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Structural basis for DNA duplex separation by a superfamily-2 helicase


To reveal the mechanism of processive strand separation by superfamily-2 (SF2) 3′→5′ helicases, we determined apo and DNA-bound crystal structures of archaeal Hel308, a helicase that unwinds lagging strands and is related to human DNA polymerase θ. Our structure captures the duplex-unwinding reaction, shows that initial strand separation does not require ATP and identifies a prominent β-hairpin loop as the unwinding element. Similar loops in hepatitis C virus NS3 helicase and RNA-decay factors support the idea that this duplex-unwinding mechanism is applicable to a broad subset of SF2 helicases. Comparison with ATP-bound SF2 enzymes suggests that ATP promotes processive unwinding of 1 base pair by ratchet-like transport of the 3′ product strand. Our results provide a first structural framework for strand separation by processive SF2 3′→5′ helicases and reveal important mechanistic differences from SF1 helicases.

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Figure 1: Structure of the Hel308–DNA complex.
Figure 2: Details of selected DNA-recognition sites.
Figure 3: Model of processive unwinding by archaeal Hel308.
Figure 4: Test for large-scale conformational changes by limited proteolyis.
Figure 5: Comparison of Hel308 to other SF2 and SF1 helicases.

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We thank S. Cui for comments on the manuscript, F. Uhlmann for collaborative efforts and scientific discussions, and the staff of the Swiss Light Source (beamline PXI) and the European Synchrotron Radiation Facility for generous beamtime allocation and help with data collection. K.-P.H. acknowledges grant support from the German research council (Deutsche Forschungsgemeinschaft HO2489/3-1 and SFB455), Human Frontiers of Science and the European Commission (integrated project 'DNA Repair').

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Authors and Affiliations



K.B. crystallized DNA-bound Hel308, determined structures and designed research; S.N. cloned the gene for A. fulgidus Hel308 and crystallized apo-Hel308; K.-P.H. designed research, participated in model building and evaluation, and wrote manuscript.

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Correspondence to Karl-Peter Hopfner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Sequence alignment of A. fulgidus Hel308 with selected SF2/Ski2 family helicases. (PDF 629 kb)

Supplementary Fig. 2

Composit simulated annealed omit electron density shows how strands of a DNA duplex are separated by archaeal Hel208. (PDF 749 kb)

Supplementary Fig. 3

Comparison of apo- and DNA-bound archaeal Hel308. (PDF 688 kb)

Supplementary Fig. 4

Test for large-scale DNA- and ATP-induced conformational changes in archaeal Hel308 by limited proteolysis digestion using trypsin and chymotrypsin. (PDF 339 kb)

Supplementary Methods (PDF 68 kb)

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Büttner, K., Nehring, S. & Hopfner, KP. Structural basis for DNA duplex separation by a superfamily-2 helicase. Nat Struct Mol Biol 14, 647–652 (2007).

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