The effect of roadblocks on DNA target search by NdeI

DNA binding proteins use cycles of three-dimensional and one-dimensional search to locate their target binding sites. During one-dimensional search, proteins can be blocked by other DNA binding proteins. These roadblocks are bypassed by hopping, jumping, intersegmental transfer or dislodging the roadblocks. Because roadblocks affect target search kinetics their introduction is a useful way to probe DNA target search. Here we report the use of a single molecule DNA tethering method to characterize the target search kinetics of the type II restriction endonuclease NdeI in the presence of dCas9 roadblocks. We find that target search with roadblocks is sensitive to salt concentration, a result that holds for bare DNA also. Additionally, roadblocks inhibit target search at low salt concentration. However, at higher, more in vivo-like salt concentrations, the search rate is insensitive to roadblocks. This search rate is shown to be the same as for shorter unblocked DNAs, indicating that the protein does not hop over the roadblocks. We develop a model of target search that includes sliding only with no hopping. The model explains our data and demonstrates that NdeI can bypass roadblocks at in vivo salt concentrations by employing multiple cycles of three-dimensional search and sliding, a search method termed jumping. Our data also suggests hopping could play a role at mid-range salt concentrations.