Abstract
Bacterial DNA primase DnaG is an attractive target for antibiotic discovery since it plays an essential role in DNA replication. Over the last 10 years, we have developed and optimized a robust colorimetric assay that enabled us to identify and validate inhibitors of bacterial primases. Here, we provide a detailed protocol for this colorimetric assay for DnaG from three different pathogenic bacteria (Mycobacterium tuberculosis, Bacillus anthracis, and Staphylococcus aureus), which can be performed in high throughput. We also describe secondary assays to characterize hits from this high-throughput screening assay. These assays are designed to identify inhibitors of the coupled enzyme inorganic pyrophosphatase, DNA binding agents, and elucidate the mode of inhibition of primase inhibitors.
Original language | English |
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Title of host publication | Methods in Molecular Biology |
Pages | 283-301 |
Number of pages | 19 |
DOIs | |
State | Published - 2023 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2601 |
ISSN (Print) | 1064-3745 |
ISSN (Electronic) | 1940-6029 |
Bibliographical note
Publisher Copyright:© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Keywords
- DNA intercalation
- DNA replication
- Drug discovery
- High-throughput assay
- Inorganic pyrophosphatase
- Mode of inhibition
ASJC Scopus subject areas
- Molecular Biology
- Genetics