Cognate DNAs Research Articles

Conformational tuning of a DNA-bound transcription factor.

Transcription factors are involved in many cellular processes that take place remote from their cognate DNA sequences. The efficiencies of these activities are thus in principle counteracted by high binding affinities of the factors to their cognate DNAs. Models such as facilitated diffusion or dissociation address this apparent contradiction. We show that the MYC associated transcription factor X (MAX) undergoes nanoscale conformational fluctuations in the DNA-bound state, which is consistent with facilitated dissociation from or diffusion along DNA strands by transiently reducing binding energies. An integrative approach involving EPR, NMR, crystallographic and molecular dynamics analyses demonstrates that the N-terminal domain of MAX constantly opens and closes around a bound DNA ligand thereby dynamically tuning the binding epitope and the mode of interaction.

The modular mechanism of chromocenter formation in Drosophila.

A central principle underlying the ubiquity and abundance of pericentromeric satellite DNA repeats in eukaryotes has remained poorly understood. Previously we proposed that the interchromosomal clustering of satellite DNAs into nuclear structures known as chromocenters ensures encapsulation of all chromosomes into a single nucleus (Jagannathan et al., 2018). Chromocenter disruption led to micronuclei formation, resulting in cell death. Here we show that chromocenter formation is mediated by a 'modular' network, where associations between two sequence-specific satellite DNA-binding proteins, D1 and Prod, bound to their cognate satellite DNAs, bring the full complement of chromosomes into the chromocenter. D1 prod double mutants die during embryogenesis, exhibiting enhanced phenotypes associated with chromocenter disruption, revealing the universal importance of satellite DNAs and chromocenters. Taken together, we propose that associations between chromocenter modules, consisting of satellite DNA binding proteins and their cognate satellite DNA, package the Drosophila genome within a single nucleus.

Transcription Regulation and Membrane Stress Management in Enterobacterial Pathogens.

Transcription regulation in a temporal and conditional manner underpins the lifecycle of enterobacterial pathogens. Upon exposure to a wide array of environmental cues, these pathogens modulate their gene expression via the RNA polymerase and associated sigma factors. Different sigma factors, either involved in general 'house-keeping' or specific responses, guide the RNA polymerase to their cognate promoter DNAs. The major alternative sigma54 factor when activated helps pathogens manage stresses and proliferate in their ecological niches. In this chapter, we review the function and regulation of the sigma54-dependent Phage shock protein (Psp) system-a major stress response when Gram-negative pathogens encounter damages to their inner membranes. We discuss the recent development on mechanisms of gene regulation, signal transduction and stress mitigation in light of different biophysical and biochemical approaches.

The length of glycine-rich linker in DNA-binding domain is critical for optimal functioning of quorum-sensing master regulatory protein HapR.

HapR is a quorum-sensing master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of this wild-type protein. While unraveling the underlying cause of functional inertness of a natural variant (HapRV2), the significance of a conserved glycine residue at position 39 in a glycine-rich linker in DNA-binding domain comes into light. This work aims at investigating how the length of glycine-rich linker (R(33)GIGRGG(39)) bridging helices α1 and α2 modulates the functionality of HapR. In pursuit of our interest, glycine residues were inserted after terminal glycine (G39) of the linker in a sequential manner. To evaluate functionality, all the glycine linker variants were subjected to a battery of performance tests under various conditions. Combined in vitro and in vivo results clearly demonstrated a gradual functional impairment of HapR linker variants coupled with increasing length of glycine-rich linker and finally, linker variant harboring four glycine residues resulted in a functionally compromised protein with significant loss of communication with cognate DNAs. Molecular dynamics studies of modeled HapR linker variants in complex with cognate promoter region show that residues namely Ser50, Thr53 and Asn56 are involved in varying degree of interactions with different nucleotides of HapR-DNA complex. The diminished functionality between variants and DNA appears to result from reduced or no interactions between Phe55 and nucleotides of cognate DNA as observed during simulations.

Physical and Structural Basis for the Strong Interactions of the -ImPy- Central Pairing Motif in the Polyamide f-ImPyIm

The polyamide f-ImPyIm has a higher affinity for its cognate DNA than either the parent analogue, distamycin A (10-fold), or the structural isomer, f-PyImIm (250-fold), has for its respective cognate DNA sequence. These findings have led to the formulation of a two-letter polyamide "language" in which the -ImPy- central pairings associate more strongly with Watson-Crick DNA than -PyPy-, -PyIm-, and -ImIm-. Herein, we further characterize f-ImPyIm and f-PyImIm, and we report thermodynamic and structural differences between -ImPy- (f-ImPyIm) and -PyIm- (f-PyImIm) central pairings. DNase I footprinting studies confirmed that f-ImPyIm is a stronger binder than distamycin A and f-PyImIm and that f-ImPyIm preferentially binds CGCG over multiple competing sequences. The difference in the binding of f-ImPyIm and f-PyImIm to their cognate sequences was supported by the Na(+)-dependent nature of DNA melting studies, in which significantly higher Na(+) concentrations were needed to match the ability of f-ImPyIm to stabilize CGCG with that of f-PyImIm stabilizing CCGG. The selectivity of f-ImPyIm beyond the four-base CGCG recognition site was tested by circular dichroism and isothermal titration microcalorimetry, which shows that f-ImPyIm has marginal selectivity for (A.T)CGCG(A.T) over (G.C)CGCG(G.C). In addition, changes adjacent to this 6 bp binding site do not affect f-ImPyIm affinity. Calorimetric studies revealed that binding of f-ImPyIm, f-PyImIm, and distamycin A to their respective hairpin cognate sequences is exothermic; however, changes in enthalpy, entropy, and heat capacity (DeltaC(p)) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of DeltaC(p) for binding of f-ImPyIm to CGCG were in good agreement (-142 and -177 cal mol(-)(1) K(-)(1), respectively). (1)H NMR of f-ImPyIm and f-PyImIm complexed with their respective cognate DNAs confirmed positively cooperative formation of distinct 2:1 complexes. The NMR results also showed that these triamides bind in the DNA minor groove and that the oligonucleotide retains the B-form conformation. Using minimal distance restraints from the NMR experiments, molecular modeling and dynamics were used to illustrate the structural complementarity between f-ImPyIm and CGCG. Collectively, the NMR and ITC experiments show that formation of the 2:1 f-ImPyIm-CGCG complex achieves a structure more ordered and more thermodynamically favored than the structure of the 2:1 f-PyImIm-CCGG complex.

Forces Driving the Binding of Homeodomains to DNA

Homeodomains are helix-turn-helix type DNA-binding domains that exhibit sequence-specific DNA binding by insertion of their "recognition" alpha helices into the major groove and a short N-terminal arm into the adjacent minor groove without inducing substantial distortion of the DNA. The stability and DNA binding of four representatives of this family, MATalpha2, engrailed, Antennapedia, and NK-2, and truncated forms of the last two lacking their N-terminal arms have been studied by a combination of optical and microcalorimetric methods at different temperatures and salt concentrations. It was found that the stability of the free homeodomains in solution is rather low and, surprisingly, is reduced by the presence of the N-terminal arm for the Antennapedia and NK-2 domains. Their stabilities depend significantly upon the presence of salt: strongly for NaCl but less so for NaF, demonstrating specific interactions with chloride ions. The enthalpies of association of the homeodomains with their cognate DNAs are negative, at 20 degrees C varying only between -12 and -26 kJ/mol for the intact homeodomains, and the entropies of association are positive; i.e., DNA binding is both enthalpy- and entropy-driven. Analysis of the salt dependence of the association constants showed that the electrostatic component of the Gibbs energy of association resulting from the entropy of mixing of released ions dominates the binding, being about twice the magnitude of the nonelectrostatic component that results from dehydration of the protein/DNA interface, van der Waals interactions, and hydrogen bonding. A comparison of the effects of NaCl/KCl with NaF showed that homeodomain binding results in a release not only of cations from the DNA phosphates but also of chloride ions specifically associated with the proteins. The binding of the basic N-terminal arms in the minor groove is entirely enthalpic with a negative heat capacity effect, i.e., is due to sequence-specific formation of hydrogen bonds and hydrophobic interactions rather than electrostatic contacts with the DNA phosphates.

A Functional Histidine-Tagged Replication Initiator Protein: Implications for the Study of Single-Stranded DNA Virus Replication In Planta

Replication initiation of nanoviruses, plant viruses with a multipartite circular single-stranded DNA genome, is triggered by the master Rep (M-Rep) protein. To enable the study of interactions between M-Rep and viral or host factors involved in replication, we designed oligohistidine-tagged variants of the nanovirus Faba bean necrotic yellows virus (FBNYV) M-Rep protein that allow affinity purification of enzymatically active M-Rep from plant tissue. The tagged M-Rep protein was able to initiate replication of its cognate and other FBNYV DNAs in Nicotiana benthamiana leaf disks and plants. The replicon encoding the tagged M-Rep protein multiplied and moved systemically in FBNYV-infected Vicia faba plants and was transmitted by the aphid vector of the virus. Using the tagged M-Rep protein, we demonstrated the in planta interaction between wild-type M-Rep and its tagged counterpart. Such a tagged and fully functional replication initiator protein will have bearings on the isolation of protein complexes from plants.

The Role of the C-terminal Extension (CTE) of the Estrogen Receptor α and β DNA Binding Domain in DNA Binding and Interaction with HMGB

HMGB-1/-2 are coregulatory proteins that facilitate the DNA binding and transcriptional activity of steroid receptor members of the nuclear receptor family of transcription factors. We investigated the influence and mechanism of action of HMGB-1/-2 (formerly known as HMG-1/-2) on estrogen receptor alpha (ERalpha) and ERbeta. Both ER subtypes were responsive to HMGB-1/-2 with respect to enhancement of receptor DNA binding affinity and transcriptional activity in cells. Responsiveness to HMGB-1/-2 was dependent on the C-terminal extension (CTE) region of the ER DNA binding domain (DBD) and correlated with a direct protein interaction between HMGB-1/-2 and the CTE. Thus the previously reported higher DNA binding affinity and transcription activity of ERalpha as compared with ERbeta is not due to a lack of ERbeta interaction with HMGB-1/-2. Using chimeric receptor DBDs, the higher intrinsic DNA binding affinity of ERalpha than ERbeta was shown to be due to a unique property of the ERalpha CTE, independent of HMGB-1/-2. The CTE of both ER subtypes was also shown to be required for interaction with ERE half-sites. These studies reveal the importance of the CTE and HMGB-1/-2 for ERalpha and ERbeta interaction with their cognate target DNAs.

Specificity of DNA triple helix formation analyzed by a FRET assay

BackgroundA third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis.ResultsWe have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET). Under our conditions, the TFOs bind to their cognate double strand DNAs with binding constants of 2.6 × 105 and 2.3 × 106 M-1. Our data confirm that the polypurine TFO binds in an antiparallel orientation with respect to the polypurine DNA strand and that triplex formation requires Mg2+ions whereas it is inhibited by K+ions. The rate of formation of triple helices is slow with bimolecular rate constants of 5.6 × 104 and 8.1 × 104 min-1 M-1. Triplex dissociation was not detectable over at least 30 hours. Triplex formation is sequence specific; alteration of a single base pair within the 13 base pairs long TFOs prevents detectable triplex formation.ConclusionWe have applied a FRET assay to investigate the specificity of DNA triple helix formation. This assay is homogeneous, continuous and specific, because the appearance of the FRET signal is directly correlated to triplex formation. We show that polypurine TFOs bind highly specifically to polypurine stretches in double stranded DNA. This is a prerequisite for biotechnical applications of triple helices to mediate sequence specific recognition of DNA.

Target-Sequence Recognition by Separate-type Cys2/His2 Zinc Finger Proteins in Plants

The EPF family is a group of transcription factors containing canonical Cys2/His2-type zinc finger motifs that were first discovered in plants. These zinc finger proteins are characterized by two zinc fingers that are separated by spacers of various lengths, which are much longer than typical spacers (HC-link) in cluster-type zinc finger proteins. We describe here direct evidence that the two zinc fingers make contact with two tandemly repeated AGT core sequences that are separated by about 13 base pairs, by contrast to the cluster-type zinc finger proteins that bind to contiguous triplet sequences. DNA binding affinities were sensitive to the spaces between the core sequences, and the sensitivity to the spacing was greatly affected by the DNA sequence between the core sequences, with GC-rich sequences endowing much higher specificity than AT-rich sequences. Among the members of the EPF family, EPF1 was less sensitive to the spacing than EPF2-5. These results suggest that EPFs recognize their cognate target DNAs not only by the sequence of the core sites but also by the spacing between the core sites and, moreover, that different members in the EPF family distinguish their specific target genes by reference to these two parameters. This represents a unique type of target-sequence recognition among Cys2/His2-type zinc finger transcription factors. In addition, site-directed mutagenesis studies demonstrated that the two zinc fingers contribute synergistically to the binding to DNA, indicating that both fingers are necessary for the high affinity DNA binding.

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs

Specific and non-specific interactions SsoII restriction endonuclease (R· SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R· SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R· SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg 2+ ions.

Patterns of allele losses suggest the existence of five distinct regions of LOH on chromosome 17 in breast cancer.

Chromosome 17 is a frequent target during breast-cancer formation and progression. It has been shown to be affected by allele losses at multiple sites, as well as by DNA amplification. Our aim was to delineate a map of the genetic alterations on chromosome 17 in a given set of breast tumors. To this end we analyzed 151 pairs of tumor and cognate lymphocyte DNAs by Southern blotting with 5 RFLP or VNTR probes and by PCR at 8 CA repeat polymorphic loci for LOHs. Moreover, we studied DNA amplification of the evi2, erbB2, thraI, gcsf and rara genes. Data presented here point strongly to the existence of 5 distinct regions of allele losses on chromosome 17:2 on 17p, 3 on 17q. Of the 2 regions on 17p, one involves tp53 while the second is located more distally toward the telomere. LOH was found in 45.9% and 58.8% respectively. The 3 regions on 17q are located: (i) on the proximal portion of the long arm band q21, corresponding to the brcaI region; (ii) in a central region defined by the marker D17S74; (iii) on the distal part of 17q (band q25) characterized by losses of the marker D17S24. Each of these regions presented respectively allele losses in 47.5%, 33.3% and 40.8% of the informative tumors. Whereas some tumors presented patterns of LOH consistent with the loss of a complete chromosomal arm or of large portions of the chromosome, a high proportion of the analyzed tumors showed interstitial losses. Amplifications were found in 15% of the tumors and were centered around erbB2.(ABSTRACT TRUNCATED AT 250 WORDS)

Six amino acids determine the sequence-specific DNA binding and replication specificity of the initiator proteins of the pT181 family.

The replication of pT181 and related plasmids of Staphylococcus aureus proceeds by a rolling circle mechanisms. The initiator proteins encoded by the plasmids of the pT181 family have sequence-specific DNA binding and topoisomerase activities. These proteins nick one strand of the DNA at the origin of replication. The free 3'-hydroxyl end at the nick is then used as a primer for the replication of the leading strand of the DNA. Although these initiator proteins are highly homologous, they show specificity in DNA binding and replication for their cognate DNAs. In this study, we have generated hybrid initiator proteins and studied their various biochemical activities in vitro. Our results show that 6 amino acids are sufficient to determine the DNA binding and replication specificities of such initiator proteins.

Further analyses of the orthopoxviruses volepox virus and raccoon poxvirus

Volepox virus (VPX) from skin lesions on a vole and a piñon mouse caught in California and raccoon poxvirus (RCN) from raccoons trapped in Maryland were examined to begin elucidating their relationship to other orthopoxviruses, most of which are not known to be indigenous to the Americas. VPX and RCN produced pinpoint, nonhemorrhagic pocks on chick embryo chorioallantoic membranes. In cell cultures both viruses produced 1-mm diameter, irregular plaques, A-type inclusions (ATIs), and despite production of hemagglutinin, both viruses caused syncytia formation. Considerable cross-hybridization was seen between VPX and RCN DNA and the DNAs of other orthopoxviruses; however, HindIII cleavage site maps showed marked central and terminal region differences between VPX (222.8 kbp) and RCN (224.8 kbp) DNA and mapped DNAs of other orthopoxviruses. Cognate DNAs of the ATI 160-kDa protein and 38-kDa serine protease inhibitor homologue of cowpox virus (CPV) and the 14-kDa fusion protein of vaccinia virus (VAC) were present within the right end of VPX and RCN DNA, matching their location in CPV and VAC. VPX and RCN, respectively, expressed a 150- and a 155-kDa ATI major protein and a 20- and an 18-kDa fusion protein. Low stringency annealing suggested that cognate DNAs for the VAC growth factor and the α-amanitin target protein were present within the left end of VPX and RCN DNA, matching their location in VAC. Terminal tandem repeat sequences of VAC and RCN did not cross-hybridize with each other or with VPX DNA end fragments. Together, the data suggested that VPX and RCN are phylogenetically rather distant from orthopoxviruses not indigenous to the Americas, although genetic information is arranged as in other examined orthopoxviruses.

Evidence for a chromosomal location of polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus.

Evidence is presented in support of a chromosomal location for sequences homologous to polydnavirus DNA in the ichneumonid parasitoid Hyposoter fugitivus. In this study, four different viral genome segments were cloned and used as probes against genomic DNA extracted from male parasitoids and digested with a variety of restriction enzymes. Each probe typically identified a single off-size fragment (OSF) in the case of enzymes not cutting viral genome segments, while two OSFs were generated by enzymes cutting at one and two sites. While extra OSFs were occasionally observed, these were invariably found to be due to the presence of polymorphic restriction sites in flanking chromosomal DNA. Analysis of these data suggests that a single, stable chromosomal locus exists for sequences homologous to each viral genome segment; the data also indicate that viral and cognate parasitoid genomic DNAs are largely if not entirely colinear.

Osmoregulatory expression of the porin genes in Escherichia coli: evidence for signal titration in the signal transduction through EnvZ-OmpR phosphotransfer.

The OmpR protein of Escherichia coli is a positive regulator specific for the ompF and ompC genes. The function of OmpR is modulated through phosphotransfer signaling mediated by the kinase, EnvZ. We previously demonstrated that OmpR contains two functional domains, which are physically separable; one is responsible for the interaction with EnvZ, whereas the other participates in interactions with cognate promoter DNAs. In this study, these domains of OmpR were overproduced in wild-type cells harboring the endogenous intact ompR gene on their chromosome. It was found that when the N-terminal domain of OmpR, which contains the phosphorylation site, was overproduced, expression of the ompF and ompC genes was markedly inhibited, irrespective of the osmolarity of the growth medium. Based on our current model for the molecular mechanism underlying signal transduction through Envz-OmpR phosphotransfer (T. Mizuno and S. Mizushima, Mol. Microbiol. 4, (1990), 1077-1082), we provide evidence that this phenomenon is best interpreted by the concept of 'signal titration' in the phosphotransfer signaling pathway.

Osmoregulatory expression of the porin genes inEscherichia coli: evidence for signal titration in the signal transduction through EnvZ-OmpR phosphotransfer

The OmpR protein of Escherichia coli is a positive regulator specific for the ompF and ompC genes. The function of OmpR is modulated through phosphotransfer signaling mediated by the kinase, EnvZ. We previously demonstrated that OmpR contains two functional domains, which are physically separable; one is responsible for the interaction with EnvZ, whereas the other participates in interactions with cognate promoter DNAs. In this study, these domains of OmpR were overproduced in wild-type cells harboring the endogenous intact ompR gene on their chromosome. It was found that when the N-terminal domain of OmpR, which contains the phosphorylation site, was overproduced, expression of the ompF and ompC genes was markedly inhibited, irrespective of the osmolarity of the growth medium. Based on our current model for the molecular mechanism underlying signal transduction through Envz-OmpR phosphotransger (T. Mizuno and S. Mizushima, Mol. Microbiol. 4, (1990), 1077–1082), we provide evidence that this phenomenon is best interpreted by the concept of ‘signal titration’ in the phosphotransfer signaling pathway.

Signal transduction and osmoregulation in Escherichia coli. A novel type of mutation in the phosphorylation domain of the activator protein, OmpR, results in a defect in its phosphorylation-dependent DNA binding

The transcriptional factors, OmpR and EnvZ, are crucially involved in the osmotic regulation of ompF and ompC expression in Escherichia coli. The DNA binding ability of the positive regulator, OmpR, is modulated through its phosphorylation and dephosphorylation mediated by EnvZ in response to the medium osmolarity. In this study, two examples of a novel type of mutant ompR allele, ompR96A and ompR115S, whose phenotype is OmpF- OmpC- irrespective of the medium osmolarity, were characterized. These mutations result in amino acid conversions, Glu96 to Ala and Arg115 to Ser, respectively, within the phosphorylation domain of OmpR. Nevertheless, these mutant proteins were capable of undergoing phosphorylation and dephosphorylation normally, just like wild-type OmpR. However, the phosphorylation-dependent enhancement of their in vitro DNA binding ability was found to be severely affected. It was thus revealed that these mutant OmpR represent a novel type in terms of the mechanism of phosphorylation-dependent activation of the function of OmpR, i.e. those are normally phosphorylated but not activated to bind to the cognate promoter DNAs. In this respect, it was further suggested that OmpR oligomerization may be involved in the mechanism underlying the phosphorylation-dependent enhancement of the DNA binding ability of OmpR. The mutant proteins characterized in this study seem to be defective in this particular oligomerization process observed in vitro.