Monomeric DNA-binding Domain Research Articles

DNA binding and bridging by human CtIP in the healthy and diseased states.

The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed.

1H, 13C, and 15N resonance assignments of SarA monomer from Staphylococcus aureus in complex with DNA.

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarAΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1H, 13C and 15N NMR assignment of SarAΔN19/DNA complex which is the first step towards further structure and function analysis.

A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria.

Engineered bacteria promise to revolutionize diagnostics and therapeutics, yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-binding domains (split-DBDs). Using E. coli as a model system, we engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of our platform by using the DBDs of two different transcriptional regulators. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various hosts to detect ligands for which no receptor is found in nature.

Time-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS.

The virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NSctd), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The ‘fast’, ‘intermediate’ and ‘slow’ H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all ‘fast’ protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps.

A bistable genetic switch based on designable DNA-binding domains.

Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains. However, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability in mammalian cells. Here, the challenge of engineering a bistable switch based on monomeric DNA-binding domains is solved via the introduction of a positive feedback loop composed of activators based on the same TALE domains as their opposing repressors and competition for the same DNA operator site. This design introduces nonlinearity and results in epigenetic bistability. This principle could be used to employ other monomeric DNA-binding domains such as CRISPR for applications ranging from reprogramming cells to building digital biological memory.

Why Hofmeister effects of many salts favor protein folding but not DNA helix formation.

The majority ( approximately 70%) of surface buried in protein folding is hydrocarbon, whereas in DNA helix formation, the majority ( approximately 65%) of surface buried is relatively polar nitrogen and oxygen. Our previous quantification of salt exclusion from hydrocarbon (C) accessible surface area (ASA) and accumulation at amide nitrogen (N) and oxygen (O) ASA leads to a prediction of very different Hofmeister effects on processes that bury mostly polar (N, O) surface compared to the range of effects commonly observed for processes that bury mainly nonpolar (C) surface, e.g., micelle formation and protein folding. Here we quantify the effects of salts on folding of the monomeric DNA binding domain (DBD) of lac repressor (lac DBD) and on formation of an oligomeric DNA duplex. In accord with this prediction, no salt investigated has a stabilizing Hofmeister effect on DNA helix formation. Our ASA-based analyses of model compound data and estimates of the surface area buried in protein folding and DNA helix formation allow us to predict Hofmeister effects on these processes. We observe semiquantitative to quantitative agreement between these predictions and the experimental values, obtained from a novel separation of coulombic and Hofmeister effects. Possible explanations of deviations, including salt-dependent unfolded ensembles and interactions with other types of surface, are discussed.

Keap1/Nrf2 Signaling Regulates Oxidative Stress Tolerance and Lifespan in Drosophila

Keap1/Nrf2 signaling defends organisms against the detrimental effects of oxidative stress and has been suggested to abate its consequences, including aging-associated diseases like neurodegeneration, chronic inflammation, and cancer. Nrf2 is a prominent target for drug discovery, and Nrf2-activating agents are in clinical trials for cancer chemoprevention. However, aberrant activation of Nrf2 by keap1 somatic mutations may contribute to carcinogenesis and promote resistance to chemotherapy. To evaluate potential functions of Keap1 and Nrf2 for organismal homeostasis, we characterized the pathway in Drosophila. We demonstrate that Keap1/Nrf2 signaling in the fruit fly is activated by oxidants, induces antioxidant and detoxification responses, and confers increased tolerance to oxidative stress. Importantly, keap1 loss-of-function mutations extend the lifespan of Drosophila males, supporting a role for Nrf2 signaling in the regulation of longevity. Interestingly, cancer chemopreventive drugs potently stimulate Drosophila Nrf2 activity, suggesting the fruit fly as an experimental system to identify and characterize such agents.

DNA-induced conformational changes are the basis for cooperative dimerization by the DNA binding domain of the retinoid X receptor

Dimerization of the DNA-binding domains of nuclear hormone receptors occurs in a manner that is highly cooperative with DNA binding. We have investigated the molecular basis for this cooperativity through an NMR study of the interaction between the monomeric DNA-binding domain (DBD) of the retinoid-X-receptor (RXR) and a single DNA half-site. Major changes were observed in the chemical shifts of the backbone resonances and in the pattern of medium-range nuclear Overhauser enhancement connectivities of the RXR upon binding to DNA, indicating that the DNA induces conformational changes in the monomer. Binding to DNA induces and stabilizes the structure in a region of the second zinc binding domain that forms the dimerization interface when RXR binds as a dimer to a direct repeat recognition element. These studies provide direct experimental evidence that DNA-induced protein conformational changes constitute the molecular basis for cooperative enhancement of dimer formation and DNA binding by the nuclear hormone receptor DBDs. In contrast to the localized folding induced in the dimerization interface, DNA binding leads to unfolding of the C-terminal helix found in the free RXR DBD. Unwinding of this helix may facilitate homodimer formation by maximizing interactions between the two DNA-bound RXR domains.

Solution structure of the DNA-binding domain and model for the complex of multifunctional hexameric arginine repressor with DNA.

The structure of the monomeric DNA-binding domain of the Escherichia coli arginine repressor, ArgR, determined by NMR spectroscopy, shows structural homology to the winged helix-turn-helix (wHTH) family, a motif found in a diverse class of proteins including both gene regulators and gene organizers from prokaryotes and eukaryotes. Biochemical data on DNA binding by intact ArgR are used as constraints to position the domain on its DNA target and to derive a model for the hexamer-DNA complex using the known structure of the L-arginine-binding domain. The structural independence of the wHTH fold may be important for multimeric DNA-binding proteins that contact extended DNA regions with imperfect match to consensus sequences, a feature of many wHTH-domain proteins.

Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure.

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure.

Heat stress regulation of human heat shock genes is mediated by human heat shock transcription factor hHSF1, which contains three 4-3 hydrophobic repeats (LZ1 to LZ3). In unstressed human cells (37 degrees C), hHSF1 appears to be in an inactive, monomeric state that may be maintained through intramolecular interactions stabilized by transient interaction with hsp70. Heat stress (39 to 42 degrees C) disrupts these interactions, and hHSF1 homotrimerizes and acquires heat shock element DNA-binding ability. hHSF1 expressed in Xenopus oocytes also assumes a monomeric, non-DNA-binding state and is converted to a trimeric, DNA-binding form upon exposure of the oocytes to heat shock (35 to 37 degrees C in this organism). Because endogenous HSF DNA-binding activity is low and anti-hHSF1 antibody does not recognize Xenopus HSF, we employed this system for mapping regions in hHSF1 that are required for the maintenance of the monomeric state. The results of mutagenesis analyses strongly suggest that the inactive hHSF1 monomer is stabilized by hydrophobic interactions involving all three leucine zippers which may form a triple-stranded coiled coil. Trimerization may enable the DNA-binding function of hHSF1 by facilitating cooperative binding of monomeric DNA-binding domains to the heat shock element motif. This view is supported by observations that several different LexA DNA-binding domain-hHSF1 chimeras bind to a LexA-binding site in a heat-regulated fashion, that single amino acid replacements disrupting the integrity of hydrophobic repeats render these chimeras constitutively trimeric and DNA binding, and that LexA itself binds stably to DNA only as a dimer but not as a monomer in our assays.

Formation of a monomeric DNA binding domain by Skn-1 bZIP and homeodomain elements.

Maternally expressed Skn-1 protein is required for the correct specification of certain blastomere fates in early Caenorhabditis elegans embryos. Skn-1 contains a basic region similar to those of basic leucine zipper (bZIP) proteins but, paradoxically, it lacks a leucine zipper dimerization segment. Random sequence selection methods were used to show that Skn-1 binds to specific DNA sequences as a monomer. The Skn-1 basic region lies at the carboxyl terminus of an 85-amino acid domain that binds preferentially to a bZIP half-site and also recognizes adjacent 5' AT-rich sequences in the minor groove, apparently with an amino (NH2)-terminal "arm" related to those of homeodomain proteins. The intervening residues appear to stabilize interactions of these two subdomains with DNA. The Skn-1 DNA binding domain thus represents an alternative strategy for promoting binding of a basic region segment recognition helix to its cognate half-site. The results point to an underlying modularity in subdomains within established DNA binding domains.

Yeast heat shock transcription factor contains a flexible linker between the DNA-binding and trimerization domains. Implications for DNA binding by trimeric proteins.

All heat shock transcription factors (HSFs) share two regions of homology, identified as the DNA binding and trimerization regions. The DNA binding region consists of two parts, an 89-amino-acid minimal DNA-binding domain and an additional 21 amino acids which are not necessary for specific DNA binding of a monomeric DNA-binding domain. These 21 amino acids may act as a flexible linker between the DNA-binding and trimerization domains. Saccharomyces cerevisiae HSF has an additional 52 amino acids between the proposed flexible linker and the trimerization domain. Deletion of this unique region has no effect on the structural integrity or essential in vivo functions of HSF. To investigate the role of the 21-amino-acid proposed linker, a series of internal deletions was created in fragments containing the DNA-binding and trimerization domains. The deletions have no effect on the structural integrity of the protein as assayed by circular dichroism spectroscopy. However, alterations of the linker do affect affinity of trimeric HSF binding to its target DNA. In addition, deletion of part or all of the proposed linker from full-length yeast HSF, an essential protein, disrupts growth of yeast.