[Expansion of trinucleotide repeats]. / Ekspansiia trinukleotidnykh povtorov.

This review describes a novel type of genome instability, expansion of trinucleotide repeats. Originally discovered in 1991 upon cloning the gene responsible for the fragile X syndrome, it appeared to be a general phenomenon responsible for a growing number of human neurological disorders. Besides apparent medical importance, the discovery of trinucleotide repeat expansion unraveled a fundamental problem of human genetics: a non-Mendelian type of inheritance called anticipation. Understanding the mechanisms of repeat expansion and the molecular pathways leading from these expansions to human diseases became a formidable task for modern biology and one of its spectacular achievements. Here we discuss the major breakthroughs in this field made during the last decade with an emphasis on molecular models of repeat expansion.

A new trick for an old dog: TraY binding to a homopurine-homopyrimidine run attenuates DNA replication.

The effects of the d(GA)(n).d(TC)(n) repeat on plasmid replication in Escherichia coli cells were analyzed using electrophoretic analysis of replication intermediates. This repeat appeared to stall the replication fork progression in E. coli strains carrying F' episomes. The potency of replication stalling increased with the repeat's length but did not depend on its orientation relative to the replication origin, or transcription through the repeat. Treatment of E. coli cells with the protein synthesis inhibitor chloramphenicol abolished replication blockage, indicating that protein binding might be responsible for the repeat-caused replication blockage. Concordantly, dimethylsulfate footprinting in vivo revealed methylation protection of all guanine residues within the d(GA)(n).d(TC)(n). Gel retardation assays with crude cell extracts confirmed the presence of a d(GA)(n).d(TC)(n) -binding activity in F', but not F(-), strains. Further, strains cured from the F' episome lost this activity, while F(-) strains that acquired the F' factor via conjugation, acquired d(GA)(n).d(TC)(n)-binding activity as well. Thus, this d(GA)(n).d(TC)(n)-binding protein is encoded by the F' factor. Purification of this protein by affinity chromatography revealed a single polypeptide with an apparent molecular mass of 15.2 kDa. Microsequencing of its two tryptic peptides revealed two perfect matches with the TraY protein, which is encoded by the F factor. Overexpression of an individual TraY protein in the F(-) E. coli strain conveyed d(GA)(n).d(TC)(n)-binding activity in vitro and replication stalling at d(GA)(n).d(TC)(n) repeats in vivo. We conclude that TraY binding to a homopurine-homopyrimidine repeat is responsible for stalling DNA replication. Biological applications of this phenomenon are discussed.

Expansion of the (CTG)(n) repeat in the 5'-UTR of a reporter gene impedes translation.

Effects of d(CAG)(n).d(CTG)(n) repeats on expression of a reporter gene in human cell culture were studied using transient transfection, RNase protection and coupled transcription/translation assays. Cloning these repeats into the reporter 3'-UTR did not affect gene functioning. In contrast, placing the repeats in the reporter 5'-UTR led to strong inhibition of expression. This inhibition depended on the repeat orientation, being prominent only when the (CTG)(n) tracts were in the sense strand for transcription. Further, the strength of inhibition increased exponentially with an increase in repeat length. Our data indicate that expanded (CTG)(n) repeats prevent efficient translation of the reporter mRNA both in vitro and in vivo. We suggest that formation of stable hairpins by (CUG)(n) runs of increasing length in the 5'-UTR of a mRNA progressively inhibits the scanning step of translation initiation. This points to a novel mechanism of regulating gene expression by expandable d(CTG)(n) repeats.

Large-scale effects of transcriptional DNA supercoiling in vivo.

The scale of negative DNA supercoiling generated by transcription in Top(+) Escherichia coli cells was assessed from the efficiency of cruciform formation upstream of a regulated promoter. An increase in negative supercoiling upon promoter induction led to cruciform formation, which was quantitatively measured by chemical probing of intracellular DNA. By placing a cruciform-forming sequence at varying distances from the promoter, we found that the half-dissociation length of transcription supercoiling wave is approximately 800 bp. This is the first proof that transcription can affect DNA structure on such a remarkably large scale in vivo. Moreover, cooperative binding of the cI repressor to the upstream promoter DNA did not preclude efficient diffusion of transcriptional supercoiling. Finally, our plasmids appeared to contain discrete domains of DNA supercoiling, defined by the features and relative orientation of different promoters.

Transcription through a simple DNA repeat blocks replication elongation.

The influence of d(G)n.d(C)n repeats on plasmid replication in Escherichia coli cells was analyzed using electrophoretic analysis of replication intermediates. These repeats impeded the replication fork in a length- and orientation-dependent manner. Unexpectedly, the replication arrest relied primarily on the repeats' transcription. When the d(C)n sequence served as the transcriptional template, both transcription and replication were blocked. This was true for transcription driven by either bacterial or phage RNA polymerases. We hypothesize that the replication fork halts after it encounters a stalled ternary complex of the RNA polymerase, the DNA template and the r(G)n transcript. This constitutes a novel mechanism for the regulation of replication elongation. The effects of this mechanism on repeat length polymorphism and genome rearrangements are discussed.

Trinucleotide repeats affect DNA replication in vivo.

(CGG)n.(CCG)n and (CTG)n.(CAG)n repeats of varying length were cloned into a bacterial plasmid, and the progression of the replication fork through these repeats was followed using electrophoretic analysis of replication intermediates. We observed stalling of the replication fork within repeated DNAs and found that this effect depends on repeat length, repeat orientation relative to the replication origin and the status of protein synthesis in a cell. Interruptions within repeated DNAs, similar to those observed in human genes, abolished the replication blockage. Our results suggest that the formation of unusual DNA structures by trinucleotide repeats in the lagging-strand template may account for the observed replication blockage and have relevance to repeat expansion in humans.

Characteristic enrichment of DNA repeats in different genomes.

Using computer programs developed for this purpose, we searched for various repeated sequences including inverted, direct tandem, and homopurine-homopyrimidine mirror repeats in various prokaryotes, eukaryotes, and an archaebacterium. Comparison of observed frequencies with expectations revealed that in bacterial genomes and organelles the frequency of different repeats is either random or enriched for inverted and/or direct tandem repeats. By contrast, in all eukaryotic genomes studied, we observed an overrepresentation of all repeats, especially homopurine-homopyrimidine mirror repeats. Analysis of the genomic distribution of all abundant repeats showed that they are virtually excluded from coding sequences. Unexpectedly, the frequencies of abundant repeats normalized for their expectations were almost perfect exponential functions of their size, and for a given repeat this function was indistinguishable between different genomes.

Mechanisms of triplex-caused polymerization arrest.

Pyrimidine/purine/purine triplexes are known to inhibit DNA polymerization. Here we have studied the mechanisms of this inhibition by comparing the efficiency of Vent DNA polymerase on triplex- and duplex-containing templates at different temperatures, Mg2+concentrations and time intervals with the thermal stability of the corresponding structures. Our results show that triplexes can only be by-passed at temperatures where thermal denaturation initiates, while duplexes, in contrast, are overcome at temperatures where they are quite stable. These results show that DNA polymerase cannot untangle triplex regions within DNA templates and seems to entirely depend on their thermal fluctuations. The high stability of triplexes at physiological temperatures and ambient conditions make them a barrier to polymerization.

Triplex DNA structures.

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.

Trapping DNA polymerases using triplex-forming oligodeoxyribonucleotides.

Triplexes (triple helices) formed within DNA templates prior to or during DNA synthesis cause DNA polymerase to terminate [Samadashwily et al., EMBO J. 13 (1993) 4975-4983]. Here, we show that triplex-forming oligodeoxyribonucleotides (oligos) efficiently trap DNA polymerases at target DNA sequences within single-stranded (ss) templates. This was observed for all studied DNA polymerases, including Sequenase and the thermophilic Taq and Vent polymerases. The termination rate depends on the fine structure of a triplex, as well as on ambient conditions such as temperature and the concentration of magnesium ions. Inhibition of DNA synthesis was observed not only when triplexes blocked the path of DNA polymerase, but also when a polymerization primer was involved in triplex formation. Escherichia coli ss-binding (SSB) protein helps DNA polymerase overcome the triplex barrier, but with an efficiency dramatically dependent on the triplex configuration. These results describe a novel method for blocking DNA replication at target homopurine-homopyrimidine sequences by means of triplex-forming oligos in direct analogy with similar results during transcription.

Transcriptional activity of the homopurine-homopyrimidine repeat of the c-Ki-ras promoter is independent of its H-forming potential.

The mouse c-Ki-ras protooncogene promoter contains an unusual DNA element consisting of a 27 bp-long homopurine-homopyrimidine mirror repeat (H-motif) adjacent to a d(C-G)5 repeat. We have previously shown that in vitro these repeats may adopt H and Z conformations, respectively, causing nuclease and chemical hypersensitivity. Here we have studied the functional role of these DNA stretches using fine deletion analysis of the promoter and a transient transcription assay in vivo. We found that while the H-motif is responsible for approximately half of the promoter activity in both mouse and human cell lines, the Z-forming sequence exhibits little, if any, such activity. Mutational changes introduced within the homopurine-homopyrimidine stretch showed that its sequence integrity, rather than its H-forming potential, is responsible for its effect on transcription. Electrophoretic mobility shift assays revealed that the putative H-motif tightly binds several nuclear proteins, one of which is likely to be transcription factor Sp1, as determined by competition experiments. Southwestern hybridization studies detected two major proteins specifically binding to the H-motif: a 97 kD protein which presumably corresponds to Sp1 and another protein of 60 kD in human and 64 kD in mouse cells. We conclude that the homopurine-homopyrimidine stretch is required for full transcriptional activity of the c-Ki-ras promoter and at least two distinct factors, Sp1 and an unidentified protein, potentially contribute to the positive effect on transcription.

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.

Intramolecular DNA triplexes: unusual sequence requirements and influence on DNA polymerization.

Homopurine-homopyrimidine mirror repeats are known to form intramolecular DNA triplexes in vitro. By probing with chemical agents specific for unusual DNA conformations, we have now demonstrated the formation of intramolecular triplexes consisting of G.G.C and T.A.T base triplets by DNA sequences that are neither homopurine-homopyrimidine nor mirror repeats. This finding significantly enlarges the number of sequences that could form DNA triplexes. The observed triplexes are stable under the conditions that are optimal for DNA polymerases in vitro. We found that triplex formation causes specific termination of DNA polymerization in vitro. This effect is detected for different DNA polymerases and may have implications for the regulation of DNA replication in vivo.

Transcriptionally driven cruciform formation in vivo.

We studied the formation of d(A-T)n cruciforms in E.coli cells by probing intracellular plasmid DNA with chloroacetaldehyde followed by fine analysis of modified DNA bases. d(A-T)16 sequences were inserted into specifically designed plasmids either upstream of a single trc promoter, or between two divergent trc promoters. We found that in both cases, induction of transcription by IPTG leads to the transition of the d(A-T)16 stretch into a cruciform state. In the case of two divergent promoters, we observed cruciform formation even without IPTG. Enhanced cruciform formation correlates with the elevation in promoter activity as defined by the opening of the promoter at the -10 to +2 positions. We conclude that transcriptionally driven negative supercoiling provokes cruciform formation in vivo.

H-DNA and Z-DNA in the mouse c-Ki-ras promoter.

The mouse c-Ki-ras protooncogene promoter contains a homopurine-homopyrimidine domain that exhibits S1 nuclease sensitivity in vitro. We have studied the structure of this DNA region in a supercoiled state using a number of chemical probes for non-B DNA conformations including diethyl pyrocarbonate, osmium tetroxide, chloroacetaldehyde, and dimethyl sulfate. The results demonstrate that two types of unusual DNA structures formed under different environmental conditions. A 27-bp homopurine-homopyrimidine mirror repeat adopts a triple-helical H-DNA conformation under mildly acidic conditions. This H-DNA seems to account for the S1 hypersensitivity of the promoter in vitro, since the observed pattern of S1 hypersensitivity at a single base level fits well with the H-DNA formation. Under conditions of neutral pH we have detected Z-DNA created by a (CG)5-stretch, located adjacent to the homopurine-homopyrimidine mirror repeat. The ability of the promoter DNA segment to form non-B structures has implications for models of gene regulation.

[Primary structure of two natural IS1-flanking transposons Tn9* and Tn9' coding for stability to chloramphenicol]. / Pervichnaia struktura dvukh prirodnykh IS1-flankirovannykh transposonov Tn9* i Tn9', kodiruiushchikh ustoichivost' k khloramfenicolu.

The DNA nucleotide sequence from the central region of the composite transposons Tn9* and Tn9' at the junction with the right copy of IS1 was determined. From the data obtained it follows that both transposons are members of the Tn9 family, although they contain additional DNA segments with regard to Tn9 of the length about 320 and 290 b.p. respectively lying distal to the cat gene. It was proposed that all the transposons of the Tn9 family have been formed as a result of IS1-mediated deletions of the plasmid R100 r-determinant. It was revealed from the data of computer analysis that in the sequenced DNA there are two potential promotors with transcription directed opposite in relation to the transcription of the cat gene.

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.

[Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression]. / Vstraivanie (dA-dT)n-blokov v reguliatornuiu oblast' gena pho5 narushaet ego ékspressiiu.

We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression. These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's). A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured. We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state. (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal.