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. 2017 Apr 22;6:e23932. doi: 10.7554/eLife.23932

Figure 2. Real-time fluorescence imaging of coupled DNA replication.

(A) Kymograph of an individual DNA molecule undergoing coupled leading and lagging strand replication. The gray scale indicates the fluorescence intensity of stained DNA. (B) Single-molecule trajectory obtained from the kymograph in (A), used to quantify the rates and processivities of replication events. The magenta box represents an example line segment used to determine rates. (C) Kymograph of the dynamics of red-labeled Pol IIIs on an individual DNA molecule. The Pol III moves with the replisome in the direction of flow as it elongates the DNA, visible as a bright magenta spot moving away from the surface anchor point. Additional Pol IIIs are left behind the moving replisome, seen as horizontal lines on the kymograph. (D) Histograms of the rate of replication for wild-type Pol III (492 ± 23 bp/s) and red Pol III (561 ± 27 bp/s) fit to Gaussian distributions. (E) Kymograph of the distribution of red Pol III on an individual DNA molecule in the presence of 150 nM Pol I and 100 nM DNA ligase. Prolonged Pol III spots behind the replisome are no longer observed due to the action of Pol I in Okazaki fragment processing. (F) Fluorescence intensity as a function of time of individual red Pol IIIs immobilized on the surface of a coverslip (lower trace; black line is an exponential fit with lifetime = 14.1 ± 0.4 s), and of the replisomal spot in (C) (upper trace). The fluorescence lifetime of red Pol III at the replisome is much longer than the photobleaching lifetime of the dye. The errors represent the standard errors of the mean.

DOI: http://dx.doi.org/10.7554/eLife.23932.004

Figure 2.

Figure 2—figure supplement 1. Representative field of view of SYTOX orange-stained dsDNA from the single-molecule rolling-circle DNA replication assay.

Figure 2—figure supplement 1.

(A) Efficient DNA replication proceeds in the presence of the full complement of replication reaction mix, including the reconstituted E. coli replisome, NTPs and dNTPs. Note both the length and number of products. (B) No DNA products are evident in the entire flow cell in the absence of dNTPs from the replication reaction mix. Note some rolling-circle templates become linearized due to photodamage, visible as lines shorter than 7 kb (2.1 μm).
DOI: http://dx.doi.org/10.7554/eLife.23932.005
Figure 2—figure supplement 2. Separation of proteolytic fragments of SNAP-α from full-length SNAP-α.

Figure 2—figure supplement 2.

(A) SDS-PAGE of the final fraction from the τC16 affinity chromatography, pooled from successive samples from the peak in the chromatography profile in (B).
DOI: http://dx.doi.org/10.7554/eLife.23932.006
Figure 2—figure supplement 3. Comparison of activities of wild-type and SNAP-labeled Pol III cores.

Figure 2—figure supplement 3.

(a) Agarose gel of products of Pol III strand-displacement (SD) DNA synthesis, a demanding assay for Pol III* activity (Jergic et al., 2013). The time course of flap-primer extension on M13 ssDNA depicts products larger than unit length of dsDNA (TFII) generated by SD DNA synthesis. (B) Alkaline agarose gel of coupled DNA replication. Reactions were performed on a 2 kb circular dsDNA template with wild-type (WT) Pol III*, WT Pol III* + SYTOX orange, red SNAP-labeled Pol III* and green SNAP-labeled Pol III*. (Left panel) The gel was stained with SYBR-Gold. (Right panel) Intensity profiles of lanes 2–5 of the left panel. The Okazaki fragment size distribution is centered at 1.3 ± 0.4 kb. Intensity profiles have been corrected for the difference in intensity of different size fragments using the ladder as a standard.
DOI: http://dx.doi.org/10.7554/eLife.23932.007
Figure 2—figure supplement 4. Representative kymograph of simultaneous staining of double-stranded DNA and fluorescence imaging of Pol III in real time.

Figure 2—figure supplement 4.

Double-stranded DNA was stained with SYTOX orange (gray scale) and Pol III was labeled with a red fluorophore (magenta). The kymograph demonstrates the fluorescent spot corresponding to Pol III co-localizes with the tip of the growing DNA product (evident as a white spot) where the replication fork is located.
DOI: http://dx.doi.org/10.7554/eLife.23932.008