Alterations In DNA Structure Research Articles

Substrate structure influences binding of the non-histone protein HMG-I(Y) to free nucleosomal DNA.

High mobility group protein HMG-I(Y) selectively binds to stretches of A.T-rich B-form DNA in vitro by recognition of substrate structure rather nucleotide sequence. Recognition of altered DNA structures has also been proposed to explain the preferential binding of this non-histone protein to four-way junction DNA as well as to restricted regions of DNA on random-sequence nucleosome core particles. Here we describe experiments that examine the influence of intrinsic DNA structure, and of structure imposed by folding of DNA around histone cores, on the binding of HMG-I(Y). As substrates for binding, we chose defined-sequence DNA molecules containing A.T-rich segments demonstrated previously to have very different structures in solution. These segments are either intrinsically bent (phase A.T tracts), flexible (oligo[d(A-T)]), or straight and rigid [oligo(dA).oligo(dT)]. DNase-I and hydroxyl radical footprinting techniques were employed to analyze protein binding to these DNAs either free in solution or when they were reconstituted into monomer or dinucleosomes in vitro. Results indicate that the DNA structure exerts a significant influence on HMG-I(Y) binding both when substrates are free in solution and when they are wrapped into nucleosomal structures. For example, when DNA is free in solution, HMG-I(Y) prefers to bind to the narrow minor groove of A.T sequences but sometimes also binds to certain GpC residues having narrowed major grooves that are embedded in such sequences. On the other hand, depending on the structure and/or orientation assumed by particular A.T-rich segments on the surface of reconstituted histone octamers, HMG-I(Y) binding site selection on individual nucleosomes differs considerably. Two observations are of particular importance: (i) HMG-I(Y) can preferentially bind to certain types of A.T-DNA located on the surface of nucleosomes; and (ii) HMG-I(Y) binding can induce localized alterations in the helical periodicity and/or rotational setting of DNA on the surface of some nucleosomes. The abilities of HMG-I(Y) suggests that in vivo the protein may play an important role in recognizing and altering the structure of localized regions of chromatin.

SARs are cis DNA elements of chromosome dynamics: Synthesis of a SAR repressor protein

SARs are candidate DNA elements for defining the bases of chromatin loops and possibly for serving as cis elements of chromosome dynamics. SARs contain numerous A tracts, whose altered DNA structure is recognized by cooperatively interacting proteins such as topoisomerase II. We constructed multi-AT hook (MATH) proteins and demonstrate that they specifically bind the clustered A tracts of SARs in chromatin and chromosomes. They are also potent inhibitors of chromosome assembly in mitotic Xenopus extracts, demonstrating the importance of SARs in this process. Titration of SARs with MATH20 (20 hooks) blocks shape determination of chromatids but not chromatin condensation per se. SARs are also required for shape maintenance of chromosomes. If MATH20 is added after formation of chromatids, they collapse and are reshaped by an active, mitotic process into spherical chromatid balls.

In Vivo Binding of Trimethylpsoralen Detects DNA Structural Alterations Associated with Transcribing Regions in the Human β-Globin Cluster

In order to increase our knowledge about the mechanisms that regulate expression of human beta-like globin genes, we have used a novel technique to analyze the chromatin structure in living cells. This approach allowed us to detect specific DNA regions in vivo where nucleosome folding or unconstrained DNA supercoiling in erythroid cells differs from that in non-erythroid cells. In this method, we use 4,5',8-trimethylpsoralen (TMP) as a probe capable of detecting altered chromatin conformations. Our results show that TMP binds to DNA with a higher affinity over the regions in the locus that are actively expressed, including both the promoter and the transcribed region. This higher affinity detected when comparing erythroid cells with non-erythroid cells does not extend to other regions inside the beta-globin cluster. Our data suggest that the observed effect is likely due to nucleosome displacement. Alternatively, it could result from localized DNA supercoiling, but not from widespread torsional stress across the entire beta-like globin locus as hypothesized previously.

The reduced expression of endogenous duplications (REED) in the maize R gene family is mediated by DNA methylation.

The duplicated R and Sn genes regulate the maize anthocyanin biosynthetic pathway and encode tissue-specific products that are homologous to helix-loop-helix transcriptional activators. As a consequence of their coupling in the genome, Sn is partially silenced. Genomic restriction analysis failed to reveal gross structural DNA alterations between the strong original phenotype and the weak derivatives. However, the differences in pigmentation were inversely correlated with differences in the methylation of the Sn promoter. Accordingly, treatment with 5-azacytidine (AZA), a demethylating agent, restored a strong pigmentation pattern that was transmitted to the progeny and that was correlated with differential expression of the Sn transcript. Genomic sequencing confirmed that methylation of the Sn promoter was more apparent in the less pigmented seedlings and was greatly reduced in the AZA revertants. In addition, some methylcytosines were located in non-symmetrical C sequences. These findings provide an insight into Sn and R interaction, a process that we have termed Reduced Expression of Endogenous Duplications (REED). We propose that increasing the copy number of regulatory genes by endogenous duplication leads to such epigenetic mechanisms of silencing. Further understanding of the REED process may have broader implications for gene regulation and may identify new levels of regulation within eukaryotic genomes.

The role of chromatin-associated protein Hbsu in beta-mediated DNA recombination is to facilitate the joining of distant recombination sites.

The beta recombinase is unable to mediate in vitro DNA recombination between two directly oriented recombination sites unless a bacterial chromatin-associated protein (Bacillus subtilis Hbsu or Escherichia [correction of Eschrichia] coli HU] is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between beta recombinase dimers through a protein-protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate beta-mediated recombination. These chromatin-associated proteins share the property of binding to DNA in a relatively non-specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing-over region, since at limiting protein-DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of beta protein to DNA, but inhibited the formation of the synaptic complex.

TheBacillus subtilisFlagellar Regulatory Protein σD: Overproduction, Domain Analysis and DNA-binding Properties

Flagellar biosynthesis requires an alternative sigma (σ) subunit of RNA polymerase to allow recognition of the promoters for flagellin and other late genes of the flagellar regulon. We have now overproduced and characterizedBacillus subtilisσD: the prototype of the S28family of flagellar σ factors. Limited protease digestion studies indicate that σDcontains an amino-terminal domain, comprising conserved regions 1.2 and 2, and a carboxyl-terminal domain containing conserved regions 3.2 and 4. The protease-sensitive region between these two domains correlates with a region of very low sequence conservation among bacterial σ factors. Unlike the primary σ factor, σDbinds to DNA. In non-denaturing polyacrylamide gel electrophoresis the σD-DNA complex has an apparent equilibrium dissociation constant of 1 μM. Binding of σDto the promoter for flagellin, PD-6, appears to lead to an altered DNA structure near the −35 and −10 recognition elements as detected by DNase I footprinting and by the enhanced reactivity of several bases to dimethylsulfate.f2f2Abbreviations used: DMS, dimethylsulfate; GST, glutathione-S-transferase; PCR, polymerase chain reaction; IPTG, isopropyl-β-d-thiogalactoside; PMSF, phenylmethylsulfonyl fluoride; HPLC, high pressure liquid chromatography.

A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells.

We recently identified and enriched a protein (CBP) from HeLa cells with binding specificity for cruciform-containing DNA. We have now studied the interaction of CBP with stable cruciform DNA molecules containing the 27 bp palindrome of SV40 on one strand and an unrelated 26 bp palindrome on the other strand by hydroxyl radical footprinting. The CBP-DNA interaction is localized to the four-way junction at the base of the cruciforms. CBP appears to interact with the elbows of the junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed in the cruciform stems and in a DNA region adjacent to the junction. These features distinguish CBP from other cruciform binding proteins, which bind symmetrically and display exclusively either contacts with the DNA backbone or structural alterations in the DNA.

Parameters That Influence the Extent of Site Occupancy by a Candidate Telomere End-binding Protein

The MF3 protein specifically recognizes telomeric and non-telomeric DNA probes that can form G.G base-paired structures (Gualberto, A., Patrick, R. M., and Walsh, K. (1992) Genes & Dev. 6, 815-824). Here we further characterize the nucleic acid recognition properties of MF3 and present a mathematical analysis that evaluates the potential extent of telomere site occupancy by this factor. The substitution of dI at dG positions in telomeric DNA probes revealed that a single dG at any position within the internal repeat was sufficient for high affinity binding to MF3. The RNA analogs of high affinity DNA sites were not bound specifically by MF3, but the substitution of dU for dT in a DNA probe had little or no effect on binding. These data demonstrate that ribose ring structure is a critical feature of nucleoprotein complex formation, and this ribose specificity may enable MF3 to occupy sites of unusual DNA structure while minimizing interactions with cellular RNAs. Collectively, the nucleic acid binding properties of MF3 suggest that it may occupy a significant fraction of sites at telomere ends or other G-rich regions of altered DNA structure in vivo.

NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human beta-globin locus control region.

The beta-like globin genes require the upstream locus control region (LCR) for proper expression. The active elements of the LCR coincide with strong erythroid-specific DNase I-hypersensitive sites (HSs). We have used 5' HS4 as a model to study the formation of these HSs. Previously, we identified a 101 bp element that is required for the formation of this HS. This element binds six proteins in vitro. We now report a mutational analysis of the HS4 HS-forming element (HSFE). This analysis indicates that binding sites for the hematopoietic transcription factors NF-E2 and GATA-1 are required for the formation of the characteristic chromatin structure of the HS following stable transfection into murine erythroleukemia cells. Similarly arranged NF-E2 and GATA binding sites are present in the other HSs of the human LCR, as well as in the homologous mouse and goat sequences and the chicken beta-globin enhancer. A combination of DNase I and micrococcal nuclease sensitivity assays indicates that the characteristic erythroid-specific hypersensitivity of HS4 to DNase I is the result of tissue-specific alterations in both nucleosome positioning and tertiary DNA structure.

Correlation of doxorubicin footprints with deletion endpoints in lacO of E. coli

This study explored the possibility that the sequence location of doxorubicin-induced deletion endpoints might relate to DNA structural alterations caused by doxorubicin binding to DNA. The 3′-OH endpoints of doxorubicin-induced deletions terminating in the 35-bp region of lacO appear to distribute differently from spontaneous deletion endpoints. Doxorubicin-induced deletions focus in the 26-bp palindrome which is separated by a 9-bp region with no reverse complementary, whereas spontaneous deletion 3′-OH endpoints are found distributed throughout the operator region. In order to explore the mechanism of deletion induction by doxorubicin, drug footprinting studies were carried out with DNA labeled at the 5′ end of each of the complementary DNA strands encompassed by lacO. Doxorubicin protected the 9-bp region between the palindromic sequences from DNase I cutting and caused enhanced DNase I cleavage at symmetrical sites in the palindrome, which were inherently resistant to the nuclease in the absence of the drug. These symmetrical sites also define regions in which the occurrence of deletion endpoints is enhanced 6-fold in the presence of doxorubicin. This enhanced cutting and mutation occur in regions of the palindrome that are flanked by expected doxorubicin binding sites, but are not themselves binding sites of the drug. Similarly, other sites where the frequency of deletion endpoints increased in response to doxorubicin occurred directly adjacent to regions where doxorubicin appeared to inhibit cutting by DNase I. These results suggest that the binding of doxorubicin in the palindrome directs both the frequency and the specificity of deletion formation in this gene region.

The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure.

Insertion of the gypsy retrotransposon of Drosophila melanogaster into a gene control region can repress gene expression. The zinc finger protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy and prevents gene enhancers from activating transcription. SUHW blocks an enhancer only when positioned between the enhancer and promoter. Although position dependent, SUHW enhancer blocking is distance independent. These properties indicate that SUHW does not interact with the transcription activator proteins that bind to enhancers. To explore if DNA distortions are involved in enhancer blocking, the ability of SUHW to alter DNA structure was examined in gel mobility assays. Indeed, SUHW induces an unusual change in the structure of the binding-site DNA. The change is not a directed DNA bend but correlates with loss of sequence-directed bends in the unbound DNA. The DNA distortion requires a SUHW protein domain not required for DNA binding, and mutant proteins that fail to alter DNA structure also fail to eliminate the sequence-directed bends. These results suggest that SUHW increases DNA flexibility. The DNA distortion is not sufficient to block enhancers, and therefore it is suggested that increased DNA flexibility may help SUHW interact and interfere with proteins that support long-distance enhancer-promoter interactions.

The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure.

Insertion of the gypsy retrotransposon of Drosophila melanogaster into a gene control region can repress gene expression. The zinc finger protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy and prevents gene enhancers from activating transcription. SUHW blocks an enhancer only when positioned between the enhancer and promoter. Although position dependent, SUHW enhancer blocking is distance independent. These properties indicate that SUHW does not interact with the transcription activator proteins that bind to enhancers. To explore if DNA distortions are involved in enhancer blocking, the ability of SUHW to alter DNA structure was examined in gel mobility assays. Indeed, SUHW induces an unusual change in the structure of the binding-site DNA. The change is not a directed DNA bend but correlates with loss of sequence-directed bends in the unbound DNA. The DNA distortion requires a SUHW protein domain not required for DNA binding, and mutant proteins that fail to alter DNA structure also fail to eliminate the sequence-directed bends. These results suggest that SUHW increases DNA flexibility. The DNA distortion is not sufficient to block enhancers, and therefore it is suggested that increased DNA flexibility may help SUHW interact and interfere with proteins that support long-distance enhancer-promoter interactions.

Facilitated binding of TATA-binding protein to nucleosomal DNA.

BINDING of the TATA-binding protein (TBP) to the TATA box is required for transcription from many eukaryotic promoters in gene expression. Regulation of this binding is therefore likely to be an important determinant of promoter activity. Incorporation of the TATA sequence into nucleosomes dramatically reduces transcription initiation, presumably because of stereochemical constraints on binding of general transcription factors. Biochemical and genetic studies imply that cellular factors such as yeast SWI/SNF are required for activator function and might alter chromatin structure. One step that could be regulated during the activation process is TBP binding in chromatin 12, 13. We show here that binding of TBP to the TATA sequence is severely inhibited by incorporation of this sequence into a nucleosome. Inhibition can be overcome by ATP-dependent alterations in nucleosomal DNA structure mediated by hSWI/SNF, a putative human homologue of the yeast SWI/SNF complex. Additionally, the orientation of the TATA sequence relative to the surface of the histone core affects the access of TBP. We propose that the dynamic remodelling of chromatin structure to allow TBP binding is a key step in the regulation of eukaryotic gene expression.

A second high affinity HU binding site in the phage Mu transpososome.

The bacteriophage Mu in vitro transposition reaction proceeds through several higher order nucleoprotein intermediates (transpososomes). One of the requirements for complex assembly is the Escherichia coli sequence-independent DNA-binding protein, HU. This protein has an affinity for Mu transpososomes, which is at least 100 times greater than for supercoiled DNA (Lavoie and Chaconas, 1990). We have recently identified one such high affinity binding site at the Mu left end by converting HU into a chemical nuclease (Lavoie and Chaconas, 1993). Using immunoelectron microscopy, we now report high affinity HU binding to a region(s) distinct from the previously characterized left end site. HU can be removed from this region by a 0.5 M NaCl wash and subsequently reassembled into the complex with high efficiency. Furthermore, chemical modification of the Mu A protein in the Type 1 complex does not block HU reassembly into the transpososome; the high affinity HU binding observed is therefore unlikely to result from A-HU interactions. These findings are corroborated by the ability of eukaryotic HMG-1 to functionally replace HU in transpososome formation and to efficiently assemble into HU-depleted complexes. We propose that HU recognition of an altered DNA structure, rather than protein-protein interactions, mediates high affinity HU binding to Mu transpososomes.

Quantitative footprinting analysis

This review outlines the steps for obtaining relative binding constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA site. Slopes of plots outside of drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netropsin to a 139-base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netropsin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.

Protein tracking-induced supercoiling of DNA: a tool to regulate DNA transactions in vivo?

An interplay between DNA-dependent biological processes appears to be crucial for cell viability. At the molecular level, this interplay relies heavily on the communication between DNA-bound proteins, which can be facilitated and controlled by the dynamic structure of double-stranded DNA. Hence, DNA structural alterations are recognized as potential tools to transfer biological information over some distance within a genome. Until recently, however, direct evidence for DNA structural information as a mediator between cellular processes was lacking. This changed when the concept of transient waves of DNA supercoiling, induced by proteins tracking along the right-handed DNA double helix, came into the limelight. Indeed, a number of observations now suggest that helix tracking-induced DNA structural information might be exploited to participate in the regulation of a variety of DNA transactions in vivo.

Light-activated cleavage of DNA by cobalt-bleomycin

We have studied the light-activated cleavage of DNA by cobalt-bleomycin using a series of synthetic DNA fragments containing (AT)n and (GC)n. This cleavage reaction requires high concentrations of the antibiotic and appears to be a stoichiometric process rather than a catalytic process. We find that, in common with the iron-complex, cobalt-bleomycin can cleave at ApT steps within regions of alternating AT residues; ApT steps within other sequences including (AAT)n. (ATP)n are not good substrates for cobalt-bleomycin cleavage. Some repetitive regions display an alternating pattern of cleavage products, revealing the preferred arrangement of ligand molecules along a saturated DNA lattice. A similar repetitive pattern is found for diethylpyrocarbonate modification and hydroxyl-radical cleavage. Although cleavage of ApT and GpC proceeds at equivalent rates, the data suggest that bleomycin binds more tightly to the latter. Adenine residues on the 3' side of both GpC-cleavage and ApT-cleavage sites are rendered more reactive to diethylpyrocarbonate, consistent with a ligand-induced alteration in local DNA structure. The cobalt-bleomycin-binding site consists of not more than four base pairs, and may be as small as three base pairs.

Cis-DDP induced alteration of DNA structure studied by scanning tunneling microscopy.

Scanning tunneling microscopy (STM) was used to study the structural changes of DNA induced by the antitumor drug cis-diamminedichloroplatinum. The STM image showed a dramatic structural perturbation of the DNA by complexed Pt with the characteristic bend of the double helix.

Trp repressor interaction with bromodeoxyuridine-substituted operators alters UV-induced perturbation pattern in a sequence-dependent manner

In order to explore DNA sites influenced by the trp repressor-operator interaction, bromodeoxyuridine (BrdU) was chemically incorporated into the TrpEDCBA, TrpR, and aroH operators at selected thymidine positions. Different patterns of repressor protection from strand scission in the two halves of complexes with the TrpEDCBA, TrpR, and aroH operators suggest different local environments despite the highly symmetric sequences. Although protection was observed at multiple sites in the operators in the presence of repressor, UV irradiation did not lead to a cross-linked repressor-operator complex. This result indicates the absence of close contacts in the major groove between suitable repressor residues and the 5-methyl of thymidines. Upon trp repressor binding and UV irradiation, in addition to protection from strand scission, multiplets were observed at some sites, notably within CTAG sequences in the BrdU-substituted operators. This phenomenon (termed band migration) may result from distortion by the trp repressor of the BrdU-substituted operator DNA and consequent exposure of different sites along the backbone to strand scission. Interestingly, UV footprinting of two BrdU-substituted TrpEDCBA variant operators showed different patterns when base pair symmetry was matched to each side of the symmetry axis. These observations suggest that alterations in the UV photolysis pattern in response to protein binding result from DNA structural alterations that are sequence dependent.

The embryonic lethality of homozygous lethal yellow mice (Ay/Ay) is associated with the disruption of a novel RNA-binding protein.

Lethal yellow (Ay) is a mutation at the mouse agouti (a) locus that is associated with an all-yellow coat color, obesity, diabetes, tumors in heterozygotes, and preimplantation embryonic lethality in homozygotes. Previously, we cloned and characterized the wild-type agouti gene and demonstrated that it expresses a 0.8-kb mRNA in neonatal skin. In contrast, Ay expresses a 1.1-kb transcript that is ectopically overexpressed in all tissues examined. The Ay mRNA is identical to the wild-type a transcript for the entire coding region, but the 5'-untranslated sequence of the a gene has been replaced by novel sequence. Here, we demonstrate that the novel 5' sequence in the Ay mRNA corresponds to the 5'-untranslated sequence of another gene that is normally tightly linked to a in mouse chromosome 2. This other gene (Raly) has the potential to encode a novel RNA-binding protein that is normally expressed in the preimplantation embryo, throughout development, and in all adult tissues examined. Importantly, the Ay mutation disrupts the structure and expression of the Raly gene. The data suggest that the Ay mutation arose from a DNA structural alteration that affects the expression of both agouti and Raly. We propose that the dominant pleiotropic effects associated with Ay may result from the ectopic overexpression of the wild-type a gene product under the control of the Raly promoter and that the recessive embryonic lethality may be the result of the lack of Raly gene expression in the early embryo.