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Real-time Observation of Bacteriophage T4 gp41 Helicase Reveals Unwinding Mechanism

Wednesday, September 19, 2007 - 9:00am - 10:00am
EE/CS 3-180
Vincent Croquette (École Normale Supérieure)
Joint work with Timothée Lionnet1, Michelle M. Spiering2, Stephen J. Benkovic2, and David Bensimon1

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
ssDNA
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; second, this weak helicase cannot efficiently unwind
the T4 genome
alone. Future experiments on the full replisome will be
useful to understand how fast and processive replication is
achieved.

1LPS, ENS, Paris, France.;
2Dept.of Chemistry, Penn State
University, USA.