Force-driven unbinding of proteins HU and Fis from DNA quantified using a thermodynamic Maxwell relation

Abstract

Determining numbers of proteins bound to large DNAs is important for understanding their chromosomal functions. Protein numbers may be affected by physical factors such as mechanical forces generated in DNA, e.g. by transcription or replication. We performed single-DNA stretching experiments with bacterial nucleoid proteins HU and Fis, verifying that the force–extension measurements were in thermodynamic equilibrium. We, therefore, could use a thermodynamic Maxwell relation to deduce the change of protein number on a single DNA due to varied force. For the binding of both HU and Fis under conditions studied, numbers of bound proteins decreased as force was increased. Our experiments showed that most of the bound HU proteins were driven off the DNA at 6.3 pN for HU concentrations lower than 150 nM; our HU data were fit well by a statistical-mechanical model of protein-induced bending of DNA. In contrast, a significant amount of Fis proteins could not be forced off the DNA at forces up to 12 pN and Fis concentrations up to 20 nM. This thermodynamic approach may be applied to measure changes in numbers of a wide variety of molecules bound to DNA or other polymers. Force-dependent DNA binding by proteins suggests mechano-chemical mechanisms for gene regulation.

ParB spreading requires DNA bridging

“…HU proteins condense DNA by introducing flexible local bends, each of which causes only a small change in the end-to-end length of DNA (Swinger and Rice 2004). As a result, condensation of DNA by B. subtilis HBsu is not predicted to be strongly suppressed by the <1 pN forces exerted on DNA in our assay, and compaction by HBsu therefore occurs along the entire DNA molecule (Xiao et al 2010(Xiao et al , 2011Wagner et al 2011;TGW Graham and JJ Loparo, unpubl.). In contrast, the end bias in compaction by Spo0J and H-NS suggests that each compaction step involves trapping a much larger loop of DNA.…”

A stochastic reaction-diffusion model for protein aggregation on DNA

“…This mechanism of local bends formation also interprets the force dependent unbinding rate of HU protein observed in experiments. 46,9 (ii) HU-induced DNA compaction and sti®ening. At low HU concentration, the bending rigidity of the DNA always is reduced irrespective of whether the HUinduced°exible hinges result in rigid bends.…”

“…This e®ect suggests that the bimodal DNA softening/sti®ening behavior does not survive for moderate salt concentrations, in agreement with the experimental observation. 9 At an even higher salt concentration, the attractive HU-DNA electrostatic interaction vanishes, resulting in the nonbinding of the HU proteins to DNA. Consequently, the°exibility of the DNA is not a®ected by the presence of HU in solution as observed in the experiment of Ref.…”

“…In other words, low HU concentration promotes DNA compaction, whereas high HU concentration results in the spontaneous formation of rigid HU¯laments that reduce the°exibility of the molecule. 6,7,9 Experiments have also shown that high salt concentration eliminates rigid¯laments and the bimodal behavior is not observed. 9 Recent experimental observation revealed that, even at moderate salt concentrations, the bimodal behavior does not survive for long time scales (&2 h) due to the ability of the HU proteins to rearrange around the DNA.…”

“…6,7,9 Experiments have also shown that high salt concentration eliminates rigid¯laments and the bimodal behavior is not observed. 9 Recent experimental observation revealed that, even at moderate salt concentrations, the bimodal behavior does not survive for long time scales (&2 h) due to the ability of the HU proteins to rearrange around the DNA. 10 Interestingly, HU¯laments tend to form spiral con¯gurations that excite supercoiling, which could potentially promote DNA compaction and regulate the promoter activity.…”