Significance: Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity for bacteria and archaea. They use a protein complex that targets invading nucleic acids using a bound RNA that hybridizes to complementary sequence stretches. On DNA targets, the resulting structure is an R-loop. By twisting single DNA molecules, we can directly observe R-loop formation and dissociation in two distinct types of CRISPR-Cas system. This allows us to investigate the role of different elements of the target sequence. Whereas upstream elements control the actual R-loop formation, downstream elements ensure R-loop stability, suggesting a directional R-loop formation. This provides insight into the recognition process by these enzymes, which is the crucial step to understand targeting in the rapidly emerging genetic engineering applications of CRISPR-Cas systems.

Proteins that interact with specific DNA sites bind to DNA at random and then translocate to the target site. This may occur by one‐dimensional diffusion along the DNA, or through three‐dimensional space via multiple dissociation/re‐associations. To distinguish these routes, reactions of the Eco RV endonuclease were studied on substrates with two Eco RV sites separated by varied distances. The fraction of encounters between the DNA and the protein that resulted in the processive cleavage of both sites decreased as the length of intervening DNA was increased, but not in the manner demanded for one‐dimensional diffusion. The variation in processivity with inter‐site spacing shows instead that protein moves from one site to another through three‐dimensional space, by successive dissociation/re‐associations, though each re‐association to a new site is followed by a search of the DNA immediately adjacent to that site. Although DNA‐binding proteins are usually thought to find their target sites by one‐dimensional pathways, three‐dimensional routes may be more common than previously anticipated.

Read the full review for this F1000Prime recommended article: Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme.

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▪ Abstract Most reactions on DNA are carried out by multimeric protein complexes that interact with two or more sites in the DNA and thus loop out the DNA between the sites. The enzymes that cataly...