Altered DNA Conformation Research Articles

PIF4 enhances DNA binding of CDF2 to co-regulate target gene expression and promote Arabidopsis hypocotyl cell elongation

How specificity is conferred within gene regulatory networks is an important problem in biology. The basic helix–loop–helix PHYTOCHROME-INTERACTING FACTORs (PIFs) and single zinc-finger CYCLING DOF FACTORs (CDFs) mediate growth responses of Arabidopsis to light and temperature. We show that these two classes of transcription factor (TF) act cooperatively. CDF2 and PIF4 are temporally and spatially co-expressed, they interact to form a protein complex and act in the same genetic pathway to promote hypocotyl cell elongation. Furthermore, PIF4 substantially strengthens genome-wide occupancy of CDF2 at a subset of its target genes. One of these, YUCCA8, encodes an auxin biosynthesis enzyme whose transcription is increased by PIF4 and CDF2 to contribute to hypocotyl elongation. The binding sites of PIF4 and CDF2 in YUCCA8 are closely spaced, and in vitro PIF4 enhances binding of CDF2. We propose that this occurs by direct protein interaction and because PIF4 binding alters DNA conformation. Thus, we define mechanisms by which PIF and CDF TFs cooperate to achieve regulatory specificity and promote cell elongation in response to light.

A non-canonical promoter element drives spurious transcription of horizontally acquired bacterial genes.

RNA polymerases initiate transcription at DNA sequences called promoters. In bacteria, the best conserved promoter feature is the AT-rich -10 element; a sequence essential for DNA unwinding. Further elements, and gene regulatory proteins, are needed to recruit RNA polymerase to the -10 sequence. Hence, -10 elements cannot function in isolation. Many horizontally acquired genes also have a high AT-content. Consequently, sequences that resemble the -10 element occur frequently. As a result, foreign genes are predisposed to spurious transcription. However, it is not clear how RNA polymerase initially recognizes such sequences. Here, we identify a non-canonical promoter element that plays a key role. The sequence, itself a short AT-tract, resides 5 base pairs upstream of otherwise cryptic -10 elements. The AT-tract alters DNA conformation and enhances contacts between the DNA backbone and RNA polymerase.

Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme—The spore photoproduct lyase

DNA repair enzymes typically recognize their substrate lesions with high affinity to ensure efficient lesion repair. In UV irradiated endospores, a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, termed the spore photoproduct (SP), is the dominant DNA photolesion, which is rapidly repaired during spore outgrowth mainly by spore photoproduct lyase (SPL) using an unprecedented protein-harbored radical transfer process. Surprisingly, our in vitro studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01–0.2min−1. The faster turnover numbers are reached under single turnover conditions, and SPL activity is low with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3–0.4min−1, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from other specialized DNA repair enzymes. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins, helicases or an altered DNA conformation may cooperate with SPL to enable efficient SP repair in germinating spores. Therefore, both SP formation and SP repair are likely to be tightly controlled by the unique cellular environment in dormant and outgrowing spore-forming bacteria, and thus SP repair may be extremely slow in non-spore-forming organisms.

Sensing of DNA conformation based on change in FRET efficiency between laser dyes

This communication reports the effect of DNA conformation on fluorescence resonance energy transfer (FRET) efficiency between two laser dyes in layer by layer (LbL) self assembled film. The dyes Acraflavine and Rhodamine B were attached onto the negative phosphate backbones of DNA in LbL film through electrostatic attraction. Then FRET between these dyes was investigated. Increase in pH or temperature causes the denaturation of DNA followed by coil formation of single stranded DNA. As a result the FRET efficiency also changed along with it. These observations demonstrated that by observing the change in FRET efficiency between two laser dyes in presence of DNA it is possible to detect the altered DNA conformation in the changed environment.

Locating the binding sites of antitumor drug tamoxifen and its metabolites with DNA

We located the binding sites of antitumor drugs tamoxifen, 4-hydroxytamoxifen and endoxifen with calf-thymus DNA. FTIR, CD, UV–vis and fluorescence spectroscopic methods as well as molecular modeling were used to characterize the drug binding sites, binding constant and the effect of drug binding on DNA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bind DNA via hydrophobic and hydrophilic interactions with overall binding constants of Ktam-DNA=3.5 (±0.2)×104M−1, K4-hydroxytam-DNA=3.3 (±0.4)×104M−1 and Kendox-DNA=2.8 (±0.8)×104M−1. The number of binding sites occupied by drug is 1 (tamoxifen), 0.8 (4-hydroxitamoxifen) and 1.2 (endoxifen). Docking showed the participation of several nucleobases in drug–DNA complexes with the free binding energy of −3.85 (tamoxifen), −4.18 (4-hydroxtamoxifen) and −3.74kcal/mol (endoxifen). The order of binding is 4-hydroxy-tamoxen>tamoxifen>endoxifen. Drug binding did not alter DNA conformation from B-family structure, while major biopolymer aggregation occurred at high drug concentrations. The drug binding mode is correlated with the mechanism of action of antitumor activity of tamoxifen and its metabolites.

CD and DSC Investigation of Individual and Complex Influence of Meso-Tetra(4-Oxiethylpyridil) Porphyrin (TOEPyP4) and Its Zn-Complex on DNA

CD spectra of (DNA-TOEPyP4) + ZnTOEPyP4, (DNA-ZnTOEPyP4) + TOEPyP4, and DNA + (TOEPyP4-ZnTO- EPyP4) complexes have been studied. It is shown that CD spectra of these triple complexes significantly differ from the DNA-TOEPyP4 and DNA-ZnTOEPyP4 double complex spectra, and they are not sum of these double complexes. Especially some strong differences in CD spectra of the triple and double complexes were observed when both porphyrins were added simultaneously into the DNA solution. In this case, ZnTOEPyP4 revealed a dominant influence on CD spectrum form. Zn-porphyrin also caused a strong intensity of positive band at 416 nm and a negative band at 437 nm when it was added into solution containing the DNA-TOEPyP4 complex. On the basis of obtained data, it was supposed that the observed significant changes in CD spectra of triple complexes were connected to an altered DNA conformation initiated by intercalation of porphyrin TOEPyP4 into GC-rich sites. The melting process analysis of the double complexes was carried out. The mechanisms of individual and joint influence of the porphyrins on DNA, and influence of binding modes on stability of these complexes are also discussed.

The Load-Bearing Mechanosome Revisited

We introduced the mechanosome hypothesis in 2003 as a heuristic model for investigating mechanotransduction in bone (Pavalko et al., J Cell Biochem, 2003, 88(1):104-112). This model suggested specific approaches for investigating how mechanical information is conveyed from the membrane of the sensor bone cell to the target genes and how this transmitted information from the membrane is converted into changes in transcription. The key concepts underlying the mechanosome hypothesis are that load-induced deformation of bone deforms the sensor cell membrane; embedded in the membrane are the focal adhesion and cadherin-catenin complexes, which in turn are physically connected to the chromatin via a solid-state scaffold. The physical stimulation of the membrane launches multiprotein complexes (mechanosomes) from the adhesion platforms while concomitantly tugging target genes into position for contact with the incoming mechanosomes, the carriers of the mechanical information to the nucleus. The mechanosome is comprised of an adhesion-associated protein and a nucleocytoplasmic shuttling transcription factor. Upon arrival at the target gene, mechanosomes alter DNA conformation and thus influence the interactions between trans-acting proteins along the gene, changing gene activity. Here, we update significant progress related to the mechanosome concept since publication of our original hypothesis. The launching of adhesion- and cytoskeletal-associated proteins into the nucleus toward target genes appears to be a common mechanism for regulating cell response to changes in its mechanical microenvironment.

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDDeltaN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (DeltaL(k) = -4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDDeltaN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.

Huntingtin Modulates Transcription, Occupies Gene PromotersIn Vivo, and Binds Directly to DNA in a Polyglutamine-Dependent Manner

Transcriptional dysregulation is a central pathogenic mechanism in Huntington's disease, a fatal neurodegenerative disorder associated with polyglutamine (polyQ) expansion in the huntingtin (Htt) protein. In this study, we show that mutant Htt alters the normal expression of specific mRNA species at least partly by disrupting the binding activities of many transcription factors which govern the expression of the dysregulated mRNA species. Chromatin immunoprecipitation (ChIP) demonstrates Htt occupation of gene promoters in vivo in a polyQ-dependent manner, and furthermore, ChIP-on-chip and ChIP subcloning reveal that wild-type and mutant Htt exhibit differential genomic distributions. Exon 1 Htt binds DNA directly in the absence of other proteins and alters DNA conformation. PolyQ expansion increases Htt-DNA interactions, with binding to recognition elements of transcription factors whose function is altered in HD. Together, these findings suggest mutant Htt modulates gene expression through abnormal interactions with genomic DNA, altering DNA conformation and transcription factor binding.

P1 nuclease cleavage is dependent on length of the mismatches in DNA

P1 nuclease is one of the most extensively used single-strand DNA specific nucleases in molecular biology. In modern biology, it is used as an enzymatic probe to detect altered DNA conformations. It is well documented that P1 cleaves single-stranded nucleic acids and single-stranded DNA regions. The fact that P1 can act under a wide range of conditions, including physiological pH and temperature make it the most commonly used enzymatic probe in DNA structural studies. Surprisingly, to this date, there is no study to characterize the influence of length of mismatches on P1 sensitivity. Using a series of radioactively labeled oligomeric DNA substrates-containing mismatches, we find that P1 nuclease cleavage is dependent on the length of mismatches. P1 does not cleave DNA when there is a single-base mismatch. P1 cleavage efficiency is optimum when mismatch length is 3nt or more. Changing the position of the mismatches also does not make any difference in cleavage efficacy. These novel findings on P1 properties have implications for its use in DNA structure and DNA repair studies.

Acenaphtho[1,2- b]pyrrole derivatives as new family of intercalators: Various DNA binding geometry and interesting antitumor capacity

A series of acenaphtho[1,2- b]pyrrole derivatives were synthesized and their intercalation geometries with DNA and antitumor activities were investigated in detail. From combination of SYBR Green–DNA melt curve, fluorescence titration, absorption titration, and circular dichroism (CD) studies, it was identified that to different extent, all the compounds behaved as DNA intercalators and transformed B form DNA to A-like conformation. The different intercalation modes for the compounds were revealed. The compounds containing a methylpiperazine substitution (series I) intercalated in a fashion that the long axis of the molecule paralleled to the base-pair long axis, while the alkylamine- substituted compounds (series II and III) located vertically to the long axis of DNA base pairs. Consequently, the DNA binding affinity of these compounds was obtained with the order of II > III > I, which attributed to the role of the substitution in binding geometry. Further, cell-based studies showed all the compounds exhibited outstanding antitumor activities against two human tumor cell lines with IC 50 ranging from 10 −7 to 10 −6 M. Interestingly, compound 1 a (a compound in series I), whose binding affinity was one of the lowest but altered DNA conformation most significantly, showed much lower IC 50 value than other compounds. Moreover, it could induce tumor cells apoptosis, while the compounds 2 a and 3 a (in series II and III, respectively) could only necrotize tumor cells. Their different mechanism of killing tumor cells might lie in their different DNA binding geometry. It could be concluded that the geometry of intercalator–DNA complex contributed much more to the antitumor property than binding affinity.

Topo IIβ Gets to Work

DNA topoisomerases regulate conformational changes in DNA topology by catalyzing DNA strand breakage and rejoining. Topoisomerase IIβ (Topo IIβ) alters DNA conformation near gene promoters and associates with sequence-specific transcription factors and chromatin modifying and remodeling factors. Ju et al. now show that DNA Topo IIβ generates double-strand breaks at transcriptional promoters when these genes are activated in a signal-dependent manner. Subsequently, DNA repair enzymes are activated and there is a resultant exchange in histone composition and local chromatin structure. B.-G. Ju, V. V. Lunyak, V. Perissi, I. Garcia-Bassets, D. W. Rose, C. K. Glass, M. G. Rosenfeld, A topoisomerase IIβ-mediated dsDNA break required for regulated transcription. Science 312 , 1798-1802 (2006). [Abstract] [Full Text] J.-F. Haince, M. Rouleau, G. G. Poirier, Gene expression needs a break to unwind before carrying on. Science 312 , 1752-1753 (2006). [Summary] [Full Text]

317. DNA Damage and Homologous Recombination Regulate the Frequency and Process of Targeted Gene Repair

The pathway followed by targeted gene repair is not fully elucidated. In its simplest iteration, the process is viewed as being initiated by the introduction of short (25–75 bases) synthetic single-stranded oligonucleotides into a mammalian cell. The oligonucleotide aligns in homologous register with the target gene using the underlying sequence complementarity as a guide. Once positioned properly, a single mismatched base pair is created between the oligonucleotide and the target site. The structural changes and altered DNA conformation that clearly take place during the pairing phase attract proteins involved in DNA repair pathway. Here we demonstrate the involvement of the homologous recombination (HR) process in gene repair. By using a phenotypic readout assay system, where an eGFP gene containing a single point mutation is integrated into DLD-1 cells, we observe between 0.7 and 1.5% correction of the mutation. Gene repair is assessed by FACS analyses, cell sorting and confirmatory DNA sequencing of the target gene. When the cells are arrested in S phase as a result of DNA damage incurred by the presence of hydroxyurea or thymidine, the level of gene repair increases 3 to 4 fold. Addition of the anticancer drug VP16 (etopside) or Camptothecin (CPT) induces a similar response for both episomal or chromosomal targets. Synchronized cells are also more amenable to the gene repair reaction once released into S phase and a synergistic effect is seen when cell synchronization is coupled to the addition of HU or thymidine or CPT. These results suggest that DNA damage at the replication fork stimulates the homologous recombination pathway to act upon strand lesions (double strand breaks, etc.), enabling a more efficient gene repair reaction to take place. Agents or enzymes that induce the double strand breaks repair response are also quite effective in regulating and elevating the frequency of gene repair. Taken together, we propose that the factors and/or protein activated by recombination induced by replication inhibition (RIRI), particularly those involved in HR, are capable of enhancing the frequency of gene repair in mammalian cells.

First evidence to show the topological change of DNA from B-dNA to Z-DNA conformation in the hippocampus of Alzheimer's brain.

Alzheimer's disease (AD) is a complex neurodegenerative disorder. Our studies for the first time showed evidence for altered DNA conformation in the hippocampus of Alzheimer's disease affected brain. The Circular dichroism spectra of severely affected AD DNA showed a typical left-handed Z-DNA conformation, whereas normal, young, and aged brain DNA have the usual B-DNA conformation. Moderately affected AD DNA has modified B-DNA conformation (probable B-Z intermediate form). The ELISA, ethidium bromide binding pattern to DNA and melting temperature (Tm) profiles also revealed the conformational transition from B to Z DNA in AD brain DNA. The altered conformation of DNA will have tremendous implications in gene expressions.

DNA as a Possible Target for Antitumor Ruthenium(III) Complexes: A Spectroscopic and Molecular Biology Study of the Interactions of Two Representative Antineoplastic Ruthenium(III) Complexes with DNA

The interaction of two experimental ruthenium(III)-containing antitumor complexes—Na[trans-RuCl4(DMSO)(Im)] (NAMI) and dichloro(1,2-propylendiaminetetraacetate)ruthenium(III) (RAP)—with DNA was investigated through a number of spectroscopic and molecular biology techniques, including spectrophotometry, circular dichroism, gel shift analysis, and restriction enzyme inhibition. It was found that both complexes slightly alter DNA conformation, modify its electrophoretic mobility, and inhibit DNA recognition and cleavage by some restriction enzymes, though they were less effective than cisplatin in producing such effects. Notably, the effects produced by NAMI on DNA were much larger than those induced by RAP. Implications of these results for the mechanism of action of ruthenium(III) antitumor complexes are discussed.

DNA Bending and Twisting Properties of Integration Host Factor Determined by DNA Cyclization

The binding of many proteins to DNA is profoundly affected by DNA bending, twisting, and supercoiling. When protein binding alters DNA conformation, interaction between inherent and induced DNA conformation can affect protein binding affinity and specificity. Integration host factor (IHF), a sequence-specific, DNA-binding protein of Escherichia coli, strongly bends the DNA upon binding. To assess the influence of inherent DNA bending on IHF binding, we took advantage of the high degree of natural static curvature associated with an IHF site on a 163-bp minicircle and measured the binding affinity of IHF for its recognition site contained on this DNA in both circular and linear form. IHF showed a higher affinity for the circular form of the DNA when compared to the linear form. In addition, the presence of IHF during DNA cyclization changed the topology of cyclization products and their ability to bind IHF, consistent with IHF untwisting DNA. These results show that inherent DNA conformation anisotropy is an important determinant of IHF binding affinity and suggests a mechanism for modulation of IHF activity by local DNA conformation.

Binding of HMG-I(Y) elicits structural changes in a silencer of the human beta-globin gene.

Proteins involved in repression of the human beta-globin gene may be useful in the treatment of sickle cell anemia, in conjunction with therapy to reactivate fetal globin genes. If there is a reciprocal elevation of gamma-globin expression upon repression, this approach could be useful in additional hemoglobinopathies. We previously showed that repression of the beta-globin gene appears to be mediated through two DNA sequences, silencers I and II, and identified a protein termed BP1 which binds to both silencer sequences. In this study, we cloned two cDNAs encoding proteins which bind to an oligonucleotide in silencer I containing a BP1 binding site. These cDNAs correspond to HMG-I and HMG-Y, isoforms regarded as architectural proteins. We demonstrate that binding of HMG-I(Y) to this oligonucleotide causes bending/flexure of the DNA. HMG-I(Y) also binds to a second oligonucleotide containing a BP1 binding site located in a negative control region upstream of the delta-globin gene, suggesting a role for HMG-I(Y) in repression of adult globin genes. Expression studies revealed that HMG-I(Y) is ubiquitously expressed in human tissues that do not express beta-globin, being present in 48 of 50 tissues and six hematopoietic cell lines examined. Furthermore, HMG-I(Y) expression is down-regulated during differentiation of primary erythroid cells. We present a model in which HMG-I(Y) alters DNA conformation to allow binding of repressor proteins, and in which the relative amount of HMG-I(Y) helps to determine the repressive state of the beta-globin gene.

Minor groove-binding architectural proteins: structure, function, and DNA recognition.

To date, high-resolution structures have been solved for five different architectural proteins complexed to their DNA target sites. These include TATA-box-binding protein, integration host factor (IHF), high mobility group I(Y)[HMG I(Y)], and the HMG-box-containing proteins SRY and LEF-1. Each of these proteins interacts with DNA exclusively through minor groove contacts and alters DNA conformation. This paper reviews the structural features of these complexes and the roles they play in facilitating assembly of higher-order protein-DNA complexes and discusses elements that contribute to sequence-specific recognition and conformational changes.

In vitro DNA binding of the archaeal protein Sso7d induces negative supercoiling at temperatures typical for thermophilic growth.

The topological state of DNA in hyperthermophilic archaea appears to correspond to a linking excess in comparison with DNA in mesophilic organisms. Since DNA binding proteins often contribute to the control of DNA topology by affecting DNA geometry in the presence of DNA topoisomerases, we tested whether the histone-like protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus alters DNA conformation. In ligase-mediated supercoiling assays carried out at 37, 60, 70, 80 and 90 degrees C we found that DNA binding of increasing amounts of Sso7d led to a progressive decrease in plasmid linking number (Lk), producing negative supercoiling. Identical unwinding effects were observed when recombinant non-methylated Sso7d was used. For a given Sso7d concentration the DNA unwinding induced was augmented with increasing temperature. However, after correction for the overwinding effect of high temperature on DNA, plasmids ligated at 60-90 degrees C exhibited similar sigma values at the highest Sso7d concentrations assayed. These results suggest that Sso7d may play a compensatory role in vivo by counteracting the overwinding effect of high temperature on DNA. Additionally, Sso7d unwinding could be involved in the topological changes observed during thermal stress (heat and cold shock), playing an analogous role in crenarchaeal cells to that proposed for HU in bacteria.

HMGI family proteins: architectural transcription factors in mammalian development and cancer.

The HMGI proteins, a subfamily of the high mobility group (HMG) proteins, play an important role in the regulation of gene expression. They bind to AT-rich DNA sequences and alter DNA conformation to modulate the binding affinity of transcription factors to their cognate sites. They are expressed almost exclusively during embryogenesis and Hmgi-c null mice have the mouse pygmy phenotype. Studies have revealed the disruption of HMGI family genes in a variety of mesenchymal-derived benign tumors. Therefore, the HMGI genes function in the coordination of cell proliferation and differentiation during mammalian development.