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Real-time single-molecule observation of rolling-circle DNA replication.

Tanner NA, Loparo JJ, Hamdan SM, Jergic S, Dixon NE, van Oijen AM - Nucleic Acids Res. (2009)

Bottom Line: We present a simple technique for visualizing replication of individual DNA molecules in real time.By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA.This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

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Related in: MedlinePlus

Kymographs of example DNA molecules from (a) T7 and (b) E. coli replication experiments. Endpoint trajectories are plotted vs. time to obtain rates of synthesis by fitting with linear regression (c). Rates of shown traces are: 99.4 bp s−1 (T7) and 467.1 bp s−1 (E. coli).
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Figure 2: Kymographs of example DNA molecules from (a) T7 and (b) E. coli replication experiments. Endpoint trajectories are plotted vs. time to obtain rates of synthesis by fitting with linear regression (c). Rates of shown traces are: 99.4 bp s−1 (T7) and 467.1 bp s−1 (E. coli).

Mentions: A single-stranded, 7.2-kb circular M13mp18 DNA is primed and its complementary strand synthesized to form dsDNA. Upon completion of synthesis of the entire circular substrate, the polymerase acting at the 3′-end will encounter the 5′-end of the original primer and synthesis will continue by displacing the previously synthesized DNA as ssDNA (Figure 1a). In the presence of protein activities required for priming and other lagging-strand processes, the displaced ssDNA tail will be effectively converted into dsDNA by repetitive priming and synthesis of lagging-strand Okazaki fragments. To allow surface immobilization and single-molecule observation of the replication substrates, we used a 5′-biotinylated ‘tail’ primer in constructing the M13 rolling-circle substrate. This substrate is purified and introduced into a flow chamber constructed with a biotin–streptavidin-functionalized coverslip (12). The filled, surface-attached M13 can serve as a replication template upon addition of nucleotides and proteins. Flowing low picomolar concentrations of M13 DNA into the chamber results in hundreds of DNA molecules in a single field of view (125 μm × 125 μm), each of which can serve as a substrate for the introduced proteins (Figure 1b). As the replication reaction proceeds, the DNA attaching the M13 circle to the surface is extended and stretched fully by hydrodynamic flow of buffer. We are able to visualize individual dsDNA molecules through total-internal reflection fluorescence (TIRF) microscopy by adding SYTOX Orange dsDNA intercalating stain to the reaction buffer, allowing for observation of coupled leading- and lagging-strand synthesis as a processively lengthening, stained dsDNA molecule. Addition of the necessary replication proteins resulted in the lengthening of the M13 template indicating processive DNA synthesis (see Supplementary Data). Trajectories of DNA length as a function of time reported on rates of DNA synthesis (Figure 2), and total product length at the end of synthesis was used to measure processivity.Figure 1.


Real-time single-molecule observation of rolling-circle DNA replication.

Tanner NA, Loparo JJ, Hamdan SM, Jergic S, Dixon NE, van Oijen AM - Nucleic Acids Res. (2009)

Kymographs of example DNA molecules from (a) T7 and (b) E. coli replication experiments. Endpoint trajectories are plotted vs. time to obtain rates of synthesis by fitting with linear regression (c). Rates of shown traces are: 99.4 bp s−1 (T7) and 467.1 bp s−1 (E. coli).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651787&req=5

Figure 2: Kymographs of example DNA molecules from (a) T7 and (b) E. coli replication experiments. Endpoint trajectories are plotted vs. time to obtain rates of synthesis by fitting with linear regression (c). Rates of shown traces are: 99.4 bp s−1 (T7) and 467.1 bp s−1 (E. coli).
Mentions: A single-stranded, 7.2-kb circular M13mp18 DNA is primed and its complementary strand synthesized to form dsDNA. Upon completion of synthesis of the entire circular substrate, the polymerase acting at the 3′-end will encounter the 5′-end of the original primer and synthesis will continue by displacing the previously synthesized DNA as ssDNA (Figure 1a). In the presence of protein activities required for priming and other lagging-strand processes, the displaced ssDNA tail will be effectively converted into dsDNA by repetitive priming and synthesis of lagging-strand Okazaki fragments. To allow surface immobilization and single-molecule observation of the replication substrates, we used a 5′-biotinylated ‘tail’ primer in constructing the M13 rolling-circle substrate. This substrate is purified and introduced into a flow chamber constructed with a biotin–streptavidin-functionalized coverslip (12). The filled, surface-attached M13 can serve as a replication template upon addition of nucleotides and proteins. Flowing low picomolar concentrations of M13 DNA into the chamber results in hundreds of DNA molecules in a single field of view (125 μm × 125 μm), each of which can serve as a substrate for the introduced proteins (Figure 1b). As the replication reaction proceeds, the DNA attaching the M13 circle to the surface is extended and stretched fully by hydrodynamic flow of buffer. We are able to visualize individual dsDNA molecules through total-internal reflection fluorescence (TIRF) microscopy by adding SYTOX Orange dsDNA intercalating stain to the reaction buffer, allowing for observation of coupled leading- and lagging-strand synthesis as a processively lengthening, stained dsDNA molecule. Addition of the necessary replication proteins resulted in the lengthening of the M13 template indicating processive DNA synthesis (see Supplementary Data). Trajectories of DNA length as a function of time reported on rates of DNA synthesis (Figure 2), and total product length at the end of synthesis was used to measure processivity.Figure 1.

Bottom Line: We present a simple technique for visualizing replication of individual DNA molecules in real time.By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA.This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

Show MeSH
Related in: MedlinePlus