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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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(a) Rates of T7 DNA synthesis at various ddGTP concentrations. (b) Lengths of T7 replication products at various ddGTP concentrations. Values are means and error bars represent standard error of the mean.
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Figure 4: (a) Rates of T7 DNA synthesis at various ddGTP concentrations. (b) Lengths of T7 replication products at various ddGTP concentrations. Values are means and error bars represent standard error of the mean.

Mentions: The search for novel antibacterial and antiviral agents remains an important field of research, and DNA replication is an obvious target for pharmacological investigation (30,31). The ease of performing our experiment and the availability of automated fluorescence imaging systems make the assay reported here an ideal method for screening and characterizing replication inhibitors. We demonstrated this concept by measuring rates and processivities of the T7 replication reaction with varying amounts of dideoxyGTP (ddGTP), a chain-terminating nucleotide that can be removed by the exonuclease activity of gp5 (32). Obviously, the incorporation of a single ddGTP depends upon concentration, and each inclusion requires the polymerase to switch between polymerase and exonuclease activities. The relative portion of time needed to excise the dideoxynucleotides increases with the ddGTP concentration and results in a decrease in aggregate rate of DNA synthesis. We observed a rate decrease from 75.9 ± 4.8 bp s−1 in the absence of ddGTP to 49.2 ± 3.1 bp s−1 at 1 μM ddGTP (Figure 4a). In addition, product length decreased from 25.3 ± 1.7 kbp without ddGTP to 4.8 ± 0.5 kbp at 1 μM ddGTP (Figure 4b). It is interesting to note that the decrease in rate and product length scale differently with ddGTP concentration. Product length decreases by ∼5-fold over the concentration range compared to ∼1.5-fold for rate. These results suggest that each ddGTP incorporation increases the probability of replisome dissociation (‘off’ rate), leading to a decrease in total replication processivity. Translating our assay to larger-scale screening or testing would present a simple method for easily analyzed, multiplexed studies of the effects of small molecules on replisomal DNA replication.Figure 4.


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)

(a) Rates of T7 DNA synthesis at various ddGTP concentrations. (b) Lengths of T7 replication products at various ddGTP concentrations. Values are means and error bars represent standard error of the mean.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: (a) Rates of T7 DNA synthesis at various ddGTP concentrations. (b) Lengths of T7 replication products at various ddGTP concentrations. Values are means and error bars represent standard error of the mean.
Mentions: The search for novel antibacterial and antiviral agents remains an important field of research, and DNA replication is an obvious target for pharmacological investigation (30,31). The ease of performing our experiment and the availability of automated fluorescence imaging systems make the assay reported here an ideal method for screening and characterizing replication inhibitors. We demonstrated this concept by measuring rates and processivities of the T7 replication reaction with varying amounts of dideoxyGTP (ddGTP), a chain-terminating nucleotide that can be removed by the exonuclease activity of gp5 (32). Obviously, the incorporation of a single ddGTP depends upon concentration, and each inclusion requires the polymerase to switch between polymerase and exonuclease activities. The relative portion of time needed to excise the dideoxynucleotides increases with the ddGTP concentration and results in a decrease in aggregate rate of DNA synthesis. We observed a rate decrease from 75.9 ± 4.8 bp s−1 in the absence of ddGTP to 49.2 ± 3.1 bp s−1 at 1 μM ddGTP (Figure 4a). In addition, product length decreased from 25.3 ± 1.7 kbp without ddGTP to 4.8 ± 0.5 kbp at 1 μM ddGTP (Figure 4b). It is interesting to note that the decrease in rate and product length scale differently with ddGTP concentration. Product length decreases by ∼5-fold over the concentration range compared to ∼1.5-fold for rate. These results suggest that each ddGTP incorporation increases the probability of replisome dissociation (‘off’ rate), leading to a decrease in total replication processivity. Translating our assay to larger-scale screening or testing would present a simple method for easily analyzed, multiplexed studies of the effects of small molecules on replisomal DNA replication.Figure 4.

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