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SYSM: Symposium Single Molecules

SYSM 1: Single molecules

SYSM 1.4: Hauptvortrag

Dienstag, 26. Februar 2008, 16:00–16:30, H 0105

Real-time observation of bacteriophage T4 gp41 helicase reveals unwinding mechanism — M. Manosa, T. Lionnet, M. M. Spiering, S. J. Benkovic, D. Bensimon, and •V. Croquette — Laboratoire de Physique Statistique, Ecole Normale Supérieure, 24 rue Lhomond, 75005 Paris, France

Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-bp resolution. Whereas gp41 translocates on ssDNA as fast as the in vivo replication fork (~400 bp/s), its unwinding rate extrapolated to zero force is much slower (~30 bp/s). Taken together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; and second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome in order to achieve rapid and processive replication. We have also investigate Rec-Q from E-coli which is a monomeric helicase involved in DNA repair. This enzyme behaves very differently from gp41 clearly demonstrating an active unwinding behavior.