Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism.
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 ( approximately 400 bp/s), its unwinding rate extrapolated to zero force is much slower ( approximately 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.
Lionnet
Timothée
T
Laboratoire de Physique Statistique, Ecole Normale Supérieure, Centre National de la Recherche Scientifique-UMR8550, 24 Rue Lhomond, 75005 Paris, France. tlionnet@aecom.yu.edu
Spiering
Michelle M
MM
Benkovic
Stephen J
SJ
Bensimon
David
D
Croquette
Vincent
V
D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins; C063983:gene 41 protein, Enterobacteria phage T4