ja408656y_si_002.avi (1.59 MB)
DNA Origami Based Visualization System for Studying Site-Specific Recombination Events
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posted on 2014-01-08, 00:00 authored by Yuki Suzuki, Masayuki Endo, Yousuke Katsuda, Keiyu Ou, Kumi Hidaka, Hiroshi SugiyamaSite-specific
recombination involves reciprocal exchange between
defined DNA sites. The reaction initiates from the formation of a
recombinase–DNA synaptic complex, in which two recombination
sites arrange in an appropriate configuration. However, there is incomplete
information about how the topological state of the substrate influences
the synapsis and outcome of the reaction. Here, we show that Cre-mediated
recombination can be regulated by controlling the orientation and
topology of the loxP substrate in a DNA frame nanoscaffold.
High-speed atomic force microscopy analyses revealed that the loxP-containing substrate strands in the antiparallel orientation
can be recombined only through formation of synaptic complexes. By
tethering Holliday junction (HJ) intermediates to DNA frames in different
connection patterns and using them as a starting substrate, we found
that the topological state of the HJ intermediates dictates the outcome
of the resolution. Our approach should provide a new platform for
structural–functional studies of various DNA targeting enzymes,
especially which require formation of synaptic complexes.