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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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Translocation of MDCC-PcrA along ssDNA. (A) MDCC-PcrA was premixed with different lengths of oligo(dT), then rapidly mixed with ATP in the solution conditions (buffer and temperature, 22°C) of the TIRF measurements. The time to minimum fluorescence for each length represents the time for the last PcrA molecules to translocate to the 5′-end. The curves are offset from each other for clarity. (B) These time were plotted against length (squares) to give the translocation rate of 125 (±2) bases s−1 from the gradient. These plots are also shown for measurements under the conditions used by Dillingham et al. (21) for translocation (circles, 20°C, 50 mM Tris–HCl, pH 7.5, 150 mM KCl and 3 mM MgCl2), giving 99 (±2) bases s−1, and the solution plasmid unwinding conditions of Slatter et al. (14) (triangles, 30°C, 50 mM Tris–HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2 and 1 mM EDTA), giving 244 (±4) bases s−1.
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gkt194-F2: Translocation of MDCC-PcrA along ssDNA. (A) MDCC-PcrA was premixed with different lengths of oligo(dT), then rapidly mixed with ATP in the solution conditions (buffer and temperature, 22°C) of the TIRF measurements. The time to minimum fluorescence for each length represents the time for the last PcrA molecules to translocate to the 5′-end. The curves are offset from each other for clarity. (B) These time were plotted against length (squares) to give the translocation rate of 125 (±2) bases s−1 from the gradient. These plots are also shown for measurements under the conditions used by Dillingham et al. (21) for translocation (circles, 20°C, 50 mM Tris–HCl, pH 7.5, 150 mM KCl and 3 mM MgCl2), giving 99 (±2) bases s−1, and the solution plasmid unwinding conditions of Slatter et al. (14) (triangles, 30°C, 50 mM Tris–HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2 and 1 mM EDTA), giving 244 (±4) bases s−1.

Mentions: This signal was then used to monitor PcrA translocation to the 5′-end of ssDNA, driven by ATP hydrolysis (Figure 2A). MDCC-PcrA was pre-mixed with different lengths of oligo(dT), rapidly mixed with ATP and then the fluorescence was followed with time. In all cases, the fluorescence decreased as PcrA translocated from an initial random binding along the ssDNA to the 5′-end (21). The decrease is completed when all PcrA molecules have reached that end, including those initially bound at or near the 3′-end. The time taken to reach the final fluorescence level, therefore, reports the translocation time for PcrA to move along the full length of the template. The duration of the fluorescence change increased linearly with ssDNA length, and a plot of translocation time against ssDNA template length gives the translocation rate (Figure 2B). To validate the method, the rate was measured under the solution conditions used previously at 20°C (21) and gave a similar rate [99 (±6) bases s−1] here compared with 80 bases s−1. The rate was also measured under the solution conditions used for plasmid unwinding in solution at 30°C (14) [244 (±4) bases s−1] and conditions similar to that used in the single-molecule unwinding assays at 23°C [133 (±5) bases s−1]. In all cases, the ssDNA translocation rate was significantly higher than values measured for PcrA-RepD moving along and unwinding dsDNA.Figure 2.


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)

Translocation of MDCC-PcrA along ssDNA. (A) MDCC-PcrA was premixed with different lengths of oligo(dT), then rapidly mixed with ATP in the solution conditions (buffer and temperature, 22°C) of the TIRF measurements. The time to minimum fluorescence for each length represents the time for the last PcrA molecules to translocate to the 5′-end. The curves are offset from each other for clarity. (B) These time were plotted against length (squares) to give the translocation rate of 125 (±2) bases s−1 from the gradient. These plots are also shown for measurements under the conditions used by Dillingham et al. (21) for translocation (circles, 20°C, 50 mM Tris–HCl, pH 7.5, 150 mM KCl and 3 mM MgCl2), giving 99 (±2) bases s−1, and the solution plasmid unwinding conditions of Slatter et al. (14) (triangles, 30°C, 50 mM Tris–HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2 and 1 mM EDTA), giving 244 (±4) bases s−1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643603&req=5

gkt194-F2: Translocation of MDCC-PcrA along ssDNA. (A) MDCC-PcrA was premixed with different lengths of oligo(dT), then rapidly mixed with ATP in the solution conditions (buffer and temperature, 22°C) of the TIRF measurements. The time to minimum fluorescence for each length represents the time for the last PcrA molecules to translocate to the 5′-end. The curves are offset from each other for clarity. (B) These time were plotted against length (squares) to give the translocation rate of 125 (±2) bases s−1 from the gradient. These plots are also shown for measurements under the conditions used by Dillingham et al. (21) for translocation (circles, 20°C, 50 mM Tris–HCl, pH 7.5, 150 mM KCl and 3 mM MgCl2), giving 99 (±2) bases s−1, and the solution plasmid unwinding conditions of Slatter et al. (14) (triangles, 30°C, 50 mM Tris–HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2 and 1 mM EDTA), giving 244 (±4) bases s−1.
Mentions: This signal was then used to monitor PcrA translocation to the 5′-end of ssDNA, driven by ATP hydrolysis (Figure 2A). MDCC-PcrA was pre-mixed with different lengths of oligo(dT), rapidly mixed with ATP and then the fluorescence was followed with time. In all cases, the fluorescence decreased as PcrA translocated from an initial random binding along the ssDNA to the 5′-end (21). The decrease is completed when all PcrA molecules have reached that end, including those initially bound at or near the 3′-end. The time taken to reach the final fluorescence level, therefore, reports the translocation time for PcrA to move along the full length of the template. The duration of the fluorescence change increased linearly with ssDNA length, and a plot of translocation time against ssDNA template length gives the translocation rate (Figure 2B). To validate the method, the rate was measured under the solution conditions used previously at 20°C (21) and gave a similar rate [99 (±6) bases s−1] here compared with 80 bases s−1. The rate was also measured under the solution conditions used for plasmid unwinding in solution at 30°C (14) [244 (±4) bases s−1] and conditions similar to that used in the single-molecule unwinding assays at 23°C [133 (±5) bases s−1]. In all cases, the ssDNA translocation rate was significantly higher than values measured for PcrA-RepD moving along and unwinding dsDNA.Figure 2.

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