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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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Distribution of intensity change for unwinding events with linear DNA. (A–C) solution PcrA and (D–F) immobilized bioPcrA with the stated DNA lengths. The distributions of ΔItot are presented as percentage frequency, normalized to the maximum.
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gkt194-F5: Distribution of intensity change for unwinding events with linear DNA. (A–C) solution PcrA and (D–F) immobilized bioPcrA with the stated DNA lengths. The distributions of ΔItot are presented as percentage frequency, normalized to the maximum.

Mentions: In the presence of RepD, many unwinding events were observed for all dsDNA template lengths (Table 1). The mean value of both Δttot and ΔItot (Figures 4A–C and 5A–C) showed a linear dependence with the dsDNA length (Figure 6A), indicating complete unwinding of the dsDNA templates up to the length that is likely to remain in the evanescent field (27). A linear fit to the plot of mean Δttot versus dsDNA template length gave an unwinding rate of 31 (±7) bp s−1, in good agreement with the stopped-flow measurement (30 bp s−1).Figure 4.


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)

Distribution of intensity change for unwinding events with linear DNA. (A–C) solution PcrA and (D–F) immobilized bioPcrA with the stated DNA lengths. The distributions of ΔItot are presented as percentage frequency, normalized to the maximum.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt194-F5: Distribution of intensity change for unwinding events with linear DNA. (A–C) solution PcrA and (D–F) immobilized bioPcrA with the stated DNA lengths. The distributions of ΔItot are presented as percentage frequency, normalized to the maximum.
Mentions: In the presence of RepD, many unwinding events were observed for all dsDNA template lengths (Table 1). The mean value of both Δttot and ΔItot (Figures 4A–C and 5A–C) showed a linear dependence with the dsDNA length (Figure 6A), indicating complete unwinding of the dsDNA templates up to the length that is likely to remain in the evanescent field (27). A linear fit to the plot of mean Δttot versus dsDNA template length gave an unwinding rate of 31 (±7) bp s−1, in good agreement with the stopped-flow measurement (30 bp s−1).Figure 4.

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