Limits...
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.

Show MeSH

Related in: MedlinePlus

(a) Length distributions of replication products, with means of 25.3 ± 1.7 kbp (T7) and 85.3 ± 6.1 kbp (E. coli). (b) Rate distributions of single molecules, with means of 75.9 ± 4.8 bp s−1 (T7) and 535.5 ± 39 bp s−1 (E. coli). The log of the rates are plotted to allow simultaneous display of both broad distributions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651787&req=5

Figure 3: (a) Length distributions of replication products, with means of 25.3 ± 1.7 kbp (T7) and 85.3 ± 6.1 kbp (E. coli). (b) Rate distributions of single molecules, with means of 75.9 ± 4.8 bp s−1 (T7) and 535.5 ± 39 bp s−1 (E. coli). The log of the rates are plotted to allow simultaneous display of both broad distributions.

Mentions: Our single-molecule rolling-circle assay is broadly applicable to general DNA replication. We examined two common model systems of DNA replication, the bacteriophage T7 and E. coli replisomes. After binding our rolling-circle substrate to the surface of the chamber, we introduced the replisomal proteins in replication buffer containing the intercalating dye SYTOX Orange and observed replication as the solution entered the field of view. The T7 replisome can be reconstituted with four proteins: gp4, helicase/primase; gp5 + thioredoxin, polymerase; gp2.5, ssDNA-binding protein (1,13,21) At room temperature (22°C), we observed T7 replication at 75.9 ± 4.8 bp s−1 with an average processivity of 25.3 ± 1.7 kbp (Figure 3). Rates of synthesis were consistent with the previously reported value of 80 bp s−1 at room temperature (13).Figure 3.


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) Length distributions of replication products, with means of 25.3 ± 1.7 kbp (T7) and 85.3 ± 6.1 kbp (E. coli). (b) Rate distributions of single molecules, with means of 75.9 ± 4.8 bp s−1 (T7) and 535.5 ± 39 bp s−1 (E. coli). The log of the rates are plotted to allow simultaneous display of both broad distributions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: (a) Length distributions of replication products, with means of 25.3 ± 1.7 kbp (T7) and 85.3 ± 6.1 kbp (E. coli). (b) Rate distributions of single molecules, with means of 75.9 ± 4.8 bp s−1 (T7) and 535.5 ± 39 bp s−1 (E. coli). The log of the rates are plotted to allow simultaneous display of both broad distributions.
Mentions: Our single-molecule rolling-circle assay is broadly applicable to general DNA replication. We examined two common model systems of DNA replication, the bacteriophage T7 and E. coli replisomes. After binding our rolling-circle substrate to the surface of the chamber, we introduced the replisomal proteins in replication buffer containing the intercalating dye SYTOX Orange and observed replication as the solution entered the field of view. The T7 replisome can be reconstituted with four proteins: gp4, helicase/primase; gp5 + thioredoxin, polymerase; gp2.5, ssDNA-binding protein (1,13,21) At room temperature (22°C), we observed T7 replication at 75.9 ± 4.8 bp s−1 with an average processivity of 25.3 ± 1.7 kbp (Figure 3). Rates of synthesis were consistent with the previously reported value of 80 bp s−1 at room temperature (13).Figure 3.

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