Overcoming a barrier for DNA polymerization in triplex-forming sequences.

Folded structures in the DNA template, such as hairpins and multi-stranded structures, often serve as pause and arrest sites for DNA polymerases. DNA polymerization is particularly difficult on mirror-repeated homopurine.homopyrimidine templates where triple-stranded (triplex) structures may form between the nascent and folded template strands. In order to use a linear PCR amplification approach for the structural analysis of DNA in mirror-repeated sequences we modified a conventional protocol. The barrier for DNA synthesis can be eliminated using an oligonucleotide that hybridizes with the template to prevent its folding and is subsequently displaced by the progressing polymerase. The described approach is potentially useful for sequencing and analysis of chemical adducts and point mutations in a variety of sequences prone to the formation of folded structures, such as long hairpins and quadruplexes.     

Unusual Protonated Structure in the Homopurine · Homopyrimidine Tract of Supercoiled and Linearized Plasmids Recognized by Chemical Probes

Abstract

Plasmid pEJ4, which is a derivative of pUC19 containing an insert with 60-bp-long · homopurine homopyrimidine tract from sea urchin P. miliaris histone gene spacer, was studied by chemical probes of the DNA structure osmium tetroxide and glyoxal. The former probe reacts with pyrimidine bases, while the latter forms a stable product only with guanine residues. These probes can thus be applied as specific probes for the homopyrimidine and homopurine strands, respectively.

At pH 6.0 the site-specific modification of the homopurine · homopyrimidine tract by both probes was observed at native superhelical density of the plasmid. In the linear plasmid under the same conditions this modification was absent; it appeared, however, at more acid pH values. In supercoiled DNA the hypersensitivity of the homopurine homopyrimidine tract to osmium tetroxide did not substantially change when pH was decreased from 6.0 to 4.0. Changes in NaCl concentration at pH 4.5 did not influence the hypersensitivity to osmium tetroxide; at pH 6.0 this hypersensitivity decreased with increasing NaCl concentration. These results thus show that the chemical probes recognize an unusual protonated structure containing unpaired bases or non-Watson-Crick base pairs. At pH 5.6 the site-specific modification occurred at or near to the middle of the homopurine · homopyrimidine tract, suggesting that a hairpin may be involved in the unusual structure under the given conditions. From the models suggested so far for the unusual structure of homopurine · homopyrimidine tracts our results fit best the protonated triplex H form suggest by V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3, 667 (1986).     

Chemical probing of the homopurine.homopyrimidine tract in supercoiled DNA at single-nucleotide resolution

Local structure of the homopurine.homopyrimidine tract in a supercoiled plasmid pEJ4 was studied using chemical probes at single-nucleotide resolution. The conformation of the homopyrimidine strand was probed by osmium tetroxide, pyridine (Os,py) while that of the homopurine strand was tested by diethyl pyrocarbonate (DEPC), i.e. by probes reacting preferentially with single-stranded DNA. At weakly acidic pH values, a strong Os,py attack on three nucleotides at the centre of the (dC-dT)16 block and a weaker attack on two nucleotides at the end of the block were observed. DEPC modified adenines in the 5'-half of the homopurine strand. Os,py modification at the centre of the block corresponded to the loop of the hairpin formed by the homopyrimidine tract, while DEPC modification corresponded to the unstructured half of the homopurine strand in the model of protonated triplex H form of DNA.     

PKD1 intron 21: triplex DNA formation and effect on replication

Although autosomal dominant polycystic kidney disease is transmitted in an autosomal dominant fashion, there is evidence that the pathophysiology of cystogenesis involves a second hit somatic mutation superimposed upon the inherited germline mutation within the renal tubule cells. The polypurine·polypyrimidine (Pu·Py) tract of PKD1 intron 21 may play a role in promoting somatic mutations. To better characterize this tract and to evaluate its potential to participate in mutagenesis, we investigated the thermodynamics of intramolecular triplex formation by 15 Pu·Py mirror repeat tracts from PKD1 intron 21 by 2D gel electrophoresis. We demonstrate that intramolecular triplexes form with modest superhelical tensions for all the tracts examined. Primer extension studies demonstrated significant polymerase arrest within the Pu·Py tracts in one direction of replication only. We found correlation between polymerization arrest and both the potential length of the triplex and superhelical tension of intramolecular triplex formation. The presence of a Pu·Py tract also led to a replication blockade and double-strand breakage using an SV40 in vitro replication assay with HeLa cell extracts. During DNA replication, the G-rich template of the PKD1 Pu·Py tracts may form a triplex structure with the nascent strand, thereby blocking replication and potentially leading to recombination and mutation.     

Unusual protonated structure in the homopurine.homopyrimidine tract of supercoiled and linearized plasmids recognized by chemical probes

Plasmid pEJ4, which is a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from sea urchin P. miliaris histone gene spacer, was studied by chemical probes of the DNA structure osmium tetroxide and glyoxal. The former probe reacts with pyrimidine bases, while the latter forms a stable product only with guanine residues. These probes can thus be applied as specific probes for the homopyrimidine and homopurine strands, respectively. At pH 6.0 the site-specific modification of the homopurine.homopyrimidine tract by both probes was observed at native superhelical density of the plasmid. In the linear plasmid under the same conditions this modification was absent; it appeared, however, at more acid pH values. In supercoiled DNA the hypersensitivity of the homopurine.homopyrimidine tract to osmium tetroxide did not substantially change when pH was decreased from 6.0 to 4.0. Changes in NaCl concentration at pH 4.5 did not influence the hypersensitivity to osmium tetroxide; at pH 6.0 this hypersensitivity decreased with increasing NaCl concentration. These results thus show that the chemical probes recognize an unusual protonated structure containing unpaired bases or non-Watson-Crick base pairs. At pH 5.6 the site-specific modification occurred at or near to the middle of the homopurine.homopyrimidine tract, suggesting that a hairpin may be involved in the unusual structure under the given conditions. From the models suggested so far for the unusual structure of homopurine.homopyrimidine tracts our results fit best the protonated triplex H form suggest by V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3,667 (1986).     

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from –44 to –79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.     

Factors influencing the extent and selectivity of alkylation within triplexes by reactive G/A motif oligonucleotides.

G/A motif triplex-forming oligonucleotides (TFOs) complementary to a 21 base pair homopurine/homopyrimidine run were conjugated at one or both ends to chlorambucil. These TFOs were incubated with several synthetic duplexes containing the targeted homopurine run flanked by different sequences. The extent of mono and interstrand cross-linking was compared with the level of binding at equilibrium. Covalent modification took place within a triple-stranded complex and usually occurred at guanine residues in the flanking double-stranded DNA. The efficiency of alkylation was dependent upon the sequence of the flanking duplex, the solution conditions, and the rate of triplex formation relative to the rate of chlorambucil reaction. Self-association of the TFOs as parallel duplexes was demonstrated and this did not interfere with triple strand formation. With an optimal target, cross-linking of the triplex was very efficient when incubation was carried in a physiological buffer supplemented with the triplex selective intercalator coralyne.     

Pausing of simian virus 40 DNA replication fork movement in vivo by (dG-dA)n·(dT-dC)n tracts

We have earlier demonstrated that a sequence bordering an amplified DNA segment and containing the unusual sequence (dG-dA)_n·(dT-dC)_n could slow replication fork movement [Rao et al., Nucleic Acids Res. 16 (1988) 8077–8094]. This was done by cloning the unusual sequence in simian virus 40 (SV40) and following the rate of incorporation of radioactively labeled nucleotides into various regions of the SV40 genome. In the present study, we have analyzed the in vivo replicative intermediates of the SV40 variants containing the unusual sequences by a two-dimensional gel electrophoretic technique. We found that the technique can be used to detect minor pauses in DNA replication and demonstrated that the cloned (dG-dA)_n·(dT-dC)_n tracts, that can potentially adopt triplex structures, could slow DNA replication fork movement. A sequence from the plasmid pUC18 did not slow fork movement when cloned in the same locus of SV40. The pause caused by the alternating guanosine-adenosine repeats might play a role in the regulation of DNA replication and gene amplification in vivo.     

Electron Microscopy of Supercoiled pEJ4 DNA Containing Homopurine · Homopyrimidine Sequences

Abstract

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine · homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine · homopyridine tract assumes an unusual conformation) a single thick “stem” or a “denaturation bubble” in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC 19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The “denaturation bubbles” were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only “stems”. The “stems” were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine · homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.     

pZ189质粒DNA体外复制系统的建立

报道了含SV40复制起点的质粒DNA在真核细胞抽提物中进行复制的DNA体外复制系统的建立. 在外源性蛋白质SV40大T抗原(SV40 Tag)的参与下,穿梭质粒pZ189能在猴肾vero细胞胞浆抽提物中,利用其中参与体内DNA复制所需的蛋白质成分,有效地进行体外DNA复制. 从而为研究真核细胞DNA复制系统的结构与功能提供了简单、有效的模型.     

Noncanonical DNA elements in the lamin B2 origin of DNA replication

DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT)(n) (dA)(n) sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/GGAAAAAAAAAAG (3900-3912) and CCTTTTTTTTC/GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mm Tris base, 89 mm boric acid, and 2 mm MgCl(2)) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT)(40) do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads.     

Structural diversity of target-specific homopyrimidine peptide nucleic acid-dsDNA complexes

Sequence-selective recognition of double-stranded (ds) DNA by homopyrimidine peptide nucleic acid (PNA) oligomers can occur by major groove triplex binding or by helix invasion via triplex P-loop formation. We have compared the binding of a decamer, a dodecamer and a pentadecamer thymine–cytosine homopyrimidine PNA oligomer to a sequence complementary homopurine target in duplex DNA using gel-shift and chemical probing analyses. We find that all three PNAs form stable triplex invasion complexes, and also conventional triplexes with the dsDNA target. Triplexes form with much faster kinetics than invasion complexes and prevail at lower PNA concentrations and at shorter incubation times. Furthermore, increasing the ionic strength strongly favour triplex formation over invasion as the latter is severely inhibited by cations. Whereas a single triplex invasion complex is formed with the decameric PNA, two structurally different target-specific invasion complexes were characterized for the dodecameric PNA and more than five for the pentadecameric PNA. Finally, it is shown that isolated triplex complexes can be converted to specific invasion complexes without dissociation of the Hoogsteen base-paired triplex PNA. These result demonstrate a clear example of a ‘triplex first’ mechanism for PNA helix invasion.     

Purification and functional characterization of bovine RP-A in an<Emphasis Type="Italic">in vitro</Emphasis> SV40 DNA replication system

The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replicationin vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase α-primase complex.     

Suicidal nucleotide sequences for DNA polymerization.

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex-forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double-helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple-helical stretches within single-stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self-termination. Possible biological implications of such 'conformational suicide' are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex-forming oligonucleotides.     

Extension of the range of recognition sequences for triple helix formation by oligonucleotides containing guanines and thymines.

Oligodeoxynucleotides containing G and T can bind to homopurine.homopyrimidine sequences on double-stranded DNA by forming C.G x G and T.A x T base triplets. The orientation of the third strand in such triple helices depends on the number of GpT and TpG steps. Therefore a single oligonucleotide can be designed to bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine stretches alternate on the two strands of DNA. The oligonucleotide switches from one homopurine strand to the other at the junction between the two sequences. This result shows that it is possible to extend the range of DNA sequences that can be recognized by a single oligonucleotide.