Direct visualization of the effect of DNA structure and ionic conditions on HU–DNA interactions

Abstract

Architectural DNA–binding proteins are involved in many important DNA transactions by virtue of their ability to change DNA conformation. Histone-like protein from E. coli strain U93, HU, is one of the most studied bacterial architectural DNA–binding proteins. Nevertheless, there is still a limited understanding of how the interactions between HU and DNA are affected by ionic conditions and the structure of DNA. Here, using optical tweezers in combination with fluorescent confocal imaging, we investigated how ionic conditions affect the interaction between HU and DNA. We directly visualized the binding and the diffusion of fluorescently labelled HU dimers on DNA. HU binds with high affinity and exhibits low mobility on the DNA in the absence of Mg2+; it moves 30-times faster and stays shorter on the DNA with 8 mM Mg2+ in solution. Additionally, we investigated the effect of DNA tension on HU–DNA complexes. On the one hand, our studies show that binding of HU enhances DNA helix stability. On the other hand, we note that the binding affinity of HU for DNA in the presence of Mg2+ increases at tensions above 50 pN, which we attribute to force-induced structural changes in the DNA. The observation that HU diffuses faster along DNA in presence of Mg2+ compared to without Mg2+ suggests that the free energy barrier for rotational diffusion along DNA is reduced, which can be interpreted in terms of reduced electrostatic interaction between HU and DNA, possibly coinciding with reduced DNA bending.

Extracting and characterizing protein-free megabasepair DNA for in vitro experiments

“…For studying chromosome organization in the eukaryotic nucleus or in bacterial cells, numerous studies have been made on live or fixed cells through imaging (Bintu et al, 2018;Ricci et al, 2015), chromosome conformation capture techniques (Brandão et al, 2021;Falk et al, 2019), etc., while in vitro protein-DNA interactions are often characterized at the single-molecule level using techniques such as Atomic Force Microscopy (Dame et al, 2000;Japaridze et al, 2017;Liang et al, 2017), magnetic (Kaczmarczyk et al, 2020;Sun et al, 2013) and optical tweezers (Lin et al, 2021;Renger et al, 2022), and DNA visualization assays (Davidson et al, 2019;Ganji et al, 2018;Golfier et al, 2020;Greene et al;Kim et al, 2019). While these complementary approaches have yielded great insights, they leave a significant gap since typical single-molecule methods address the ~kilobasepair (kbp) scale while actual genomes consist of 10 5 -10 11 bp long DNA.…”

Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments

“…For studying chromosome organization in the eukaryotic nucleus or in bacterial cells, numerous studies have been made on live or fixed cells through imaging, 6 , 7 chromosome conformation capture techniques, 8 , 9 etc., while in vitro protein-DNA interactions are often characterized at the single-molecule level using techniques such as atomic force microscopy, 10 , 11 , 12 magnetic 13 , 14 and optical tweezers, 15 , 16 and DNA visualization assays. 17 , 18 , 19 , 20 , 21 While these complementary approaches have yielded great insights, they leave a significant gap since typical single-molecule methods address the ∼kilobase pair (kbp) scale, while actual genomes consist of 10 5 –10 11 base-pair (bp) long DNA.…”