HU Protein and DNA Supercoiling Dramatically Enhance Lac-Repressor-Mediated DNA Looping

Abstract

DNA loops mediated by proteins that bind with different affinity to distant sites regulate multiple aspects of DNA metabolism, such as transcription, replication and recombination. Binding of accessory proteins and DNA supercoiling are two factors that affect the formation of DNA loops. Previous studies indicate that the heat unstable protein (HU) enhances the formation of short loops perhaps by changing the flexibility of DNA upon binding non-specifically. However, for loops long enough such that DNA stiffness is not limiting, the effect of HU on loop formation is not well understood. Negative supercoiling also enhances the formation of long loops. To better understand how binding affinity, loop length, HU and negative supercoiling affect looping, single molecule experiments were performed with the lac repressor protein (LacI) as a DNA looping protein. Using tethered particle motion, a titration of the formation of Lac repressor mediated-loop was carried out using three different DNA templates, OID-900-O1, OID-400-O1, and O1-400-O2 (Ostrong-loop size (bp)-Oweak). It showed that long loops are surprisingly efficient across a broad range of concentration. In similar titrations of LacI-induced looping as a function of HU concentration, HU greatly enhanced looping in 400 but not the longer 900 bp loops. Magnetic tweezers were used to investigate the effect of supercoiling on the formation of LacI-mediated DNA loops using 2 kbp DNA molecules with a centrally located O1-400-O2. Even slight tension in the magnetic tweezer interfered with looping, however, supercoiling compensated for increased tension. Positive supercoiling enhanced loop formation but less efficiently than negative which increased the looping probability from 0 to 100%. Thus the levels of accessory proteins and supercoiling, two dynamic parameters, enable modulation of regulatory loops the baseline probability of which is established by loop size and binding site affinities.