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 DNAbound crystal structures of archaeal Hel308, a helicase that unwinds lagging strands and is related to human DNA polymerase theta. Our structure captures the duplex- unwinding reaction, shows that initial strand separation does not require ATP and identifies a prominent beta 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 ATPbound 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.