Results: 5

Figure 2

Figure 2. Real-time visualization of sister fork uncoupling during unwinding of doubly tethered DNA. From: Bypass of a protein roadblock by a replicative DNA helicase.

a, T-ag was drawn into a flow cell containing doubly tethered λori. After 45 min, RPAmKikGR was introduced and mKikGR was imaged for 60 min. b, Kymograph of mKikGR fluorescence. Minutes denote time after introduction of RPAmKikGR.

Hasan Yardimci, et al. Nature. ;492(7428):205-209.
Figure 5

Figure 5. Bypass of tandem protein adducts by T-ag depends on the inter-adduct distance. From: Bypass of a protein roadblock by a replicative DNA helicase.

a, Unwinding of DNA containing, none, one, or two M.HpaII adducts on the translocation strand by T-ag. The three templates (color coded differentially) had slightly different lengths because each template contained 240 bp before the first roadblock and 277 bp after the last roadblock but different amounts of DNA between the roadblocks. DNAs were internally labeled with [α32P]-dATP. b, Quantification of unwinding from 3 independent experiments as in a. Error bars indicate S.D.

Hasan Yardimci, et al. Nature. ;492(7428):205-209.
Figure 1

Figure 1. T-ag is not an obligate double hexamer during replication. From: Bypass of a protein roadblock by a replicative DNA helicase.

a, Models for DNA unwinding by T-ag. See text for details. b, Experimental procedure for replication of singly and doubly tethered λori DNA. c, SYTOX and anti-dig images of singly tethered (i) and doubly tethered (ii) λori DNAs that underwent replication in HeLa cell extracts. Dig-dUTP incorporated regions occasionally exhibited higher SYTOX intensity due to non-specific staining of anti-dig antibody with SYTOX. Extent of slack and replication on the doubly tethered DNA are indicated. Yellow arrowheads, estimated position of the origin. d, Length of anti-dig tracts on singly tethered (gray) and doubly tethered (black) DNA molecules after a 40 min dig-dUTP pulse. To measure the fork rate, the tract length distribution was fit to a Gaussian and the resulting average tract length was divided by the duration of dig-dUTP pulse (40 min).

Hasan Yardimci, et al. Nature. ;492(7428):205-209.
Figure 4

Figure 4. T-ag can bypass a covalent protein roadblock on the translocation strand. From: Bypass of a protein roadblock by a replicative DNA helicase.

a, T-ag-dependent unwinding of DNA containing MHlag (i) or MHlead (ii). The species corresponding to each band are depicted. Heat denaturation caused electrophoretic smearing of M.HpaII-conjugated DNA and therefore was not used for assessment of ssDNA migration (data not shown). (iii) Quantification of unwinding in (i) and (ii). b, (i) Unwinding of unmodified (lane 3), MHlag-modified (lane 6), and MHlead-modified (lane 9) DNA by pre-assembled T-ag (see text for details). The first two lanes in all samples correspond to 10% of input DNA without bead conjugation in the absence (lanes 1, 4, and 7) and presence (lanes 2, 5, and 8) of T-ag-mediated unwinding. (ii) Quantification of unwound DNA by pre-assembled T-ag (lanes 3, 6, 9 in B-i). Amount of unwinding was normalized to that of unmodified DNA. Error bars in a-iii and b-ii indicate S.D. for 3 independent experiments.

Hasan Yardimci, et al. Nature. ;492(7428):205-209.
Figure 3

Figure 3. T-ag translocates on ssDNA in the 3′ to 5′ direction. From: Bypass of a protein roadblock by a replicative DNA helicase.

a, (i) Cartoon of 518-bp long 5′-labeled (red stars) DNA templates used for SA displacement assays. Predictions of 3′ to 5′ ssDNA translocation (ii) and dsDNA translocation (iii) models. b, DNAs biotinylated on the top or bottom strands as in (A-i) undergo complete mobility shift upon SA addition, indicating that all DNA molecules are modified with biotin (lanes 2, 4). DNA was pre-incubated with buffer (lanes 5, 7) or SA (lanes 6, 8), and unwinding was initiated with T-ag and RPA (lanes 5–8). Excess biotin saturated any displaced SA. To assess the migration of ssDNA with or without SA, DNA was heat denatured, rapidly cooled down and mixed with buffer (lanes 9, 11) or SA (lanes 10, 12). Because both strands are radiolabeled, SA association with one strand shifts only half of the signal (lanes 10, 12). c, ssDNA with (gray) and without (black) SA from lanes 6 and 8 of panel b was quantified. Error bars indicate S.D. for 3 independent experiments. Some spontaneous dissociation of SA occurred in the presence of free biotin (lanes 6 and 8, ds). The extent of T-ag-independent SA dissociation was determined using the relative amounts of dsDNA that lost (ds) and retained SA (ds+SA) This fraction was then used to measure the amount of ssDNA that lost and retained SA, respectively, if no spontaneous SA dissociation had occurred. d, SYTOX, anti-dig, and Qdot images of representative molecules upon fork collision with Qdotlag (i) or Qdotlead (ii) in HeLa cell extracts (performed as in ). Because dig-dUTP was continuously present during the replication reaction, replication bubbles were fully labeled with anti-dig. The percentage of molecules exhibiting fork bypass and stalling events are indicated (see also ).

Hasan Yardimci, et al. Nature. ;492(7428):205-209.

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