ArticleNucleic acids research2013
A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG.
Article in Nucleic acids research, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 57 citations in OpenAlex.
- Elucidation of DNA Primase as a Drug Target in Leishmania donovani: Structure-Guided Inhibitor Identification and Biochemical Validation.Chemical biology & drug design · 2026Article
- Mechanism of T7 Primase Selecting Active Priming Sites Among Genome.Biomolecules · 2026Article
- Identification of apoptosis-related genes as potential biomarkers and therapeutic targets in cystic fibrosis patients progressing to pulmonary nontuberculous mycobacterial disease.Scientific reports · 2025Article
- Determination of Initial Rates of Lipopolysaccharide Transport.Biochemistry · 2024Article
- Identification of potential inhibitor against Leishmania donovani mitochondrial DNA primase through in-silico and in vitro drug repurposing approaches.Scientific reports · 2024Article
- Review
- Inhibition of Replication Fork Formation and Progression: Targeting the Replication Initiation and Primosomal Proteins.International journal of molecular sciences · 2023Review
- A Colorimetric Assay to Identify and Characterize Bacterial Primase Inhibitors.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase.Biochemistry · 2022Article
- CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation.Nature communications · 2021Article
- Single-strand DNA-scaffolded copper nanoclusters for the determination of inorganic pyrophosphatase activity and screening of its inhibitor.Mikrochimica acta · 2020Article
- Mimivirus encodes a multifunctional primase with DNA/RNA polymerase, terminal transferase and translesion synthesis activities.Nucleic acids research · 2019Article
- DnaG Primase-A Target for the Development of Novel Antibacterial Agents.Antibiotics (Basel, Switzerland) · 2018Review
- NMR-Fragment Based Virtual Screening: A Brief Overview.Molecules (Basel, Switzerland) · 2018Review
- Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase.Scientific reports · 2017Article
- DNA Replication in Mycobacterium tuberculosis.Microbiology spectrum · 2017Review
- Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins.Journal of visualized experiments : JoVE · 2017Article
- A Quantitative Spectrophotometric Assay to Monitor the tRNA-Dependent Pathway for Lipid Aminoacylation In Vitro.Journal of biomolecular screening · 2016Article
- Par-4 secretion: stoichiometry of 3-arylquinoline binding to vimentin.Organic & biomolecular chemistry · 2016Article
- Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG.The Journal of antibiotics · 2015Article
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
Bacterial DNA primase DnaG synthesizes RNA primers required for chromosomal DNA replication. Biochemical assays measuring primase activity have been limited to monitoring formation of radioactively labelled primers because of the intrinsically low catalytic efficiency of DnaG. Furthermore, DnaG is prone to aggregation and proteolytic degradation. These factors have impeded discovery of DnaG inhibitors by high-throughput screening (HTS). In this study, we expressed and purified the previously uncharacterized primase DnaG from Mycobacterium tuberculosis (Mtb DnaG). By coupling the activity of Mtb DnaG to that of another essential enzyme, inorganic pyrophosphatase from M. tuberculosis (Mtb PPiase), we developed the first non-radioactive primase-pyrophosphatase assay. An extensive optimization of the assay enabled its efficient use in HTS (Z' = 0.7 in the 384-well format). HTS of 2560 small molecules to search for inhibitory compounds yielded several hits, including suramin, doxorubicin and ellagic acid. We demonstrate that these three compounds inhibit Mtb DnaG. Both suramin and doxorubicin are potent (low-µM) DNA- and nucleotide triphosphate-competitive priming inhibitors that interact with more than one site on Mtb DnaG. This novel assay should be applicable to other primases and inefficient DNA/RNA polymerases, facilitating their characterization and inhibitor discovery.
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