RT Journal Article
SR Electronic
T1 Real-time observation of replicative helicase assembly onto single-stranded DNA
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 077800
DO 10.1101/077800
A1 David Dulin
A1 Zhongbo Yu
A1 Tao Ju Cui
A1 Bojk A. Berghuis
A1 Martin Depken
A1 Nynke H. Dekker
YR 2016
UL http://biorxiv.org/content/early/2016/09/27/077800.abstract
AB Replicative helicases load onto DNA at the start of replication, and play a vital role by driving the replication fork forward. These helicases assemble into closed multimeric rings that need to encircle single-stranded (ss)DNA to be activated. Though helicase loading on substrates with accessible free ends has been well characterized for the T7 gp4 helicase, a model system for superfamily IV replicative helicases, the physiologically more relevant loading onto exposed ssDNA without free ends remains less well understood. Here, using a label-free assay that exploits changes in the DNA hairpin hopping dynamics to detect gp4 binding and activity, we characterize loading and activation of gp4 on exposed ssDNA without free ends, and find clear evidence of stepwise assembly of the helicase at the fork at physiologically relevant concentrations. The gradual loading onto ssDNA, rather than pre-forming in solution followed by spontaneous ring opening which appears favored at higher concentrations, suggests a new paradigm of stepwise assembly for the helicases in superfamily IV that do not require a separate loading enzyme.