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Antimicrob._Agents_Chemother.-2014-Kuron-1699-706.pdf (1.36 MB)

Evaluation of DNA primase DnaG as a potential target for antibiotics

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posted on 2023-06-08, 17:50 authored by Aneta Kuron, Malgorzata Korycka-Machala, Anna Brzostek, Marcin Nowosielski, Aidan DohertyAidan Doherty, Bozena Dziadek, Jaroslaw Dziadek
Mycobacteria contain genes for several DNA-dependent RNA primases, including dnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. An in silico analysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-type dnaG cannot be deleted from the chromosome of Mycobacterium smegmatis without disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identified M. smegmatis AEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of native dnaG in M. smegmatis was tolerated only after the integration of an extra intact copy of the M. smegmatis or Mycobacterium tuberculosis dnaG gene, under the control of chemically inducible promoters, into the attB site of the chromosome. M. tuberculosis and M. smegmatis DnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably, M. smegmatis bacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target.

Funding

BBSRC

History

Publication status

  • Published

File Version

  • Published version

Journal

Antimicrobial Agents and Chemotherapy

ISSN

1098-6596

Publisher

American Society for Microbiology

Issue

3

Volume

58

Page range

1699-1706

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2014-07-15

First Open Access (FOA) Date

2014-07-15

First Compliant Deposit (FCD) Date

2014-07-15

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