Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD

Chisty, Liisa T and Toseland, Christopher P. and Fili, Natalia and Mashanov, Gregory I and Dillingham, Mark S and Molloy, Justin E and Webb, Martin R (2013) Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD. Nucleic acids research, 41 (9). pp. 5010-5023. ISSN 1362-4962. (doi:https://doi.org/10.1093/nar/gkt194) (Full text available)

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Abstract

The helicase PcrA unwinds DNA during asymmetric replication of plasmids, acting with an initiator protein, in our case RepD. Detailed kinetics of PcrA activity were measured using bulk solution and a single-molecule imaging technique to investigate the oligomeric state of the active helicase complex, its processivity and the mechanism of unwinding. By tethering either DNA or PcrA to a microscope coverslip surface, unwinding of both linear and natural circular plasmid DNA by PcrA/RepD was followed in real-time using total internal reflection fluorescence microscopy. Visualization was achieved using a fluorescent single-stranded DNA-binding protein. The single-molecule data show that PcrA, in combination with RepD, can unwind plasmid lengths of DNA in a single run, and that PcrA is active as a monomer. Although the average rate of unwinding was similar in single-molecule and bulk solution assays, the single-molecule experiments revealed a wide distribution of unwinding speeds by different molecules. The average rate of unwinding was several-fold slower than the PcrA translocation rate on single-stranded DNA, suggesting that DNA unwinding may proceed via a partially passive mechanism. However, the fastest dsDNA unwinding rates measured in the single-molecule unwinding assays approached the PcrA translocation speed measured on ssDNA.

Item Type: Article
Subjects: Q Science
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Chris Toseland
Date Deposited: 07 Apr 2015 11:55 UTC
Last Modified: 10 Apr 2015 13:53 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/47850 (The current URI for this page, for reference purposes)
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