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Bypass of a protein barrier by a replicative DNA helicase

Nature volume 492, pages 205–209 (2012)Cite this article

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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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Figure 1: Large T antigen is not an obligate double hexamer during replication.
Figure 2: Real-time visualization of sister fork uncoupling during unwinding of doubly tethered DNA.
Figure 3: Large T antigen translocates on ssDNA in the 3′-to-5′ direction.
Figure 4: Large T antigen can bypass a covalent protein barrier on the translocation strand.
Figure 5: Bypass of tandem protein adducts by large T antigen depends on the inter-adduct distance.

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Acknowledgements

We thank C. Richardson for critical reading of the manuscript, and C. Etson for help in the preparation of λori DNA. J.C.W. was supported by grants from the National Institutes of Health (NIH) (GM62267 and HL098316) and American Cancer Society (ACS) (RSG0823401GMC). A.M.v.O. was supported by grants from the NIH (GM077248), ACS (RSG0823401GMC), and the Netherlands Organization for Scientific Research (NWO; Vici 680-47-607). J.H. was funded by NIH grant GM5 R01 GM034559. X.W. was a long-term postdoctoral fellow of the Human Frontier Science Program; D.Z.R. was supported by NIH grant GM086466.

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Author notes

  1. Antoine M. van Oijen and Johannes C. Walter: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Hasan Yardimci, Anna B. Loveland & Johannes C. Walter

  2. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Xindan Wang & David Z. Rudner

  3. Program of Molecular Biology, Memorial Sloan–Kettering Cancer Center, New York, 10065, New York, USA

    Inger Tappin & Jerard Hurwitz

  4. The Zernike Institute for Advanced Materials, University of Groningen, 9747 AG Groningen, The Netherlands,

    Antoine M. van Oijen

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Contributions

H.Y., A.M.v.O. and J.C.W. designed the experiments. H.Y. performed the experiments. X.W. and D.Z.R. supervised and assisted H.Y. for construction of λori DNA. A.B.L. made fluorescently tagged RPA. I.T. and J.H. provided RPA and large T antigen. H.Y., A.M.v.O. and J.C.W. interpreted the data and wrote the paper. X.W. and A.B.L. contributed equally.

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Correspondence to Antoine M. van Oijen or Johannes C. Walter.

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Yardimci, H., Wang, X., Loveland, A. et al. Bypass of a protein barrier by a replicative DNA helicase. Nature 492, 205–209 (2012). https://doi.org/10.1038/nature11730

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