Measuring Mutator Enzyme Activity Using an E. coli-Based Colony Formation Assay

Mei Chen Liu, Sebastian D. Fugmann*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Mutator enzymes alter the nucleotide sequences of DNA or RNA molecules; immune systems utilize them to destroy the integrity of pathogen genomes and to optimize immune mediators of the host. Their dysregulation has been linked to tumorigenesis in various tissues. Defining and comparing the activities of such mutator enzymes requires a robust versatile assay that is independent of their biological context as in vivo mutation rates are typically low. Here we provide detailed protocols for two widely used E. coli-based approaches that detect the activities of ectopically expressed cytidine deaminases on two distinct reporter genes: an extrachromosomal kanamycin-resistance gene or an endogenous chromosomal substrate, the rpoB gene-encoding RNA polymerase. The generation of mutations is in both cases measured in a colony formation assay. With appropriate modifications, these assays can be extended to study other mutator enzymes.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages103-114
Number of pages12
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2421
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Publisher Copyright:
© 2022, Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

  • AID
  • APOBEC
  • Cytidine deaminase
  • Deaminase
  • Mutator enzyme
  • Somatic hypermutation

Fingerprint

Dive into the research topics of 'Measuring Mutator Enzyme Activity Using an E. coli-Based Colony Formation Assay'. Together they form a unique fingerprint.

Cite this