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Interaction of linear homologous DNA duplexes via Holliday junction formation
Abstract
Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA–DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA–protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.
... Under appropriate conditions these fragments can form different structures[1]which are obtained with low yields but are sufficiently stable to be studied. Some of these conformations are multistranded structures the conformation of which is not yet entirely clear[2][3][4], some of them being also formed with non-repetitive sequences[4][5][6]. Other alternative conformations of these fragments are double-stranded structures, called hcDNA, which are the object of the present paper. ...
We have previously isolated a stable alternative DNA structure, which was formed in vitro by reassociation of the strands of DNA fragments containing a 62 bp tract of the CA-microsatellite poly(CA).poly(TG). In the model which was proposed for this structure the double helix is folded into a loop, the base of the loop consists of a DNA junction in which one of the strands of one duplex passes between the two strands of the other duplex, forming a DNA hemicatenane in a hemiknot structure. The hemiknot DNA structures obtained with long CA/TG inserts have been imaged by AFM allowing us to directly visualize the loops.
Here we have analyzed this structure with several different techniques: high-resolution gel electrophoresis, probing by digestion with single stranded DNA-specific nucleases or with DNase I, modification with chemicals specific for unpaired bases, and atomic force microscopy. The data show a change in DNA structure localized to the CA/TG sequence and allow us to better understand the structure of this alternative conformation and the mechanism of its formation.
The present work is in good agreement with the model of hemicatenated DNA loop proposed previously. In the presence of protein HMGB1, shifted reassociation of the strands of DNA fragments containing a tract of the poly(CA).poly(TG) microsatellite leads to the formation of DNA loops maintained at their base by a hemicatenated junction located within the repetitive sequence. No mobility of the junction along the DNA molecule could be detected under the conditions used. The novel possibility to prepare DNA hemicatenanes should be useful to further study this alternative DNA structure and its involvement in replication or recombination.
... However, the (entropically favorable) release of the bound water molecules from the melted sections can facilitate the DNA melting (as well as a possible pairing of melted fragments, which is more pronounced in assemblies). 9,70 All in all, the net effect of the melted pieces on DNA melting in assemblies is not completely clear, and it is not excluded that it may interfere with the predicted effects. However, again, we see no reason this effect should be different for homologous and nonhomologous pairs, and this variance will likely be determined by the effects in the focus of this paper. ...
How does DNA melt in columnar aggregate relative to its melting in diluted solution? Is the melting temperature increased or decreased with the aggregate density? Have DNA-DNA interactions, predominantly of electrostatic nature, an effect on the character of the melting transition? In attempt to answer these questions, we have incorporated the theory of electrostatic interactions between DNA duplexes into the simplest model of DNA melting. The analysis shows that the effect of aggregate density is very different for aggregates built of homologous (or identical) DNA fragments relative to the case of DNA with random base pair sequences. The putative attraction between homologous DNA helices hampers their melting and increases the melting temperature and can even dramatically change the character of the transition. In the aggregate of nonhomologous DNAs, the pattern of electrostatic interactions is more complicated, and their effect could be opposite; in some cases we may even expect electrostatically induced melting. These findings define new directions for melting experiments in dense DNA assemblies.
Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleoticle invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleoticle concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.
The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA–DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.
The most promising approaches to detection of random point mutations are based on chemical cleavage of mismatches and other
noncomplementarities. To demonstrate the specificity of this method, a model system was obtained for the first time as sets
of 50-mer imperfect DNA duplexes containg all variants of mismatched and unpaired internal residues located in an invariant
context and flanked by either A · T or G · C base pairs. Chemical cleavage of DNA duplexes immobilized on magnetic beads via
the biotin-streptavidin interaction was accomplished using potassium permanganate or hydroxylamine, which are sensitive to
the secondary DNA structure and react with thymine and cytosine, respectively. The reactivity of different mismatches was
connected with the local duplex structure and depended on their type, orientation, and flanking nucleotides. The use of potassium
permanganate and hydroxylamine to modify a heteroduplex mixture makes it possible to unambiguously detect a mismatch and,
based on the type of reagent and the size of the cleavage products, to suppose the type and position of the mismatch and the
flanking nucleotides. The model system can be used to evaluate the sensitivity of a chemical cleavage method and to control
false-positive and false-negative results when different protocols are applied to the detection of DNA point mutations.
(CA/TG)n repeats belong to microsatellite DNA. They are the most abundant among the other dinucleotide repeats in mammals, constituting
approximately 0.25% of the entire genome. These repeats are recombination hot spots; however, the corresponding mechanisms
are yet vague. We postulated that one of the reasons underlying an increase in the recombination frequency in the repetitive
region could be the con-formational characteristics of duplex resulting from a specific geometry of base-stacking contacts,
providing for initiation of a single-stranded DNA invasion in th e duplex homologous regions. This work for the first time
demonstrates a DNA-DNA interaction of the d(CA)10 and d(TG)10 oligonucleotides with linear and circular duplexes containing (CA/TG)31 repeats during their coincubation in a protein-free water solution at 37°C. Using radioactively labeled oligonucleotides,
we demonstrated that the duplex—oligonucleotide interaction intensity depended on the molar ratio of duplex-to-oligonucleotide
at a duplex concentration of 30 nM. A decrease in this concentration to 3 nM had no effect on the intensity of oligonucleotide
invasion. It was demonstrated that over 1% of the duplexes yet much less than 10% were involved in the interaction with oligonucleotides
assuming that one oligonucleotide molecule interacted with one molecule of the duplex. Analysis of the kinetics showed that
d(CA)10 invasion commenced from the first minute of incubation with duplexes, while d(TG)10 interacted with the duplex even at a higher rate. The role of conformational plasticity of CA/TG repeats in the discovered
interaction is discussed as well as its biological significance, in particular, the role of CA microsatellites in the initiation
of homologous recombination.
Key wordsDNA-noncanonical structure-(CA/TG) repeats-duplexes-oligonucleotide invasion-CA micro-satellites
Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.
A novel biophysical approach in combination with an acoustic device is demonstrated as a sensitive, rapid, and label-free technique for characterizing various structures of the DNA Holliday Junction (J1) nanoswitch. We were successful in discriminating the "closed" from the "open" state, as well as confirming that the digestion of the J1 junction resulted in the two, anticipated, rod-shaped, 20 bp long fragments. Furthermore, we propose a possible structure for the ∼10 nm long (DNA58) component participating in the J1 assembly. This work reveals the potential of acoustic devices as a powerful tool for molecular conformation studies.
Systematic study of chemical reactivity of non-Watson-Crick base pairs depending on their type and microenvironment was performed on a model system that represents two sets of synthetic DNA duplexes with all types of mismatched and unmatched bases flanked by T.A or G.C pairs. Using comparative cleavage pattern analysis, we identified the main and additional target bases and performed quantitative study of the time course and efficacy of DNA modification caused by potassium permanganate or hydroxylamine. Potassium permanganate in combination with tetraethylammonium chloride was shown to induce DNA cleavage at all mismatched or bulged T residues, as well as at thymines of neighboring canonical pairs. Other mispaired (bulged) bases and thymine residues located on the second position from the mismatch site were not the targets for KMnO(4) attack. In contrast, hydroxylamine cleaved only heteroduplexes containing mismatched or unmatched C residues, and did not modify adjacent cytosines. However when G.C pairs flank bulged C residue, neighboring cytosines are also attacked by hydroxylamine due to defect migration. Chemical reactivity of target bases was shown to correlate strongly with the local disturbance of DNA double helix at mismatch or bulge site. With our model system, we were able to prove the absence of false-negative and false-positive results. Portion of heteroduplex reliably revealed in a mixture with corresponding homoduplex consists of 5% for bulge bases and "open" non-canonical pairs, and 10% for wobble base pairs giving minimal violations in DNA structure. This study provides a complete understanding of the principles of mutation detection methodology based on chemical cleavage of mismatches and clarifies the advantages and limitations of this approach in various biological and conformational studies of DNA.
In the present work we investigate the CTAB:DNA complexes decompaction process using beta-CD as a decompacting agent. The transition from globules (compacted DNA) to coils (decompacted DNA) was achieved without a coexistence region between coils and globules. This non first-order transition was investigated combining fluorescence microscopy (FM), cryo-transmission electron microscopy (cryo-TEM) and TEM of negatively stained samples. Additionally the presence of multi-globular aggregates in the proximity of the critical transition was elucidated. A possible mechanism for the decompaction process was suggested.
Decompaction of DNA-CTA self-assembled complexes by 2-hydroxypropyl-beta-cyclodextrin (2-HP-beta-CD) was studied and the results were compared with beta-CD. Different degrees of 2-HP substitution (0.6, 0.8 and 1.0, respectively) were used and the decompaction was successful with all degrees of substitution. Fluorescence microscopy, steady state fluorescence spectroscopy, density and sound velocity measurements, thermal melting and circular dichroism were used. Compared to previous work using alpha- and beta-CD, the fluorescence spectroscopy results showed that the 2-HP-substituted CDs more efficiently released DNA into solution. Furthermore, dissociation of macroscopically phase separated DNA-CTA complexes was achieved upon addition of 2-HP-beta-CD and the results gave strong indications on the non-equilibrium nature of the system. The globule-to-coil transition was not found to proceed through a coexistence region, which seems to be a general phenomenon in DNA decompaction using CDs.
Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the
plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations
in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase
lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection
is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This
method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and more recently, a second bacteriophage
resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and
mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable
and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation
detection.
Much interest has been shown in the use of multi-template reverse transcription-polymerase chain reaction (RT-PCR) as a quantitative instrument for low-abundance mRNAs. A desire to achieve finely-graded quantification of the stress- and hormone-related regulation of one splicing decision in an ion channel gene motivated us to test the reliability of simultaneous amplification of two splice variants with one pair of flanking constitutive primers. Unexpectedly indiscriminate heteroduplexing between the two amplification products, despite a large length difference, and their tight comigration with one homoduplex, mandated a rigorously-denaturing electrophoresis protocol. Conveniently, a new fluorescent dye with high affinity for single-stranded DNA has become available. Though the dye has a good dynamic range, we found that dye and gel saturation compounded by the length difference between products introduced an asymmetrical error into the calculation of relative abundance. Avoiding several pitfalls, dye calibration could be used to correct the error. We also found that differences in the amplification efficiency of the two templates were not constant, but dependent on the initial template ratio, requiring a non-linear correction. Together these improvements gave us very consistent quantitative results, and thus advance our analysis of hormonal mechanisms underlying the regulation of alternative splicing of an ion channel critically involved in stress responses.
Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.
Holliday junctions are four-stranded DNA complexes that are formed during recombination and related DNA repair events. Much work has focused on the overall structure and properties of four-way junctions in solution, but we are just now beginning to understand these complexes at the atomic level. The crystal structures of two all-DNA Holliday junctions have been determined recently from the sequences d(CCGGGACCGG) and d(CCGGTACCGG). A detailed comparison of the two structures helps to distinguish distortions of the DNA conformation that are inherent to the cross-overs of the junctions in this crystal system from those that are consequences of the mismatched dG.dA base-pair in the d(CCGGGACCGG) structure. This analysis shows that the junction itself perturbs the sequence-dependent conformational features of the B-DNA duplexes and the associated patterns of hydration in the major and minor grooves only minimally. This supports the idea that a DNA four-way junction can be assembled at relatively low energetic cost. Both structures show a concerted rotation of the adjacent duplex arms relative to B-DNA, and this is discussed in terms of the conserved interactions between the duplexes at the junctions and further down the helical arms. The interactions distant from the strand cross-overs of the junction appear to be significant in defining its macroscopic properties, including the angle relating the stacked duplexes across the junction.
Mutation and polymorphism detection is of increasing importance in the field of molecular genetics. This is reflected by the plethora of chemical, enzymatic, and physically based methods of mutation detection. The ideal method would detect mutations in large fragments of DNA and position them to single base-pair (bp) accuracy. Few methods are able to quickly screen kilobase lengths of DNA and position the mutation at the same time. The Enzyme Mismatch Cleavage (EMC) method of mutation detection is able to reliably detect nearly 100% of mutations in DNA fragments as large as 2 kb and position them to within 6 bp. This method exploits the activity of a resolvase enzyme from T4, T4 endonuclease VII, and, more recently, a second bacteriophage resolvase, T7 endonuclease I. The technique uses these enzymes to digest heteroduplex DNA formed by annealing wild-type and mutant DNA. Digestion fragments indicate the presence, and the position, of any mutations. The method is robust and reliable and much faster and cheaper than sequencing. These attributes have resulted in its increasing use in the field of mutation detection.
The isolation from proliferating mouse and human embryo fibroblasts of SDS-stable crosslinkage products of vimentin with DNA fragments containing inverted repeats capable of cruciform formation under superhelical stress and the competitive effect of a synthetic Holliday junction on the binding of cytoplasmic intermediate filament (cIF) proteins to supercoiled DNA prompted a detailed investigation of the proteins' capacity to associate with four-way junction DNA and to influence its processing by junction-resolving endonucleases. Electrophoretic mobility shift analysis of reaction products obtained from vimentin and Holliday junctions under varying ionic conditions revealed efficient complex formation of the filament protein not only with the unstacked, square-planar configuration of the junctions but also with their coaxially stacked X-conformation. Glial fibrillary acidic protein (GFAP) was less efficient and desmin virtually inactive in complex formation. Electron microscopy showed binding of vimentin tetramers or octamers almost exclusively to the branchpoint of the Holliday junctions under physiological ionic conditions. Even at several hundredfold molar excess, sequence-related single- and double-stranded DNAs were unable to chase Holliday junctions from their complexes with vimentin. Vimentin also stimulated bacteriophage T7 endonuclease I in introducing single-strand cuts diametrically across the branchpoint and thus in the resolution of the Holliday junctions. This effect is very likely due to vimentin-induced structural distortion of the branchpoint, as suggested by the results of hydroxyl radical footprinting of Holliday junctions in the absence and the presence of vimentin. Moreover, vimentin, and to a lesser extent GFAP and desmin, interacted with the cruciform structures of inverted repeats inserted into a supercoiled vector plasmid, thereby changing their configuration via branch migration and sensibilizing them to processing by T7 endonuclease I. This refers to both plasmid relaxation caused by unilateral scission and, particularly, linearization via bilateral scission at primary and cIF protein-induced secondary cruciform branchpoints that were identified by T7 endonuclease I footprinting. cIF proteins share these activities with a variety of other architectural proteins interacting with and structurally modulating four-way DNA junctions. In view of the known and hypothetical functions of four-way DNA junctions and associated protein factors in DNA metabolism, cIF proteins as complementary nuclear matrix proteins may play important roles in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription, with special emphasis on both the preservation and evolution of the genome.
We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion
with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies
with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that
the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling
kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or “kissing
complex”), concentrations as low as 3 μm in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation,
a majority of hairpins was even obtained at 80 μm in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic
considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than
from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure.
This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.
Although plant tissue cultures have been in use for the past hundred years, adapting them for the production of aroma compounds started only in the 1970s. The use of tissue cultures in aroma production has its advantages, because plant cells, unlike whole plants, are not limited to geographic locations or the seasons. Cell mass can be doubled relatively rapidly and can be induced for the production of compounds in a coordinated manner. Compounds can be isolated from cells or the medium with relative ease. Therefore, it would seem to be ideal to use plant cell cultures for the production of aroma compounds. Cell cultures, however, also have some problems. The production of aroma compounds or their precursors is in relatively low amounts, and thus this production method is expensive. Additional expenses are the cost of the medium and the purification of the compounds for food use. Also, cell cultures can only be used effectively in systems for which the biochemical pathway of the aroma compounds is known. In this paper the results of experiments for the use of tissue cultures in the production of vanilla, raspberry, strawberry garlic, and onion aromas is discussed.
Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.
Mutation detection and mismatch repair investigations based on heteroduplex formation require a linear DNA structure. DNA branching, described previously under physiological conditions, has been analysed in the heteroduplex formation process. Symmetrical chi-structures were detected after heteroduplex formation by gel electrophoresis and electron microscopy. Buffer composition, DNA concentration and duplex end-sequences influence DNA branching. Duplexes with homologous central regions but non-complementary ends do not form hybrid heteroduplexes or hybrid Holliday junctions. Our results explain the requirements for efficient heteroduplex formation, which were previously determined empirically: special solution composition, optimal DNA concentration and GC clamps. This provides the theoretical background for further optimisation of the procedure.
This paper deals with the nature of recombination intermediates. Using the electron microscope to study the DNA of the plasmid colicin E1, we have observed more than 800 molecules that appear to represent intermediates in the process of recombination. Specifically, after isolating colicin DNA and linearizing it with the restriction enzyme EcoRI, we find crossed molecules with twice the normal colicin DNA content. These forms consist of two genome-length elements held together at a region of DNA homology. The molecules can be recovered from wild type and Rec B-C host cells but are not present among the colicin DNA forms isolated from recombination-deficient Rec A cells. We have termed the experimentally observed molecules "chi forms" and believe that they represent the recombination intermediate of the Holliday model.
Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.
An extensive analysis of genomic DNA preparations from a number of normal and malignant tissues revealed BglII site polymorphism of the human p53 gene. Approximately 10% of p53 gene alleles were found to contain an additional BglII site localized in a region of intron I. This allelic form of p53 gene was also responsible for p53 protein having altered electrophoretic mobility. Molecular cloning and sequencing of both the alleles of p53 gene revealed a base-pair change in codon 72 causing arginine----proline substitution in the allele with the additional BglII site. Both variants of the p53 gene may occur in homozygous state and are therefore functional.
A DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double Helix forming Hoogsteen pairs with purines of the Watson-Crick basepairs. Intermolecular triplexes are formed between triplex forming oligonucleotides (TFO) and target sequences on duplex DNA. Intramolecular triplexes are the major elements of H-DNAs, unusual DNA structures, which are formed in homopurine-homopyrimidine regions of supercoiled DNAs. TFOs are promising gene-drugs, which can be used in an anti-gene strategy, that attempt to modulate gene activity in vivo. Numerous chemical modifications of TFO are known. In peptide nucleic acid (PNA), the sugar-phosphate backbone is replaced with a protein-like backbone. PNAs form P-loops while interacting with duplex DNA forming triplex with one of DNA strands leaving the other strand displaced. Very unusual recombination or parallel triplexes, or R-DNA, have been assumed to form under RecA protein in the course of homologous recombination.
Each of four possible sets of mismatches (G.A/C.T, C.C/G.G, A.A/T.T, and C.A/G.T) containing the 8 possible single-base-pair mismatches derived from isolated mutations were examined to test the ability of T4 endonuclease VII to consistently detect mismatches in heteroduplexes. At least two examples of each set of mismatches were studied for cleavage in the complementary pairs of heteroduplexes formed between normal and mutant DNA. Four deletion mutations were also included in this study. The various PCR-derived products used in the formation of heteroduplexes ranged from 133 to 1502 bp. At least one example of each set showed cleavage of at least one strand containing a mismatch. Cleavage of at least one strand of the pairs of heteroduplexes occurred in 17 of the 18 known single-base-pair mutations tested, with an A.A/T.T set not being cleaved in any mismatched strand. We propose that this method may be effective in detecting and positioning almost all mutational changes when DNA is screened for mutations.
The tandemly repeated DNA sequence poly(CA).poly(TG) is found in tracts up to 60 base pairs long, dispersed at thousands of sites throughout the genomes of eukaryotes. Double-stranded DNA fragments containing such sequences associated spontaneously with each other in vitro, in the absence of protein, forming stable four-stranded structures that were detected by gel electrophoresis and electron microscopy. These structures were recognized specifically by the nuclear nonhistone high mobility group (HMG) proteins 1 and 2 as evidenced by gel retardation. Such sequence-specific complexes might be involved in vivo in recombination or other processes requiring specific association of two double-stranded DNA molecules.
Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved fluorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.
We observed that DNA fragments in room temperature solution undergo low levels of denaturation in the presence of certain types of polypropylene tube surfaces. If the fragments contain (GT)n.(CA)n or (GA)n.(CT)n sequences, multimeric complexes are also formed. This surface activity is inhibited by addition of micromolar concentrations of an oligodeoxyribonucleotide of arbitrary sequence to the tube prior to adding the double-stranded DNA. The reaction was not observed in tubes made of borosilicate glass or in polypropylene-based tubes designed to have low-binding properties. In the case of the DNA fragments that form surfaced-induced multimers, similar complexes can be obtained by denaturation and renaturation of the fragment ("induced" association) without regard to the type of tube surface. However, induced association requires the presence of magnesium ions or polyethylene glycol (or concentration by evaporation) for efficient formation of complexes, whereas surface-dependent dissociation has no such requirements. This difference in buffer requirement suggests that association as well as denaturation takes place on the surface. We suggest models for the formation and structure of these complexes based on surface-dependent denaturation followed by misaligned renaturation of repeated sequences and intermolecular pairing of unpaired regions. This denaturation and complex formation may be important for the interpretation of protein-DNA binding experiments and might be related to hydrophobic interactions of DNA in vivo.
We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA sequencing system to analyze the cleaved fragments. © 1996 Wiley-Liss, Inc.
The enzyme mismatch cleavage (EMC) method relies on the use of the resolvase T4 Endonuclease VII to cleave and thus detect mismatches in heteroduplex DNA formed by annealing normal DNA with mutant DNA. Detection is based on cleavage 3′ to the mismatch within a few nucleotides. We report the detection of all 81 different homozygous single-basepair changes tested and present in the mouse β-globin promoter by using the EMC method with a single set of conditions. Efficiency of cleavage was rated as strong, medium, or weak based on the intensity of the cleavage product(s) compared with background bands on autoradiography. We expect this method to detect near 100% of mutations.
We have estimated the dissociation constant of the terminal base pairs of the B-DNA duplexes formed by 5'-d(CGCGATCGCG) and 5'-d(TAGCGCTA) by two methods, one based on the change in imino proton chemical shift with temperature and the other on the apparent pK shift of the imino proton, as monitored by the change in chemical shift of aromatic protons. These methods do not rely on imino proton exchange, whose rate was also measured. (1) The effect of ammonia on the imino proton exchange rate of the terminal pair of the 5'-d(CGCGATCGCG) duplex is 67 times less than on the isolated nucleoside. This provides an upper limit on the exchange rate from the closed pair. In fact, the effect is just as predicted from the dissociation constant, assuming that there is no exchange at all from the closed pair and that, as has been argued previously, external catalysts act on the open state as they do on the isolated nucleoside. The inhibition of catalyzed proton exchange in the closed pair, despite exposure of one face of the pair to solvent, is a new feature of the exchange process. It will allow determination of the dissociation constant of terminal pairs from the exchange rate. (2) Intrinsic catalysis of proton exchange is less efficient for the terminal pair than for an internal one. A possible explanation is that proton transfer across the water bridge responsible for intrinsic catalysis is slower, as expected if the open-state separation of the bases is larger in a terminal pair. This observation may lead to a direct method for the study of
Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is +1.1(±0.4) kcal mol−1 (at 18 °C, 10 mm-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 °C is +27.1(±1.3) kcal mol−1, while the entropy is +89 (±30) cal K−1 mol−1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent ΔCp is also observed for the formation of one mole of junction, +0.97(±0.05) kcal deg−1mol−1.
The usefulness of any database is dependent on the quality of its content. This is so for mutation databases and has been a continuing concern for those interested in such databases. This article discusses the critical points that determine the quality of the data, such as PCR errors, incomplete scanning, examination of the effect of the variation, and reporting and deposition of the mutations in a database. To illustrate the importance of quality control in mutation curation, nine articles reporting 34 novel phenylketonuria mutations were surveyed for the criteria believed to be important in describing mutations. Many articles were deficient in entries in several criteria, but there was still a high degree of certainty that the described mutations caused disease. Finally, strategies to eliminate errors and to enhance or indicate quality are discussed, including expression, the review process, checking deductions, and typing and encouraging compliance. Models for the future are also discussed.
Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a "tetramer". Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.
The chemical reactivity of matched T and C bases to osmium tetroxide and hydroxylamine near mismatched and unmatched bases
in a heteroduplex between two strands of DNA with multiple differences was examined. Data was available for matched bases
one or two positions away from 24 mismatches. Reactive bases were found near 16 of the mismatches and were usually one or
two bases away. This reactivity is consistant with structural studies indicating perturbation of the duplex around mismatches
and will allow another mode of study of the effect of mismatches. The reactivity of these bases was found not to be strongly
correlated with mismatch type or GC basepair content of the basepairs around the mismatches. Extra reactivity may have been
promoted by the presence of either T or C in the mismatch allowing increased reactivity of nearby T or C. The utility of the
phenomenon for the detection of mutations is discussed. Unmatched bases in the heteroduplex also gives rise to reactive matched
bases nearby.
A mechanism for gene conversion is proposed which overcomes many of the difficulties that any copy choice model encounters. It is suggested that along with general genetic pairing of homologous genomes at meiosis, effective pairing over short regions of the genetic material occurs at the molecular level by the separation of the strands of the DNA double helices, followed by the annealing of strands from two homologous chromatids. If the annealed region happens to span a heterozygous site, mispairing of bases will occur. Such a situation may be analogous to that in DNA which is damaged by mutagens; the same or similar repair mechanisms may operate, and these, by adjusting the base sequences in order to restore normal base pairing, would bring about gene conversion in the absence of any genetic replication. The model indicates how precise breakage and rejoining of chromatids could occur in the vicinity of the conversion, so that conversion would frequently be accompanied by the recombination of outside markers. The model also proposes that the distance between two mutant sites on a fine structure map depends not so much on the frequency of a recombinational event occurring between them, but rather on the degree of inhibition of the processes of genetic pairing by the mutants themselves.
The model will explain almost all the data in a formal way, and it has the advantage over copy choice mechanisms for gene conversion in (1) being compatible with semi-conservative replication of DNA, (2) not invoking DNA synthesis during or after genetic pairing, (3) providing a molecular mechanism for close specific pairing, (4) making it unnecessary to postulate sister strand exchange or a process akin to this, (5) suggesting why rates of gene conversion in opposite directions are sometimes unequal and (6) providing an explanation of the clustering of mutant sites, a basis for map expansion and for the apparently capricious departure of fine structure maps from additivity. Although the model proposed is a general rather than a specific one, it suggests that the process of conversion and intragenic recombination is more complex than is usually believed, since it depends on several interacting factors. Nevertheless, it is hoped that the introduction of a model with this complexity will help to stimulate specific experiments, and that these will provide definitive information which would never be obtained if simpler models of conversion and intragenic recombination were believed to explain the genetic data sufficiently well.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
A novel method for identifying DNA point mutations has been developed by using mismatch repair enzymes. The high specificity of the Escherichia coli MutY protein has permitted the development of a reliable and sensitive method for the detection and characterization of point mutations in the human genome. The MutY protein is involved in a repair pathway that can convert A/G or A/C mismatches to C/G or G/C basepairs, respectively. A/G or A/C mismatches formed by hybridization between two amplified genomic DNA samples or between specific DNA probes and target DNA are nicked at the mispaired adenine strand by MutY protein. As little as 1% of the mutant sequence can be detected by the mismatch repair enzyme cleavage (MREC) method in a mixture of normal and mutated DNAs (e.g., mutant cells are only present in 1% of the normal cell background). By using different probes, the assay also can determine the nucleotide sequence of the mutation. We have applied this method to detect single-base substitutions in human oncogenes.
Four-way DNA junctions are thought to be important intermediates in a number of recombination processes. Resolution of these junctions occurs by cleavage of two strands of DNA to generate two duplex molecules. The interaction between DNA junctions and resolving enzymes appears to be largely structure-specific, reflecting a molecular recognition on a significant scale. We propose a working model for this interaction that takes account of the present state of knowledge of the structure of the DNA junction, and the substrate requirements of the enzymes. We note that three different enzymes introduce cleavages at phosphodiester bonds that are presented on one side of the molecule, suggesting that the enzymes selectively interact with this face of the junction. By forcing a junction of constant sequence to adopt one or other of the two possible antiparallel isomers, we show that the junction is cleaved in such a way as to suggest a constant mode of interaction with the protein that is dependent on structure rather than sequence. We propose that the feature that is recognized is a mutual inclination of two DNA helices at approximately 120 degrees. We show that a number of DNA substrates that contain similar inclined helices, such as a three-way junction, bulged duplexes and a duplex that is curved because of repeated runs of oligoadenine sequences, are each cleaved by phage T4 endonuclease VII. This mode of DNA-protein interaction could be significant in either recombination or DNA repair processes.
Telomeric DNA consists of G- and C-rich strands that are always polarized such that the G-rich strand extends past the 3' end of the duplex to form a 12-16-base overhang. These overhanging strands can self-associate in vitro to form intramolecular structures that have several unusual physical properties and at least one common feature, the presence of non-Watson-Crick G.G base pairs. The term "G-DNA" was coined for this class of structures (Cech, 1988). On the basis of gel electrophoresis, imino proton NMR, and circular dichroism (CD) results, we find that changing the counterions from sodium to potassium (in 20 mM phosphate buffers) specifically induces conformational transitions in the G-rich telomeric DNA from Tetrahymena, d(T2G4)4 (TET4), which results in a change from the intramolecular species to an apparent multistranded structure, accompanied by an increase in the melting temperature of the base pairs of greater than 25 degrees, as monitored by loss of the imino proton NMR signals. NMR semiselective spin-lattice relaxation rate measurements and HPLC size-exclusion chromatography studies show that in 20 mM potassium phosphate (pH 7) buffer (KP) TET4 is approximately twice the length of the form obtained in 20 mM sodium phosphate (pH 7) buffer (NaP) and that mixtures of Na+ and K+ produce mixtures of the two forms whose populations depend on the ratio of the cations. Since K+ and NH4+ are known to stabilize a parallel-stranded quadruplex structure of poly[r(I)4], we infer that the multistranded structure is a quadruplex. Our results indicate that specific differences in ionic interactions can result in a switch in telomeric DNAs between intramolecular hairpin-like or quadruplex-containing species and intermolecular quadruplex structures, all of which involve G.G base pairing interactions. We propose a model in which duplex or hairpin forms of G-DNA are folding intermediates in the formation of either 1-, 2-, or 4-stranded quadruplex structures. In this model monovalent cations stabilize the duplex and quadruplex forms via two distinct mechanisms, counterion condensation and octahedral coordination to the carbonyl groups in stacked planar guanine "quartet" base assemblies. Substituting one of the guanosine residues in each of the repeats of the Tetrahymena sequence to give the human telomeric DNA, d(T2AG3)4, results in less effective K(+)-dependent stabilization. Thus, the ion-dependent stabilization is attenuated by altering the sequence. Upon addition of the Watson-Crick (WC) complementary strand, only the Na(+)-stabilized structure dissociates quickly to form a WC double helix.(ABSTRACT TRUNCATED AT 400 WORDS)
Processes of DNA rearrangement such as recombination or replication frequently have as products different subsets of the limitless
number of distinguishable catenanes or knots. The use of gel electrophoresis and electron microscopy for analysis of these
topological isomers has made it possible to deduce physical and geometric features of DNA structure and reaction mechanisms
that are otherwise experimentally inaccessible. Quantitative as well as qualitative characterization is possible for any pathway
in which the fate of a circular DNA can be followed. The history, theory, and techniques are reviewed and illustrative examples
from recent studies are presented.
The Holliday (four-way) junction is a critical intermediate in homologous genetic recombination. We have studied the structure of a series of four-way junctions, constructed by hybridization of four 80 nucleotide synthetic oligonucleotides. These molecules migrate anomalously slowly in gel electrophoresis. Each arm of any junction could be selectively shortened by cleavage at a unique restriction site, and we have studied the relative gel mobilities of species in which two arms were cleaved. The pattern of fragments observed argues strongly for a structure with two-fold symmetry, based on an X shape, the long arms of which are made from pairwise colinear association of helical arms. The choice of partners is governed by the base sequence at the junction, allowing a potential isomerization between equivalent structural forms. Resolvase enzymes can distinguish between these structures, and the resolution products are determined by the structure adopted, i.e., by the sequence at the junction. In the absence of cations, the helical arms of the junction are fully extended in a square configuration, and unstacking results in junction thymines becoming reactive to osmium tetroxide.
The chemical reactivity of thymine (T), when mismatched with the bases cytosine, guanine, and thymine, and of cytosine (C), when mismatched with thymine, adenine, and cytosine, has been examined. Heteroduplex DNAs containing such mismatched base pairs were first incubated with osmium tetroxide (for T and C mismatches) or hydroxylamine (for C mismatches) and then incubated with piperidine to cleave the DNA at the modified mismatched base. This cleavage was studied with an internally labeled strand containing the mismatched T or C, such that DNA cleavage and thus reactivity could be detected by gel electrophoresis. Cleavage at a total of 13 T and 21 C mismatches isolated (by at least three properly paired bases on both sides) single-base-pair mismatches was identified. All T or C mismatches studied were cleaved. By using end-labeled DNA probes containing T or C single-base-pair mismatches and conditions for limited cleavage, we were able to show that cleavage was at the base predicted by sequence analysis and that mismatches in a length of DNA could be readily detected by such an approach. This procedure may enable detection of all single-base-pair mismatches by use of sense and antisense probes and thus may be used to identify the mutated base and its position in a heteroduplex.
Using as substrates, 1: the replicative form (RF) of phage M13 mp8 in which the reading frame of the lacZ′ gene was disrupted by insertion of an octonucleotide, and 2: a restriction fragment one kb long, containing the functional lacZ′ gene (isolated from wild type M13 mp8), we show that nuclear extracts from human cells (3 lines tested) promote the targeted replacement of the altered sequence by the functional one. Following incubation with the extracts, the DNA's were introduced in JM 109 bacteria (rec A⁻ and lac Z′⁻) which were grown in presence of a colorimetric indicator of β-galactosidase activity. Homologous recombination gives rise to the genotypical modification : lacZ′⁺ instead of lacZ′ in the bacteriophage DNA. This is revealed by phenotypical expression of the lacZ′ gene product in replicating bacteriophage, i. e. the formation of blue instead of white plaques. The frequency of recombination (blue/total plaques) is increased by a factor of 50–80 as a function of protein concentration and of incubation time. The maximal frequency observed is 5×10⁻. There is no increase over the background when extracts are boiled.
Electrophoresis and electron microscopy of DNA's incubated with the extracts show the formation of recombination intermediates with single strand exchange. Restriction analysis of recombined DNA confirms that the process corresponds to targeted sequence exchange. These data allow to propose three steps for homologous recombination between two duplex DNA's : i) unpairing of the two duplexes; ii) single-strand exchange and synaptic pairing; iii) resolution of the cross-junctions. The three steps correspond to those predicted by the gene conversion model of Holliday.
A fibroblast culture was established from a lymph node biopsy of a patient with non-Hodgkin lymphoma, 9 months after chemotherapy and intensive therapeutic x-irradiation of the area. In contrast with blood and bone marrow, which were chromosomally normal, all cells of the lymph node were chromosomally abnormal, with numerous clones having multiple structural abnormalities. Numerical abnormalities (trisomies and monosomies) were not found. Structural abnormalities included translocations, terminal deletions, and pericentric inversions, with an excess of centromeric breakpoints being the only apparent deviation from a random distribution of breakpoints. None of the rearrangements associated with malignant lymphoma were seen, indicating that the chromosome abnormalities in the lymph stroma were radiation-associated, not disease-associated. These acquired changes may be a cause of additional malignant transformation.
Structural properties of DNA oligonucleotides corresponding to the single-stranded molecular terminus of telomeres from several organisms were analyzed. Based on physical studies including nondenaturing polyacrylamide gel electrophoresis, absorbance thermal denaturation analysis, and 1H and 31P nuclear magnetic resonance spectroscopy, we conclude that these molecules can self-associate by forming non-Watson-Crick, guanine.guanine based-paired, intramolecular structures. These structures form below 40 degrees C at moderate ionic strength and neutral pH and behave like hairpin duplexes in nondenaturing polyacrylamide gels. Detailed analysis of the hairpin structure formed by the telomeric sequence from Tetrahymena, (T2G4)4, shows that it is a unique structure stabilized by hydrogen bonds and contains G residues in the syn conformation. We propose that this novel form of DNA is important for telomere function and sets a precedent for the biological relevance of non-Watson-Crick base-paired DNA structures.
As part of a continuing effort to understand evolution on the basis of comparing the base sequences of related coliphage DNA's, the heteroduplex T7M-T3L has been constructed in vitro and directly observed in the electron microscope. The fraction of the heteroduplex observed as double-stranded decreases with an increase in denaturing conditions. This observation indicates that the DNA's of T7 and T3 have extensive sequences of partial homology. There are base changes throughout most of the genome. The terminal redundancies are non-homologous. However, there is little change in the sequence of the structural genes 8, 15, 16 and 17, and in the sequence of the early messenger RNA promoter and termination sites.
T4 endonuclease VII cleaves Holliday structures in vitro by cutting two strands of the same polarity at or near the branch point. The two unbranched duplexes produced by cleavage each contain a strand break that can be sealed by DNA ligase. This suggests that the cut sites are at the same position in the nucleotide sequence in each strand. The joint action of endonuclease VII and DNA ligase can therefore resolve Holliday structures into genetically sensible products. These observations account for the role of endonuclease VII in the DNA metabolism of phage T4, and provide the first example of an enzyme that acts specifically on branch points in duplex DNA.
The purpose of this review is to provide an up-to-date summary of the clinical application of polymerase chain reaction (PCR)-based methodologies.
We have explored the application of the bacteriophage resolvases T4 endonuclease VII and T7 endonuclease I for detecting mutations in genomic DNA. Heteroduplex DNA fragments prepared by amplification from DNA containing known mutations were cleaved by one or both enzymes at nucleotide mismatches created by 3 of 3 short deletions and 13 of 14 point mutations in fragments as large as 940 basepairs. Heteroduplexes representing all four classes of possible single nucleotide mismatches were cleaved, and the sizes of the cleavage products generated correlated with the location of the mutation. We conclude that bacteriophage resolvases may be useful reagents for the rapid screening of DNA for mutations.
An important step in genetic recombination is DNA branch migration, the movement of the Holliday junction or exchange point between two homologous duplex DNAs. We have determined kinetic parameters of spontaneous branch migration as a function of temperature and ionic conditions. The branch migration substrates consist of two homologous duplex DNAs each having two single-strand tails at one end that are complementary to the corresponding single-strand tails of the other duplex. Upon rapid annealing of the two duplex DNAs, a four-stranded intermediate is formed that has a Holliday junction at one end of the duplexes. Branch migration to the opposite end of the duplexes results in complete strand exchange and formation of two duplex products. The rate of branch migration is exceedingly sensitive to the type of metal ions present. In magnesium, branch migration is quite slow with a step time, tau, equal to 300 msec at 37 degrees C. Surprisingly, branch migration in the absence of magnesium was 1000 times faster. Despite this difference in rates, apparent activation energies for the branch migration step in the presence and absence of magnesium are similar. Since metal ions have a profound effect on the structure of the Holliday junction, it appears that the structure of the branch point plays a key role in determining the rate of spontaneous DNA branch migration. We discuss the role of proteins in promoting the branch migration step during homologous recombination.
In an accompanying paper we reported the use of differential scanning calorimetry and optical densitometry to characterize the melting and aggregation of 160 bp fragments of calf thymus DNA during heating in the presence of divalent metal cations. Aggregation is observed as thermal denaturation begins and becomes more extensive with increasing temperature until the melting temperature Tm is reached, after which the aggregates dissolve extensively. The order of effectiveness of the metals in inducing aggregation is generally consistent with their ability to induce melting: Cd > Ni > Co > Mn approximately Ca > Mg. Under our experimental conditions (50 mg/ml DNA, 100 mM MCl2, [metal]/[DNA phosphate] approximately 0.6), no measurable aggregates were observed for BaDNA or SrDNA. In this paper we show that the Shibata-Schurr theory of aggregation in the thermal denaturation region provides a good model for our observations. Free energies of cross-linking, induced by the divalent cations, are estimated to be between 34% and 38% of the free energies of base stacking. The ability of a divalent metal cation to induce DNA aggregation can be attributed to its ability to disrupt DNA base pairing and simultaneously to link two different DNA sites.
A complete mutational scan of the gene coding for the serpin C1 inhibitor, comprising all eight exons and adjacent intron sequences and 550 bp preceding the transcription start site, was rapidly accomplished in 36 unrelated angioedema patients by using fluorescence-assisted mismatch analysis (FAMA). Mutations accounting for C1 inhibitor deficiency were identified in every one of 34 patients, with two failures turning out to be spurious cases. Two new substitution dimorphisms were also detected in introns. Changes affecting the C1 inhibitor protein, distributed throughout the seven coding exons, provide new insights into the molecular pathology of serpins. Six different splice-site and two promoter mutations were also found. Among the latter, a C-->T transition within one of two putative CAAT boxes of this TATA-less promoter, the sole idiomorphic nucleotide change in this kindred, was found homozygous in the proband, at variance with the dominant mode of transmission observed for structural mutations. FAMA, in the chemical probes configuration used in this study, is a rapid and robust mutation-scanning procedure, applicable to large DNA segments or transcripts and proved capable of 100% detection. Moreover, it provides accurate positional information--and hence recognition of multiple substitutions, precise relationship with those already known, and often immediate identification of the nucleotide change.
We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA sequencing system to analyze the cleaved fragments.
In the absence of added metal ions, the four-way junction is extended with an open central region (3, 4). However, upon addition of magnesium or other metal ions the junction folds by pairwise coaxial stacking of helices into the stacked X-structure (reviewed in refs. 5 and 6) (Fig. 1). This structure is a substrate for a variety of junction-selective enzymes, most of which alter the global structure on binding (reviewed in ref. 7). In free solution, the DNA junction folds to create an antiparallel structure, probably governed by the favorable juxtaposition of backbones and grooves when the small angle is approximately 60°. The lowering of symmetry on formation of the stacked X-structure creates two inequivalent kinds of strands; the two continuous strands turn about the pseudo-continuous axes that pass through the stacked helical pairs, while the exchanging strands pass between the stacked helices at the crossover. The general features of the stacked X-structure have been confirmed by a variety of experimental methods (3, 8–11 …
Several features indicate that the low polymorphic human minisatellite MsH42 region could be involved in recombination. It contains different well-known recombination motifs, is able to generate single-stranded loops and is specifically recognized by nuclear proteins. These characteristics led us to investigate the possible recombinogenic activity of the MsH42 region in terms of intramolecular recombination. We constructed two plasmids, one of them carrying two copies of the minisatellite region and the other one containing sequences upstream of this repetitive region. We showed that MsH42 strongly stimulates intramolecular in vitro recombination, approximately 22 times more than the control sequence, solely when the source of biological extract is mouse testes, suggesting that MsH42 could be a hotspot involved in meiotic recombination. Furthermore, there is a direct relationship between the frequency of equal crossovers and the enhancement of recombination. Interestingly, the third repeat of the minisatellite array is always involved in the resolution of unequal crossovers leading to minisatellite shortening. As far as we know, our results provide the first evidence that a non-hypervariable minisatellite can enhance homologous recombination.
Detection of mutations in genes is vital throughout biology, however, this activity is time-consuming, expensive and requires a high degree of skill. This is unsatisfactory in a field which is increasing importance. Around 10-12 methods are commonly used with some predominating. All have their advantages and disadvantages and none is perfect. Sequencing is said to be the gold standard for detecting new mutations (and must be used to define it), but six or so methods have been described to make the search quicker to avoid sequencing the whole gene. These are called scanning methods. Other methods are used to detect known mutations and referred to as diagnostic methods. These methods will be briefly reviewed. Once mutations are described, they are usually published. The mutation lists are collected for convenience, analysis or research. In recent years, it has been realised that these lists are vital for research, patient care and commercial activities. These activities will be reviewed and the co-ordinating role of the HUGO Mutation Database Initiative will be outlined.
Many mutation detection techniques rely upon recognition of mismatched base pairs in DNA heteroduplexes. Potassium permanganate
in combination with tetraethylammonium chloride (TEAC) is capable of chemically modifying mismatched thymidine residues. The
DNA strand can then be cleaved at that point by treatment with piperidine. The reactivity of potassium permanganate (KMnO4) in TEAC toward mismatches was investigated in 29 different mutations, representing 58 mismatched base pairs and 116 mismatched
bases. All mismatched thymidine residues were modified by KMnO4/TEAC with the majority of these showing strong reactivity. KMnO4/TEAC was also able to modify many mismatched guanosine and cytidine residues, as well as matched guanosine, cytidine and
thymidine residues adjacent to, or nearby, mismatched base pairs. Previous techniques using osmium tetroxide (OsO4) to modify mismatched thymidine residues have been limited by the apparent lack of reactivity of a third of all T/G mismatches.
KMnO4/TEAC showed no such phenomenon. In this series, all 29 mutations were detected by KMnO4/TEAC treatment. The latest development of the Single Tube Chemical Cleavage of Mismatch Method detects both thymidine and
cytidine mismatches by KMnO4/TEAC and hydroxylamine (NH2OH) in a single tube without a clean-up step in between the two reactions. This technique saves time and material without
disrupting the sensitivity and efficiency of either reaction.