DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein — a 3′ to 5′ superfamily-2 DNA helicase — is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriAdeloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriAdeloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that the presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.
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October 2020
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The cover image showing the crystal structure of human hemoglobin (PDB: 1GZX). For more information, see the article by Natarajan et al. (pp. 3839–3850). Artwork Image courtesy of C. Natarajan.
Research Article|
October 16 2020
Characterization of Streptococcus pneumoniae PriA helicase and its ATPase and unwinding activities in DNA replication restart
Min-Guan Lin;
Min-Guan Lin
Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan
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Yi-Ching Li;
Yi-Ching Li
Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan
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Chwan-Deng Hsiao
Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan
Correspondence: Chwan-Deng Hsiao (hsiao@gate.sinica.edu.tw)
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Publisher: Portland Press Ltd
Received:
April 14 2020
Revision Received:
September 24 2020
Accepted:
September 25 2020
Accepted Manuscript online:
September 28 2020
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2020
Biochem J (2020) 477 (19): 3911–3922.
Article history
Received:
April 14 2020
Revision Received:
September 24 2020
Accepted:
September 25 2020
Accepted Manuscript online:
September 28 2020
Citation
Min-Guan Lin, Yi-Ching Li, Chwan-Deng Hsiao; Characterization of Streptococcus pneumoniae PriA helicase and its ATPase and unwinding activities in DNA replication restart. Biochem J 16 October 2020; 477 (19): 3911–3922. doi: https://doi.org/10.1042/BCJ20200269
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