Methods to Quantitatively Measure Topological Changes Induced by DNA-Binding Proteins In Vivo and In Vitro.
Methods Mol Biol
; 2819: 421-441, 2024.
Article
em En
| MEDLINE
| ID: mdl-39028517
ABSTRACT
Agarose gel electrophoresis in the presence of chloroquine (an intercalating agent) can be used to resolve and characterize the population of topoisomers present in supercoiled plasmid DNA. Here, we describe how chloroquine gel electrophoresis can capture changes in the topoisomer distribution of plasmid DNA that bears a recognition site for a given protein, if that plasmid is isolated from cells producing the protein of interest. We also describe two complementary in vitro assays, which can be used to capture transient changes in DNA supercoiling caused when the purified protein of interest engages its recognition site. These are the topoisomerase I-mediated relaxation assay (TMRA) and the ligase-mediated supercoiling assay (LMSA). Together, these in vivo and in vitro methods allow the capture and measurement of changes in DNA topology that are triggered by DNA-binding proteins, especially those that multimerize on or spread along DNA.
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Base de dados:
MEDLINE
Assunto principal:
Plasmídeos
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DNA Super-Helicoidal
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DNA Topoisomerases Tipo I
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Proteínas de Ligação a DNA
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article