Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.     

Effects of nucleoid proteins on DNA repression loop formation in Escherichia coli

The intrinsic stiffness of DNA limits its ability to be bent and twisted over short lengths, but such deformations are required for gene regulation. One classic paradigm is DNA looping in the regulation of the Escherichia coli lac operon. Lac repressor protein binds simultaneously to two operator sequences flanking the lac promoter. Analysis of the length dependence of looping-dependent repression of the lac operon provides insight into DNA deformation energetics within cells. The apparent flexibility of DNA is greater in vivo than in vitro, possibly because of host proteins that bind DNA and induce sites of flexure. Here we test DNA looping in bacterial strains lacking the nucleoid proteins HU, IHF or H-NS. We confirm that deletion of HU inhibits looping and that quantitative modeling suggests residual looping in the induced operon. Deletion of IHF has little effect. Remarkably, DNA looping is strongly enhanced in the absence of H-NS, and an explanatory model is proposed. Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling. These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.     

Oligomeric initiator protein-mediated DNA looping negatively regulates plasmid replication in vitro by preventing origin melting

Although DNA looping between the initiator binding sites (iterons) of the replication origin (ori) of a plasmid and the iterons located in a cis-acting control sequence called inc has been postulated to promote negative control of plasmid DNA replication, not only was definitive evidence for such looping lacking, but also the detailed molecular mechanism of this control had not been elucidated. Here, we present direct evidence showing that both the monomeric and the dimeric forms of the RepE initiator protein of F factor together promote pairing of incC-oriF sites by DNA looping. By using a reconstituted replication system consisting of 26 purified proteins, we show further that the DNA loop formation negatively regulates plasmid replication by inhibiting the formation of an open complex at the replication origin, thus elucidating a key step of replication control.     

Interplay of Protein Binding Interactions,DNA Mechanics,and Entropy in?DNA Looping Kinetics

DNA looping plays a key role in many fundamental biological processes, including gene regulation, recombination, and chromosomal organization. The looping of DNA is often mediated by proteins whose structural features and physical interactions can alter the length scale at which the looping occurs. Looping and unlooping processes are controlled by thermodynamic contributions associated with mechanical deformation of the DNA strand and entropy arising from thermal fluctuations of the conformation. To determine how these confounding effects influence DNA looping and unlooping kinetics, we present a theoretical model that incorporates the role of the protein interactions, DNA mechanics, and conformational entropy. We show that for shorter DNA strands the interaction distance affects the transition state, resulting in a complex relationship between the looped and unlooped state lifetimes and the physical properties of the looped DNA. We explore the range of behaviors that arise with varying interaction distance and DNA length. These results demonstrate how DNA deformation and entropy dictate the scaling of the looping and unlooping kinetics versus the J-factor, establishing the connection between kinetic and equilibrium behaviors. Our results show how the twist-and-bend elasticity of the DNA chain modulates the kinetics and how the influence of the interaction distance fades away at intermediate to longer chain lengths, in agreement with previous scaling predictions.     

Roles of DNA looping in enhancer-blocking activity

Enhancer-promoter interactions in eukaryotic genomes are often controlled by sequence elements that block the actions of enhancers. Although the experimental evidence suggests that those sequence elements contribute to forming loops of chromatin, the molecular mechanism of how such looping affects the enhancer-blocking activity is still largely unknown. In this article, the roles of DNA looping in enhancer blocking are investigated by numerically simulating the DNA conformation of a prototypical model system of gene regulation. The simulated results show that the enhancer function is indeed blocked when the enhancer is looped out so that it is separated from the promoter, which explains experimental observations of gene expression in the model system. The local structural distortion of DNA caused by looping is important for blocking, so the ability of looping to block enhancers can be lost when the loop length is much larger than the persistence length of the chain.     

基于DNA变形能的核小体定位预测方法研究进展

真核细胞中,作为染色质基本结构单元的核小体参与调控基因的转录、DNA复制、重组以及RNA剪接等诸多生物学过程。阐明核小体定位机制并准确预测核小体在染色体上的位置对解读染色质结构与功能有重要生物学意义。在过去30多年时间里,研究人员发展了多种预测核小体位置的方法。最理想的方法应考虑DNA序列、组蛋白修饰和染色质重塑等影响核小体定位的诸多因素,然而现实中,捕捉主要因素的模型也往往具有很高的鲁棒性和实用价值。DNA序列偏好性是在全基因组尺度上影响核小体定位的最重要因素之一,因此基于DNA序列的核小体定位预测方法也最常见。这种方法可大致分为两类,即基于DNA序列信息的生物信息学模型和基于DNA变形能的生物物理学模型。本文重点介绍生物物理学模型近些年取得的主要进展。     

Mechanical properties of symmetric and asymmetric DNA A-tracts: implications for looping and nucleosome positioning

A-tracts are functionally important DNA sequences which induce helix bending and have peculiar structural properties. While A-tract structure has been qualitatively well characterized, their mechanical properties remain controversial. A-tracts appear structurally rigid and resist nucleosome formation, but seem flexible in DNA looping. In this work, we investigate mechanical properties of symmetric A_nT_n and asymmetric A_2n tracts for n = 3, 4, 5 using two types of coarse-grained models. The first model represents DNA as an ensemble of interacting rigid bases with non-local quadratic deformation energy, the second one treats DNA as an anisotropically bendable and twistable elastic rod. Parameters for both models are inferred from microsecond long, atomic-resolution molecular dynamics simulations. We find that asymmetric A-tracts are more rigid than the control G/C-rich sequence in localized distortions relevant for nucleosome formation, but are more flexible in global bending and twisting relevant for looping. The symmetric tracts, in contrast, are more rigid than asymmetric tracts and the control, both locally and globally. Our results can reconcile the contradictory stiffness data on A-tracts and suggest symmetric A-tracts to be more efficient in nucleosome exclusion than the asymmetric ones. This would open a new possibility of gene expression manipulation using A-tracts.