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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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Related in: MedlinePlus

Visualization of circular plasmid DNA unwinding using immobilized PcrA. Cy3B-bioPcrA was used together with RepD and 3094-bp plasmid; see ‘Material and Methods’ section for details. Zero time was set as the time of the start of the unwinding event. (A) Visualization of individual fluorescent spots of increasing intensity with time on an area of 25 µm2. (B) Representative example of time course. (C) Distribution of event durations for one experiment (414 events). The average duration is 83 (±2) s, giving a rate of unwinding of 41 (±1) s−1 for this set of events, assuming complete unwinding. (D) Distribution of event intensity changes. The data from this set of measurements are summarized in Table 2.
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gkt194-F7: Visualization of circular plasmid DNA unwinding using immobilized PcrA. Cy3B-bioPcrA was used together with RepD and 3094-bp plasmid; see ‘Material and Methods’ section for details. Zero time was set as the time of the start of the unwinding event. (A) Visualization of individual fluorescent spots of increasing intensity with time on an area of 25 µm2. (B) Representative example of time course. (C) Distribution of event durations for one experiment (414 events). The average duration is 83 (±2) s, giving a rate of unwinding of 41 (±1) s−1 for this set of events, assuming complete unwinding. (D) Distribution of event intensity changes. The data from this set of measurements are summarized in Table 2.

Mentions: By loading RepD-nicked, 3094-bp plasmid, and then adding Cy3B-SSB and ATP, spots of increasing intensity were observed (Figure 7A and B). As with linear dsDNA, there were many such events in the field of observation, and these gave a broad range of durations and intensity increases (Figure 7C and D). Data from the set of measurements in Figure 7 are summarized in Table 2.Figure 7.


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 of circular plasmid DNA unwinding using immobilized PcrA. Cy3B-bioPcrA was used together with RepD and 3094-bp plasmid; see ‘Material and Methods’ section for details. Zero time was set as the time of the start of the unwinding event. (A) Visualization of individual fluorescent spots of increasing intensity with time on an area of 25 µm2. (B) Representative example of time course. (C) Distribution of event durations for one experiment (414 events). The average duration is 83 (±2) s, giving a rate of unwinding of 41 (±1) s−1 for this set of events, assuming complete unwinding. (D) Distribution of event intensity changes. The data from this set of measurements are summarized in Table 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt194-F7: Visualization of circular plasmid DNA unwinding using immobilized PcrA. Cy3B-bioPcrA was used together with RepD and 3094-bp plasmid; see ‘Material and Methods’ section for details. Zero time was set as the time of the start of the unwinding event. (A) Visualization of individual fluorescent spots of increasing intensity with time on an area of 25 µm2. (B) Representative example of time course. (C) Distribution of event durations for one experiment (414 events). The average duration is 83 (±2) s, giving a rate of unwinding of 41 (±1) s−1 for this set of events, assuming complete unwinding. (D) Distribution of event intensity changes. The data from this set of measurements are summarized in Table 2.
Mentions: By loading RepD-nicked, 3094-bp plasmid, and then adding Cy3B-SSB and ATP, spots of increasing intensity were observed (Figure 7A and B). As with linear dsDNA, there were many such events in the field of observation, and these gave a broad range of durations and intensity increases (Figure 7C and D). Data from the set of measurements in Figure 7 are summarized in Table 2.Figure 7.

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