Unusual DNA Structure Research Articles

Genetic instability within monotonous runs of CpG sequences in Escherichia coli.

Genetic information can be altered by base substitutions, frameshift mutations, and addition or deletion of nucleotides. Deletions represent an important class of genetic aberration occurring at DNA sequences where it is often possible to predict the existence of intermediates of mutation. Instability within tracts of repetitive sequence have recently been associated with several genetic disorders, including the so-called triplet repeat diseases and certain forms of colorectal cancers. In Escherichia coli, (GpC)n repetitive sequences have been shown to be deletion prone, but the precise mechanism of this mutagenic pathway is still unknown. We show here that interrupting the monotony of the (GpC)n run with an ApT or a GpT dinucleotide decreases the rate of deletions within these sequences. On the other hand, introducing purine-pyrimidine alternating sequences beside the GpC insert results in an increased rate of deletion. Two pathways can be envisioned: (1) (GpC)n tracts can be seen as potential Z-forming DNA sequences, and this unusual DNA structure can be processed by an unknown cellular mechanism to give rise to the observed deletions and (2) (GpC)n monotonous runs can be considered as a succession of direct or palindromic repeats, allowing formation of DNA structures that are known to participate to frameshift mutagenesis. The results presented in this article are discussed in the light of these two alternative pathways.

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.

Centromeric Dodeca-satellite DNA Sequences Form Fold-back Structures

The evolutionarily conserved centromeric dodeca-satellite DNA has an asymmetric distribution of guanine and cytosine residues resulting in one strand being relatively G-rich. This dodeca-satellite G-strand contains a GGGA-tract that is similar to the homopurine tracts found in most telomeric DNA sequences. Here, we show that the dodeca-satellite G-strand forms intramolecular hairpin structures that are stabilized by the formation of non-Watson-Crick G · A pairs' as well as regular Watson-Crick G · C pairs. Special stacking interactions are also likely to contribute significantly to the stability of this structure. This hairpin conformation melts at relatively high temperature, around 75°C, and is detected under many different ionic and pH conditions. As judged by electron microscopy visualization, these structures can be formed in a B-DNA environment. Under the same experimental conditions, neither the C-strand nor the double-stranded dodeca-satellite DNA were found to form any unusual DNA structure. A protein activity has been detected that preferentially binds to the single-stranded dodeca-satellite C-strand. The biological relevance of these results is discussed in view of the similarities to telomeric DNA.

Centromeric dodeca-satellite DNA sequences form fold-back structures

The evolutionarily conserved centromeric dodeca-satellite DNA has an asymmetric distribution of guanine and cytosine residues resulting in one strand being relatively G-rich. This dodeca-satellite G-strand contains a GGGA-tract that is similar to the homopurine tracts found in most telomeric DNA sequences. Here, we show that the dodeca-satellite G-strand forms intramolecular hairpin structures that are stabilized by the formation of non-Watson-Crick G.A pairs as well as regular Watson-Crick G.C pairs. Special stacking interactions are also likely to contribute significantly to the stability of this structure. This hairpin conformation melts at relatively high temperature, around 75 degrees C, and is detected under many different ionic and pH conditions. As judged by electron microscopy visualization, these structures can be formed in a B-DNA environment. Under the same experimental conditions, neither the C-strand nor the double-stranded dodeca-satellite DNA were found to form any unusual DNA structure. A protein activity has been detected that preferentially binds to the single-stranded dodeca-satellite C-strand. The biological relevance of these results is discussed in view of the similarities to telomeric DNA.

Visualisation of inter loop tertiary base pairing and stacking interactions in an unusual slipped loop DNA structure

The conformation of an unusual slipped loop DNA structure exhibited by the sequence d(GAATTCCCGAATTC)2 is determined using a combination of geometrical and molecular mechanics methods. This sequence is known to form a B-DNA-like duplex with the central non-complementary cytosines extruded into single stranded loop regions. The unusual feature is that the interior guanine does not pair with the cytosine across, instead, it pairs with the cytosine upstream by skipping two cytosines, leading to a slipped loop DNA structure with the loops staggered by two base pairs. The two loops, despite being very small, can fold across minor or major groove symmetrically or asymmetrically disposed, with one of the loop bases partially blocking the major or minor groove. Most interestingly, for certain conformations, the loop bases approach one another at close proximity so as to engage even in base pairing as well as base stacking interactions across the major groove. While such pairing and stacking are common in the tertiary folds of RNA, this is the first time that such an interaction is visualized in a DNA. This observation demonstrates that a W-C pair can readily be accomplished in a typical slipped loop structure postulated for DNA. Such tertiary loop interaction may prevent access to regulatory proteins across the major groove of the duplex DNA, thus providing a structure-function relation for the occurrence of slipped loop structure in DNA.

An analogue of the DnaJ molecular chaperone whose expression is controlled by σS during the stationary phase and phosphate starvation in Escherichia coli

The Escherichia coli CbpA protein appears to be an analogue of the molecular chaperone, DnaJ, as judged from not only its structure but also its possible function. The expression of cbpA, however, was not significantly affected by up-shift of the growth temperature. Remarkably, it was found that the expression of cbpA was induced under certain growth conditions, such as the entry of cells into stationary phase, or growth in a phosphate-limited medium. Such conditional expression of cbpA was regulated at the transcriptional level in a sigma s-dependent manner. The structure of this sigma s-dependent cbpA promoter was clarified by determining its transcription start site. The cbpA promoter region was found to contain an unusual DNA structure (i.e. DNA curvature). From these results, it was suggested that, in contrast to DnaJ, CbpA may function as a molecular chaperone in an adaptive response to environmental stresses other than heat shock.

KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer.

We have constructed a RP4 KorB overproducing strain and purified the protein to near homogeneity. KorB is a DNA binding protein recognizing defined palindromic 13-bp sequences (TTTAGCSGCTAAA). Inverted sequence repetitions of this type, designated OB, are present on RP4 12 times. OB-sequences are localized in replication and maintenance regions as well as in the regions Tra1 and Tra2 essential for conjugative transfer. All sites found in Tra regions by computer search act as targets for specific binding of KorB protein. KorB-DNA complexes were detected by DNA fragment retardation assay using polyacrylamide gels. The 13-bp symmetric arrangement of the consensus OB-sequence constitutes the core for binding KorB protein since any truncation of this sequence prevents complex assembly or leads to a considerable destabilization of the KorB-DNA complexes. A hydroxyl radical footprint analysis demonstrated complex formation of KorB with the OB-sequence directly and suggests the presence of an unusual DNA structure within the nucleoprotein complex.

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.

Mitochondrial DNA Structure and Expression in Specialized Subtypes of Mammalian Striated Muscle

Mitochondrial DNA (mt DNA) in cells of vertebrate organisms can assume an unusual triplex DNA structure known as the displacement loop (D loop). This triplex DNA structure forms when a partially replicated heavy strand of mtDNA (7S mtDNA) remains annealed to the light strand, displacing the native heavy strand in this region. The D-loop region contains the promoters for both heavy- and light-strand transcription as well as the origin of heavy-strand replication. However, the distribution of triplex and duplex forms of mtDNA in relation to respiratory activity of mammalian tissues has not been systematically characterized, and the functional significance of the D-loop structure is unknown. In comparisons of specialized muscle subtypes within the same species and of the same muscle subtype in different species, the relative proportion of D-loop versus duplex forms of mtDNA in striated muscle tissues of several mammalian species demonstrated marked variation, ranging from 1% in glycolytic fast skeletal fibers of the rabbit to 65% in the mouse heart. There was a consistent and direct correlation between the ratio of triplex to duplex forms of mtDNA and the capacity of these tissues for oxidative metabolism. The proportion of D-loop forms likewise correlated directly with mtDNA copy number, mtRNA abundance, and the specific activity of the mtDNA (gamma) polymerase. The D-loop form of mtDNA does not appear to be transcribed at greater efficiency than the duplex form, since the ratio of mtDNA copy number to mtRNA was unrelated to the proportion of triplex mtDNA genomes. However, tissues with a preponderance of D-loop forms tended to express greater levels of cytochrome b mRNA relative to mitochondrial rRNA transcripts, suggesting that the triplex structure may be associated with variations in partial versus full-length transcription of the heavy strand.

Mitochondrial DNA structure and expression in specialized subtypes of mammalian striated muscle.

Mitochondrial DNA (mt DNA) in cells of vertebrate organisms can assume an unusual triplex DNA structure known as the displacement loop (D loop). This triplex DNA structure forms when a partially replicated heavy strand of mtDNA (7S mtDNA) remains annealed to the light strand, displacing the native heavy strand in this region. The D-loop region contains the promoters for both heavy- and light-strand transcription as well as the origin of heavy-strand replication. However, the distribution of triplex and duplex forms of mtDNA in relation to respiratory activity of mammalian tissues has not been systematically characterized, and the functional significance of the D-loop structure is unknown. In comparisons of specialized muscle subtypes within the same species and of the same muscle subtype in different species, the relative proportion of D-loop versus duplex forms of mtDNA in striated muscle tissues of several mammalian species demonstrated marked variation, ranging from 1% in glycolytic fast skeletal fibers of the rabbit to 65% in the mouse heart. There was a consistent and direct correlation between the ratio of triplex to duplex forms of mtDNA and the capacity of these tissues for oxidative metabolism. The proportion of D-loop forms likewise correlated directly with mtDNA copy number, mtRNA abundance, and the specific activity of the mtDNA (gamma) polymerase. The D-loop form of mtDNA does not appear to be transcribed at greater efficiency than the duplex form, since the ratio of mtDNA copy number to mtRNA was unrelated to the proportion of triplex mtDNA genomes. However, tissues with a preponderance of D-loop forms tended to express greater levels of cytochrome b mRNA relative to mitochondrial rRNA transcripts, suggesting that the triplex structure may be associated with variations in partial versus full-length transcription of the heavy strand.

Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe

The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk = 0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHI, PstI, AvaI and HindIII could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as NarI and FspI, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-B conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences.

Identification of DNA-protein interactions and enhancer activity at the 5′ end of the upstream regulatory region in human papillomavirus type 11

We have examined the 5′ end of the noncoding region of the genome of a human papillomavirus, HPV-11, for regulatory elements using permissive host cells. This region of unknown function in the upstream regulatory region (URR) is known to have unusual DNA structure and frequently contains rearrangements which are associated with some more virulent isolates. This 5′ 269-bp fragment was found to exhibit both specific DNA-protein binding using laryngeal papilloma protein extracts and enhancer activity in normal and papillornatous primary laryngeal cells. The viral DNA flanks the Li open reading frame and does not contain the viral E2 binding site. Three distinct protein binding sites are contained in a 50-bp region of the fragment. This fragment, as a whole, functions as an enhancer in primary laryngeal and papilloma cells when ligated to the SV40 promoter and SV40 T-antigen gene. We conclude that this part of the noncoding region of the papillomaviruses has elements characteristic of regulatory elements in cells permissive for infection by these viruses.

An overhanging 3' terminus is a conserved feature of telomeres.

The reactivity of single-stranded thymidines with osmium tetraoxide was used to demonstrate the existence of a terminal overhang of the G-rich strand of telomeres from two distantly related eucaryotes, the ciliated protozoan Tetrahymena spp. and the acellular slime mold Didymium spp. Conservation of a G-strand overhang at the molecular terminus of telomeres is consistent with our suggestion that an unusual DNA structure formed by the G-strand overhang is important for telomere function (E. Henderson, C. C. Hardin, S. K. Wolk, I. Tinoco Jr., and E. H. Blackburn, Cell 51:899-908, 1987).

An overhanging 3' terminus is a conserved feature of telomeres.

The reactivity of single-stranded thymidines with osmium tetraoxide was used to demonstrate the existence of a terminal overhang of the G-rich strand of telomeres from two distantly related eucaryotes, the ciliated protozoan Tetrahymena spp. and the acellular slime mold Didymium spp. Conservation of a G-strand overhang at the molecular terminus of telomeres is consistent with our suggestion that an unusual DNA structure formed by the G-strand overhang is important for telomere function (E. Henderson, C. C. Hardin, S. K. Wolk, I. Tinoco Jr., and E. H. Blackburn, Cell 51:899-908, 1987).

The role of DNA sequence in the formation of Z-DNA versus cruciforms in plasmids.

The capacities of four synthetic sequences containing runs of perfectly alternating purine-pyrimidine base pairs (bp) to adopt left-handed structures were evaluated in a homologous family of recombinant plasmids. All the sequences had the same G+C content (50%) and consisted of simple tetranucleotide repeat units but differed in the relative orientations of these units. For some of the sequences, several alternate secondary structures were theoretically possible; a variety of probes (S1 nuclease, bromoacetaldehyde, OsO4, T7 gene 3 endonuclease, supercoil-induced gel relaxation studies) under a wide range of reaction conditions was used to determine which structures were adopted as a function of superhelical stress. The precise positions at the bp level of reactions with these chemical and enzymatic probes were determined. We conclude that for short (20-24 bp) sequences containing runs of alternating (T-G) and (C-A), the cruciform state is preferred over the similarly allowable left-handed form provided that symmetry constraints allow. However, these sequences can be induced to form a left-handed helix under appropriate conditions. This is the first demonstration of plasmid inserts which will adopt more than one unusual DNA structure in response to negative superhelical stress. The structural properties of a molecule containing a Z-Z junction were studied, and we conclude that the disruption caused by this feature extends over only a few bp although it requires a high energetic penalty.

The γδ resolvase induces an unusual DNA structure at the recombinational crossover point

γδ resolvase, a transposon-encoded protein active in site-specific recombination, induces a structural change in the DNA at the recombinational crossover point that results in enhanced intercalation of the foot-printing reagent MPE · Fe(II). The structural change correlates with the formation of a bend in the DNA: a mutant resolvase that binds to the crossover site but induces little or no bend does not promote intercalation. The properties of the mutant protein suggest that the induced structural change, which we propose is a localized kink, is required for recombination.

X-ray studies reveal unusual DNA structure

X-ray studies reveal unusual DNA structure

A spectroscopic and electron microscopic examination of the highly condensed DNA structures formed by denaturation in Mg(ClO 4) 2

1. 1. Thermal denaturation in 1.5 M Mg(ClO 4) 2 of the DNA from bacteriophage λ results in four well-separated subtransitions, as monitored by the accompanying increase in absorbance. The midpoint of the hyperchromic spectrum is significantly lowered compared to either 1.5 M MgCl 2 or 3.0 M NaClO 4. 2. 2. The first two subtransitions are associated with the melting of the A · T-richest regions of the λ DNA, as revealed by electron micrographs following fixation with formaldehyde. 3. 3. Commencing with the third subtransition, an unusual DNA structure is observed in electron micrographs. In this structure the A · T-rich half of the molecule appears completely condensed, whereas the G · C-rich half remains native. 4. 4. During the fourth subtransition DNA molecules condense completely and eventually aggregate to form extremely high molecular weight particles containing centers of electron density. Tendrils of DNA, primarily duplex, radiate outward from these centers. 5. 5. The aggregation may be reversed by the removal of magnesium. The intramolecular condensation may be at least partly reversed by increasing the Mg(ClO 4) 2 concentrations to saturating levels.