Bypass of a protein barrier by a replicative DNA helicase - PubMed (original) (raw)

. 2012 Dec 13;492(7428):205-9.

doi: 10.1038/nature11730. Epub 2012 Nov 28.

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Bypass of a protein barrier by a replicative DNA helicase

Hasan Yardimci et al. Nature. 2012.

Abstract

Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here we use single-molecule and ensemble assays to show that large T antigen assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3'-to-5' direction. Unexpectedly, large T antigen unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the large T antigen ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms, and reveal a new level of plasticity in the interactions of replicative helicases with DNA damage.

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Figures

Figure 1

Figure 1. T-ag is not an obligate double hexamer during replication

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).

Figure 2

Figure 2. Real-time visualization of sister fork uncoupling during unwinding of doubly tethered DNA

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.

Figure 3

Figure 3. T-ag translocates on ssDNA in the 3′ to 5′ direction

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 Fig. 1b). 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 Supplementary Fig. 4).

Figure 4

Figure 4. T-ag can bypass a covalent protein roadblock on the translocation strand

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.

Figure 5

Figure 5. Bypass of tandem protein adducts by T-ag depends on the inter-adduct distance

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.

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