Complex between a Multicrossover DNA Nanostructure, PX-DNA, and T7 Endonuclease I

Paranemic crossover DNA (PX-DNA) is a four-stranded multicrossover structure that has been implicated in recombination-independent recognition of homology. Although existing evidence has suggested that PX is the DNA motif in homologous pairing (HP), this conclusion remains ambiguous. Further investigation is needed but will require development of new tools. Here, we report characterization of the complex between PX-DNA and T7 endonuclease I (T7endoI), a junction-resolving protein that could serve as the prototype of an anti-PX ligand (a critical prerequisite for the future development of such tools). Specifically, nuclease-inactive T7endoI was produced and its ability to bind to PX-DNA was analyzed using a gel retardation assay. The molar ratio of PX to T7endoI was determined using gel electrophoresis and confirmed by the Hill equation. Hydroxyl radical footprinting of T7endoI on PX-DNA is used to verify the positive interaction between PX and T7endoI and to provide insight into the binding region. Cleava...