sequence recognition enables Vaccinia topoisomerase to act at defined DNA sites rather than cleaving indiscriminately. The enzyme uses that site preference to position a DNA phosphate beside its active-site tyrosine. Cleavage then creates a transient enzyme-DNA covalent intermediate, linking sequence recognition to a controlled topological reaction. In bioengineering, this specificity makes cleavage assays useful for examining how sequence context influences enzyme activity.
The active-site tyrosine forms a temporary covalent bond with the DNA phosphate when one strand is cleaved. This bond holds the broken strand in a controlled intermediate while the DNA undergoes rotation. The enzyme subsequently religates the strand, so the tyrosine supports both strand scission and restoration of DNA continuity rather than producing a permanent break.
Controlled rotation allows the cleaved DNA strand to relax torsional stress before religation. This links the enzyme's chemical steps to its larger role in managing DNA topology during genome replication and other DNA transactions. For bioengineering studies, examining this sequence of cleavage, rotation, and religation helps connect molecular mechanism with changes in DNA structural state.
A biochemical assay can examine sequence-specific cleavage and the associated religation reaction using DNA molecules containing recognized sites. By focusing on these defined reactions, researchers can investigate enzyme mechanism and DNA topology without treating the protein as a nonspecific nuclease. Such assays provide a foundation for studying how engineered variants alter the characterized reaction.
Engineered variants provide a way to test how changes in the protein affect sequence recognition, strand cleavage, covalent intermediate formation, controlled rotation, or religation. Because the underlying reaction is well characterized, variant behavior can be interpreted against a defined mechanistic framework. This supports protein design studies focused on modifying or evaluating topoisomerase function.
Its sequence-specific cleavage reaction can support molecular diagnostic approaches by providing a defined enzyme-DNA interaction for biochemical analysis. The same reaction can be used to screen compounds that disrupt topoisomerase activity. These applications connect the enzyme's mechanistic study with practical testing of altered activity, while retaining DNA topology as the relevant scientific context.