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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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Visualization and analysis of 1-kb linear DNA unwinding by TIRFM. (A) Images of the microscope field showing the occurrence of fluorescent spots. Data are shown with and without ATP, and RepD, for solution and immobilized bioPcrA. Discrete fluorescent spots of increasing intensity appeared as individual DNA molecules were being progressively unwound. Representative intensity time courses are shown for solution (B) and immobilized bioPcrA (C). The two parameters used to define unwinding: the total event duration (Δttot) and the total increase in intensity (ΔItot) are shown here.
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gkt194-F3: Visualization and analysis of 1-kb linear DNA unwinding by TIRFM. (A) Images of the microscope field showing the occurrence of fluorescent spots. Data are shown with and without ATP, and RepD, for solution and immobilized bioPcrA. Discrete fluorescent spots of increasing intensity appeared as individual DNA molecules were being progressively unwound. Representative intensity time courses are shown for solution (B) and immobilized bioPcrA (C). The two parameters used to define unwinding: the total event duration (Δttot) and the total increase in intensity (ΔItot) are shown here.

Mentions: Unwinding activity of wild-type PcrA was measured using biotinylated linear dsDNA that had been immobilized on a microscope coverslip surface. dsDNA substrates from 500 to 1500 bp in length, tagged with a single-biotin label at the 5′-end of one strand and containing the oriD sequence near the other end, were immobilized on a streptavidin-coated coverslip. This approach closely mimics the bulk measurements (as described earlier in the text) because the helicase is distal from the coverslip surface and binds the dsDNA while in solution. Also, in principle, this experimental geometry allows loading of multiple helicases from solution or monomer exchange, as might occur in bulk experiments. The immobilized dsDNA templates were pre-incubated with RepD, enabling PcrA to load and unwind the RepD–DNA complex. The time course of unwinding was monitored by measuring the accumulation of Cy3B-SSB on the growing ssDNA strands (Figure 3). Fluorescent spots that increased in intensity to a maximum value were observed as the dsDNA was unwound. In the majority of events, a short-lived maximum plateau was observed, followed by either a single-step intensity drop to baseline or a stepwise decrease to half maximum intensity followed by a subsequent fall to baseline (Figure 3B and Supplementary Table S1). The rationale for the decrease is shown in Supplementary Figure S5 in terms of stepwise dissociation of the two strands of ssDNA. The time course of the intensity rise for each unwinding event was characterized by two parameters, total increase in intensity (ΔItot) and total duration of unwinding (Δttot) (Figure 3). The distribution of these parameters provided information about the helicase processivity and the heterogeneity of its activity.Figure 3.


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)

Visualization and analysis of 1-kb linear DNA unwinding by TIRFM. (A) Images of the microscope field showing the occurrence of fluorescent spots. Data are shown with and without ATP, and RepD, for solution and immobilized bioPcrA. Discrete fluorescent spots of increasing intensity appeared as individual DNA molecules were being progressively unwound. Representative intensity time courses are shown for solution (B) and immobilized bioPcrA (C). The two parameters used to define unwinding: the total event duration (Δttot) and the total increase in intensity (ΔItot) are shown here.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt194-F3: Visualization and analysis of 1-kb linear DNA unwinding by TIRFM. (A) Images of the microscope field showing the occurrence of fluorescent spots. Data are shown with and without ATP, and RepD, for solution and immobilized bioPcrA. Discrete fluorescent spots of increasing intensity appeared as individual DNA molecules were being progressively unwound. Representative intensity time courses are shown for solution (B) and immobilized bioPcrA (C). The two parameters used to define unwinding: the total event duration (Δttot) and the total increase in intensity (ΔItot) are shown here.
Mentions: Unwinding activity of wild-type PcrA was measured using biotinylated linear dsDNA that had been immobilized on a microscope coverslip surface. dsDNA substrates from 500 to 1500 bp in length, tagged with a single-biotin label at the 5′-end of one strand and containing the oriD sequence near the other end, were immobilized on a streptavidin-coated coverslip. This approach closely mimics the bulk measurements (as described earlier in the text) because the helicase is distal from the coverslip surface and binds the dsDNA while in solution. Also, in principle, this experimental geometry allows loading of multiple helicases from solution or monomer exchange, as might occur in bulk experiments. The immobilized dsDNA templates were pre-incubated with RepD, enabling PcrA to load and unwind the RepD–DNA complex. The time course of unwinding was monitored by measuring the accumulation of Cy3B-SSB on the growing ssDNA strands (Figure 3). Fluorescent spots that increased in intensity to a maximum value were observed as the dsDNA was unwound. In the majority of events, a short-lived maximum plateau was observed, followed by either a single-step intensity drop to baseline or a stepwise decrease to half maximum intensity followed by a subsequent fall to baseline (Figure 3B and Supplementary Table S1). The rationale for the decrease is shown in Supplementary Figure S5 in terms of stepwise dissociation of the two strands of ssDNA. The time course of the intensity rise for each unwinding event was characterized by two parameters, total increase in intensity (ΔItot) and total duration of unwinding (Δttot) (Figure 3). The distribution of these parameters provided information about the helicase processivity and the heterogeneity of its activity.Figure 3.

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.

Show MeSH
Related in: MedlinePlus