Faculty Opinions recommendation of Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Author(s):  
Olivier Hyrien
Cell ◽  
2015 ◽  
Vol 161 (3) ◽  
pp. 513-525 ◽  
Author(s):  
Simina Ticau ◽  
Larry J. Friedman ◽  
Nikola A. Ivica ◽  
Jeff Gelles ◽  
Stephen P. Bell

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Kanokwan Champasa ◽  
Caitlin Blank ◽  
Larry J Friedman ◽  
Jeff Gelles ◽  
Stephen P Bell

Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.


2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.


2010 ◽  
Vol 63 (4) ◽  
pp. 624
Author(s):  
Michael J. Serpe ◽  
Jason R. Whitehead ◽  
Stephen L. Craig

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.


2011 ◽  
Vol 100 (3) ◽  
pp. 464a
Author(s):  
Promod R. Pratap ◽  
Gregor Heiss ◽  
Martin Sikor ◽  
Don C. Lamb ◽  
Max Burnett

2014 ◽  
Vol 106 (2) ◽  
pp. 394a
Author(s):  
Richard Janissen ◽  
Bojk A. Berghuis ◽  
Orkide Ordu ◽  
Max M. Wink ◽  
David Dulin ◽  
...  

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