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Transcriptional regulation usually requires the action of several proteins that either repress or activate a promotor of an open reading frame. These proteins can counteract each other, thus allowing tight regulation of the transcription of the corresponding genes, where tight repression is often linked to DNA looping or cross‐linking. Here, the tetramerization domain of the bacterial gene repressor Rco from Bacillus subtilis plasmid pLS20 (RcopLS20) has been identified and its structure is shown to share high similarity to the tetramerization domain of the well known p53 family of human tumor suppressors, despite lacking clear sequence homology. In RcopLS20, this tetramerization domain is responsible for inducing DNA looping, a process that involves multiple tetramers. In accordance, it is shown that RcopLS20 can form octamers. This domain was named TetDloop and its occurrence was identified in other Bacillus species. The TetDloop fold was also found in the structure of a transcriptional repressor from Salmonella phage SPC32H. It is proposed that the TetDloop fold has evolved through divergent evolution and that the TetDloop originates from a common ancestor predating the occurrence of multicellular life. [ABSTRACT FROM AUTHOR]

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here, we demonstrate a new method to see endogenous genomic loci using synthetic zinc‐finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag‐specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps: First, we develop an anti‐FLAG frankenbody that can bind FLAG‐tagged proteins in diverse live‐cell environments. The anti‐FLAG frankenbody complements the anti‐HA frankenbody, enabling two‐color signal amplification from FLAG‐ and HA‐tagged proteins. Second, we develop a pair of cell‐permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti‐FLAG and anti‐HA frankenbodies with FLAG‐ and HA‐tagged ZF probes, we simultaneously see the dynamics of the two loci in single living cells. This shows a close association between the two loci in the majority of cells, but the loci markedly separate from the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof of principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors. [ABSTRACT FROM AUTHOR]

There is growing evidence that 3D genome organization is a universal and significant mechanism of gene expression regulation. Tools to manipulate long‐range DNA interactions can advance the accurate control of chromosomal architecture. However, simple eukaryotic systems available to engineer chromosomal looping and gene expression are very limited. This study has developed a tool designated as chromosomal looping‐based expression activation system in yeast (CLEASY). Based on a modified yeast chromosome, it consists of conditionally interacting proteins, distal transcriptional regulatory elements, and a reporter gene. Exogenous chemical or light exposure induces the protein interaction, and results in the proximity of distal regulatory elements bound by these interacting proteins, and ultimately activates the reporter gene. In addition to this controllable induction, this system is compatible with the bivalent Cas9 complexes and their guide RNAs, to ensure target specificity and variability. Therefore, CLEASY can be utilized as a simplified eukaryotic model to engineer DNA looping machinery, and potentially serves as a fast platform to investigate looping mechanism and effective molecules. [ABSTRACT FROM AUTHOR]

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA -NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA -protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ('slow' trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ('fast' trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules. [ABSTRACT FROM AUTHOR]

Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. [ABSTRACT FROM AUTHOR]

ABSTRACT For much of the last three decades, Monte Carlo-simulation methods have been the standard approach for accurately calculating the cyclization probability, J, or J factor, for DNA models having sequence-dependent bends or inhomogeneous bending flexibility. Within the last 10 years approaches based on harmonic analysis of semi-flexible polymer models have been introduced, which offer much greater computational efficiency than Monte Carlo techniques. These methods consider the ensemble of molecular conformations in terms of harmonic fluctuations about a well-defined elastic-energy minimum. However, the harmonic approximation is only applicable for small systems, because the accessible conformation space of larger systems is increasingly dominated by anharmonic contributions. In the case of computed values of the J factor, deviations of the harmonic approximation from the exact value of J as a function of DNA length have not been characterized. Using a recent, numerically exact method that accounts for both anharmonic and harmonic contributions to J for wormlike chains of arbitrary size, we report here the apparent error that results from neglecting anharmonic behavior. For wormlike chains having contour lengths less than four times the persistence length, the error in J arising from the harmonic approximation is generally small, amounting to free energies less than the thermal energy, kBT. For larger systems, however, the deviations between harmonic and exact J values increase approximately linearly with size. © 2015 Wiley Periodicals, Inc. Biopolymers : 527-537, 2015. [ABSTRACT FROM AUTHOR]

ABSTRACT The 50th anniversary of Biopolymers coincides closely with the like celebration of the discovery of the Escherichia coli (lac) lactose operon, a classic genetic system long used to illustrate the influence of biomolecular structure on function. The looping of DNA induced by the binding of the Lac repressor protein to sequentially distant operator sites on DNA continues to serve as a paradigm for understanding long-range genomic communication. Advances in analyses of DNA structures and in incorporation of proteins in computer simulations of DNA looping allow us to address long-standing questions about the role of protein-mediated DNA loop formation in transcriptional control. Here we report insights gained from studies of the sequence-dependent contributions of the natural lac operators to Lac repressor-mediated DNA looping. Novel superposition of the ensembles of protein-bound operator structures derived from NMR measurements reveals variations in DNA folding missed in conventional structural alignments. The changes in folding affect the predicted ease with which the repressor induces loop formation and the ways that DNA closes between the protein headpieces. The peeling of the auxiliary operators away from the repressor enhances the formation of loops with the 92-bp wildtype spacing and hints of a structural reason behind their weak binding. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 1070-1081, 2013. [ABSTRACT FROM AUTHOR]

Snf2 related chromatin remodelling enzymes possess an ATPase subunit similar to that of the SF-II helicases which hydrolyzes ATP to track along DNA. Translocation and any resulting torque in the DNA could drive chromatin remodeling. To determine whether the ISWI protein can translocate and generate torque, tethered particle motion experiments and atomic force microscopy have been performed using recombinant ISWI expressed in E. coli. In the absence of ATP, ISWI bound to and wrapped DNA thereby shortening the overall contour length measured in atomic force micrographs. Although naked DNA only weakly stimulates ATP hydrolysis by ISWI, both atomic force microscopy and tethered particle motion data indicate that the protein generated loops in the presence of ATP. The duration of the looped state of the DNA measured using tethered particle motion was ATP-dependent. Finally, ISWI relaxed positively supercoiled plasmids visualized by atomic force microscopy. While other chromatin remodeling ATPases catalyze either DNA wrapping or looping, both are catalyzed by ISWI. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I–DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes. [ABSTRACT FROM AUTHOR]

Mismatch-repair (MMR) systems promote genomic stability by correction of DNA replication errors. Thus, MMR proteins-prokaryotic MutS and MutL homodimers or their MutSa and MutLa heterodimer homologs, plus accessory proteins-specifically couple mismatch recognition to nascent-DNA excision. In vivo excision-initiation signals-specific nicks in some prokaryotes, perhaps growing 3' ends or Okazaki-fragment 5' ends in eukaryotes-are efficiently mimicked in vitro by nicks or gaps in exogenous DNA substrates. In some models for recognition-excision coupling, MutSa bound to mismatches is induced by ATP hydrolysis, or simply by binding of ATP, to slide along DNA to excision-initiation sites, perhaps in association with MutLa and accessory proteins. In other models, MutSa·MutLa complexes remain fixed at mismatches and contact distant excision sites by DNA looping. To challenge the hypothesis that recognition complexes remain fixed, we placed biotin-streptavidin blockades between mismatches and pre-existing nicks. In human nuclear extracts, mismatch efficiently provoked the initiation of excision despite the intervening barriers, as predicted. However, excision progress and therefore mismatch correction were prevented. [ABSTRACT FROM AUTHOR]

In one family of bacterial plasmids, multiple initiator binding sites, called iterons, are used for initiation of plasmid replication as well as for the control of plasmid copy number. Iterons can also pair in vitro via the bound initiators. This pairing, called handcuffing, has been suggested to cause steric hindrance to initiation and thereby control the copy number. To test this hypothesis, we have compared copy numbers of isogenic miniP1 plasmid monomer and dimer. The dimer copy number was only one‐quarter that of the monomer, suggesting that the higher local concentration of origins in the dimer facilitated their pairing. Physical evidence consistent with iteron‐mediated pairing of origins preferentially in the dimer was obtained in vivo. Thus, origin handcuffing can be a mechanism to control P1 plasmid replication. [ABSTRACT FROM AUTHOR]

The probability that two sites on a linear DNA molecule will contact each other by looping depends on DNA flexibility. Although the flexibility of naked DNA in vitro is well characterized, looping in chromatin is poorly understood. By extending existing theory, we present a single equation that describes DNA looping over all distances. We also show that DNA looping in vitro can be measured accurately by FLP recombination between sites from 74 bp to 15 kb apart. In agreement with previous work, a persistence length of 50 nm was determined. FLP recombination of the same substrates in mammalian cells showed that chromatin increases the flexibility of DNA at short distances, giving an apparent persistence length of 27 nm. [ABSTRACT FROM AUTHOR]

The structure of aminopeptidase A (PepA), which functions as a DNA-binding protein in Xer site-specific recombination and in transcriptional control of the carAB operon in Escherichia coli, has been determined at 2.5 Å resolution. In Xer recombination at cer, PepA and the arginine repressor (ArgR) serve as accessory proteins, ensuring that recombination is exclusively intramolecular. In contrast, PepA homologues from other species have no known DNA-binding activity and are not implicated in transcriptional regulation or control of site-specific recombination. PepA comprises two domains, which have similar folds to the two domains of bovine lens leucine aminopeptidase (LAP). However, the N-terminal domain of PepA, which probably plays a significant role in DNA binding, is rotated by 19° compared with its position in LAP. PepA is a homohexamer of 32 symmetry. A groove that runs from one trimer face across the 2-fold molecular axis to the other trimer face is proposed to be the DNA-binding site. Molecular modelling supports a structure of the Xer complex in which PepA, ArgR and a second PepA molecule are sandwiched along their 3-fold molecular axes, and the accessory sequences of the two recombination sites wrap around the accessory proteins as a right-handed superhelix such that three negative super coils are trapped. [ABSTRACT FROM AUTHOR]

The sid1 and urbs1 genes encode L-ornithine N5-oxygenase and a GATA family transcription regulator, respectively, for siderophore biosynthesis in Ustilago maydis. The basic promoter and iron-regulatory sequences of the U.maydis sid1 gene were defined by fusing restriction and Bal31 nuclease-generated deletion fragments of the promoter region with the Escherichia coli β-glucuronidase (GUS) reporter gene. Sequences required for basal expression of sid1 mapped within 1043 bp upstream of the translation start site and include the first untranslated exon and first intron. Sequences needed for iron-regulated expression of sid1 were localized to a 306 bp region mapping 2.3 and 2.6 kb upstream of the ATG. The 306 bp region contains two G/TGATAA sequences, consensus DNA binding sites of GATA family transcription factors. Deletion or site-directed mutation of either or both GATA sequences resulted in deregulated expression of sid1. In vitro DNA binding studies showed that Urbs1 binds to the 3′-GATA site in the 306 bp iron-responsive region. However, deletion of 1.1 kb between the distal GATA sites and the basal promoter region led to deregulated expression of GUS, indicating that these GATA sequences are by themselves insufficient to regulate sid1. In vitro DNA binding and in vivo reporter gene analysis revealed that siderophores are not corepressors of Urbs1. [ABSTRACT FROM AUTHOR]

DNA looping, gene expression, photoreceptor, phytohormone, yeast Keywords: DNA looping; gene expression; photoreceptor; phytohormone; yeast EN DNA looping gene expression photoreceptor phytohormone yeast 1 1 1 03/18/21 20210315 NES 210315 3D genome organization is a significant mechanism of gene expression regulation, but simple eukaryotic toolkits to edit genome architecture are limited. In article number 2001135, Xinhao Ouyang, Xi Huang, and co-workers develop a chromosomal looping-based expression activation system in yeast (CLEASY), achieve its application compatible with CRISPR/Cas9 gene editing systems, and present a potential platform to explore DNA looping machinery and effective molecules. [Extracted from the article]