TY - JOUR
T1 - Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
AU - Lionnet, Timothée
AU - Spiering, Michelle M.
AU - Benkovic, Stephen J.
AU - Bensimon, David
AU - Croquette, Vincent
PY - 2007/12/11
Y1 - 2007/12/11
N2 - 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-base pair (bp) resolution. Although 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). 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. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.
AB - 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-base pair (bp) resolution. Although 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). 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. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.
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U2 - 10.1073/pnas.0709793104
DO - 10.1073/pnas.0709793104
M3 - Article
C2 - 18077411
AN - SCOPUS:38049186506
SN - 0027-8424
VL - 104
SP - 19790
EP - 19795
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 50
ER -