A novel non-radioactive primase-pyrophosphatase activity assay and its application to the discovery of inhibitors of Mycobacterium tuberculosis primase DnaG

Tapan Biswas, Esteban Resto-Roldá N, Sean K. Sawyer, Irina Artsimovitch, Oleg V. Tsodikov

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

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 (Z0 = 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-mM) DNA-and nucleotide triphosphatecompetitive 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.

Original languageEnglish
Pages (from-to)e56
JournalNucleic Acids Research
Volume41
Issue number4
DOIs
StatePublished - Feb 2013

Bibliographical note

Funding Information:
Seed award 2UL1TR000433-06 from the Michigan Institute for Clinical and Health Research (MICHR) and University of Michigan College of Pharmacy start-up funds (both to O.V.T.). Funding for open access charge: University of Michigan College of Pharmacy start-up funds.

ASJC Scopus subject areas

  • Genetics

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