DNA Looping Mediated by Site-Specific SfiI–DNA Interactions

Abstract

Interactions between distant DNA segments play important roles in various biological processes, such as DNA recombination. Certain restriction enzymes create DNA loops when two sites are held together and then cleave the DNA. DNA looping is important during DNA synapsis. Here we investigated the mechanisms of DNA looping by restriction enzyme SfiI by measuring the properties of the system at various temperatures. Different sized loop complexes, mediated by SfiI–DNA interactions, were visualized with AFM. The experimental results revealed that small loops are more favorable compared to other loop sizes at all temperatures. Our theoretical model found that entropic cost dominates at all conditions, which explains the preference for short loops. Furthermore, specific loop sizes were predicted as favorable from an energetic point of view. These predictions were tested by experiments with transiently assembled SfiI loops on a substrate with a single SfiI site.

Site-Search Process for Synaptic Protein-DNA Complexes

“…This shows that the initial loop size might influence the site-search direction, resulting in growth or shrinkage of the loop to attain the favorable loop size from an energetic point of view. This can be supported with the model proposed in our recent publication [ 17 ], where our model predicted that specific loop sizes around 300-350 bp were energetically favorable.…”

“…The protein appears as a bright feature connecting two recognition sites on the DNA substrate. According to our previous studies [ 17 ], these are highly specific synaptic complexes with high stability compared with non-specific complexes.…”

“…Their assembly is supported by AFM images ( Figure 2 ). Such synaptic complexes correspond to the most thermodynamically stable SfiI-DNA assemblies, so only looped complexes between the cognates for SfiI are visualized with the AFM of dried complexes ( Figure 2 A and [ 17 ]). However, in liquid, we were able to visualize the dynamics of such looped complexes ( Figure 3 and Figure 4 ).…”

“…The DNA substrate was adopted from our previous study [ 17 ]. Briefly, PCR was performed on a pUC19 plasmid (Bio Basic, Markham, ON, Canada) containing an 885 bp DNA segment with three SfiI cognate sites (GGCCTCGAG-GGCC [ 25 ]), to obtain the final DNA construct with a length of 1036 bp.…”

“…If the specific sites needed for the formation of the synaptosome are located on the same DNA molecule (i.e., pairing in cis ), the search for the two sites leads to the formation of DNA loops. The formation of such loops for various synaptosomes has been visualized with Atomic Force Microscopy (AFM) in our previous papers [ 2 , 14 , 15 , 16 , 17 , 18 , 19 ]. Note article [ 18 ], in which the assembly of the synaptosome with three DNA binding sites held by the EcoRII restriction enzyme was directly visualized.…”