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

Chisty LT, Toseland CP, Fili N, Mashanov GI, Dillingham MS, Molloy JE, Webb MR - Nucleic Acids Res. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

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.

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Cartoon of TIRFM assay for PcrA·RepD unwinding dsDNA. The figure represents a partially unwound plasmid with bioPcrA attached to a PEGylated surface, with the fluorescence excitation coming from below. It illustrates that the furthest distance the DNA can reach from the surface attachment is equivalent to half the plasmid length.
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gkt194-F1: Cartoon of TIRFM assay for PcrA·RepD unwinding dsDNA. The figure represents a partially unwound plasmid with bioPcrA attached to a PEGylated surface, with the fluorescence excitation coming from below. It illustrates that the furthest distance the DNA can reach from the surface attachment is equivalent to half the plasmid length.

Mentions: In the current study, we have used a single-molecule assay, previously described for use with another helicase, AddAB (27,28), to visualize DNA unwinding by PcrA in real-time, monitoring the accumulation of fluorescently labeled SSB on the unwound ssDNA. SSB from E. coli was labeled with Cy3B to give an adduct Cy3B-SSB. Briefly, either linear dsDNA fragments or whole-circular plasmids, containing oriD, were first nicked by the initiator protein RepD and then allowed to interact with PcrA. Using biotinylation, the active complex was immobilized onto a streptavidin-coated microscope coverslip and visualized by TIRFM (total internal reflection fluorescence microscopy) (Figure 1). Two experimental geometries were adopted, in which either the dsDNA template or the helicase were surface immobilized, so that evolution of the ssDNA product occurred in a fixed position within the evanescent field of the TIRF imaging system. ATP-driven unwinding of the dsDNA substrates was then followed in real time by the appearance of fluorescent spots of gradually increasing intensity. By immobilizing either PcrA or the DNA, this experimental approach allowed comparison of the activity of PcrA free in solution with that of individually immobilized helicases. The previous work showed that the immobilization procedure had little or no effect on the average rate of unwinding for another helicase, AddAB, and indicated that would also be so for RecBCD (27,29). The assay gave a direct measurement of individual helicase unwinding activity on individual dsDNA molecules, without use of intercalating or other DNA labels, which may perturb the helicase unwinding activity. To complement the single-molecule assays, we also performed conventional bulk assays using a coumarin-labeled SSB (DCC-SSB), which gives a large fluorescence increase on binding ssDNA, allowing dsDNA unwinding to be measured by stopped-flow methods. We also compared the rate of dsDNA plasmid unwinding with the rate of PcrA translocation on ssDNA, using a coumarin-labeled PcrA that gives a signal on arrival at the end of the ssDNA template.Figure 1.


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

Chisty LT, Toseland CP, Fili N, Mashanov GI, Dillingham MS, Molloy JE, Webb MR - Nucleic Acids Res. (2013)

Cartoon of TIRFM assay for PcrA·RepD unwinding dsDNA. The figure represents a partially unwound plasmid with bioPcrA attached to a PEGylated surface, with the fluorescence excitation coming from below. It illustrates that the furthest distance the DNA can reach from the surface attachment is equivalent to half the plasmid length.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643603&req=5

gkt194-F1: Cartoon of TIRFM assay for PcrA·RepD unwinding dsDNA. The figure represents a partially unwound plasmid with bioPcrA attached to a PEGylated surface, with the fluorescence excitation coming from below. It illustrates that the furthest distance the DNA can reach from the surface attachment is equivalent to half the plasmid length.
Mentions: In the current study, we have used a single-molecule assay, previously described for use with another helicase, AddAB (27,28), to visualize DNA unwinding by PcrA in real-time, monitoring the accumulation of fluorescently labeled SSB on the unwound ssDNA. SSB from E. coli was labeled with Cy3B to give an adduct Cy3B-SSB. Briefly, either linear dsDNA fragments or whole-circular plasmids, containing oriD, were first nicked by the initiator protein RepD and then allowed to interact with PcrA. Using biotinylation, the active complex was immobilized onto a streptavidin-coated microscope coverslip and visualized by TIRFM (total internal reflection fluorescence microscopy) (Figure 1). Two experimental geometries were adopted, in which either the dsDNA template or the helicase were surface immobilized, so that evolution of the ssDNA product occurred in a fixed position within the evanescent field of the TIRF imaging system. ATP-driven unwinding of the dsDNA substrates was then followed in real time by the appearance of fluorescent spots of gradually increasing intensity. By immobilizing either PcrA or the DNA, this experimental approach allowed comparison of the activity of PcrA free in solution with that of individually immobilized helicases. The previous work showed that the immobilization procedure had little or no effect on the average rate of unwinding for another helicase, AddAB, and indicated that would also be so for RecBCD (27,29). The assay gave a direct measurement of individual helicase unwinding activity on individual dsDNA molecules, without use of intercalating or other DNA labels, which may perturb the helicase unwinding activity. To complement the single-molecule assays, we also performed conventional bulk assays using a coumarin-labeled SSB (DCC-SSB), which gives a large fluorescence increase on binding ssDNA, allowing dsDNA unwinding to be measured by stopped-flow methods. We also compared the rate of dsDNA plasmid unwinding with the rate of PcrA translocation on ssDNA, using a coumarin-labeled PcrA that gives a signal on arrival at the end of the ssDNA template.Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.

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.

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