SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.     

Characterization of a porcine variable number tandem repeat sequence specific for the glucosephosphate isomerase locus

A variable number of tandem repeat from a porcine glucosephosphate isomerase intron has been isolated and sequenced. The repeat has a unit size of 39 bp, is highly conserved and is present in at least 14 copies. Flanking sequences show a sequence periodicity of 53-54 bp and some sequence homology to the 39 bp repeat. A considerable part of the genomic DNA has been lost during subcloning and is considered to be deletion prone or refractory to propagation in E. coli. The tandem repeat is locus specific and detects at least six alleles in BamHI digested porcine DNA. No homology to other tandem repeat sequences has been found.     

Non-B DNA conformations formed by long repeating tracts of myotonic dystrophy type 1, myotonic dystrophy type 2, and Friedreich's ataxia genes, not the sequences per se, promote mutagenesis in flanking regions

The expansions of long repeating tracts of CTG.CAG, CCTG.CAGG, and GAA.TTC are integral to the etiology of myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), and Friedreich's ataxia (FRDA). Essentially all studies on the molecular mechanisms of this expansion process invoke an important role for non-B DNA conformations which may be adopted by these repeat sequences. We have directly evaluated the role(s) of the repeating sequences per se, or of the non-B DNA conformations formed by these sequences, in the mutagenic process. Studies in Escherichia coli and three types of mammalian (COS-7, CV-1, and HEK-293) fibroblast-like cells revealed that conditions which promoted the formation of the non-B DNA structures enhanced the genetic instabilities, both within the repeat sequences and in the flanking sequences of up to approximately 4 kbp. The three strategies utilized included: the in vivo modulation of global negative supercoil density using topA and gyrB mutant E. coli strains; the in vivo cleavage of hairpin loops, which are an obligate consequence of slipped-strand structures, cruciforms, and intramolecular triplexes, by inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating sequence inserts that do, and do not, adopt non-B DNA structures in vitro. Hence, non-B DNA conformations are critical for these mutagenesis mechanisms.     

Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.     

Color-coding reveals tandem repeats in the Escherichia coli genome

Genomic DNA contains a wide variety of repetitive sequences. In Escherichia coli, there have been several classes of repetitive sequences reported, some of which cluster as tandem repeats. We propose a novel method for analyzing symbolic sequences by two-dimensional pattern formation with color-coding. We applied this method for searching tandem repeats in the E. coli genome and found approximately 50 repeats with periods longer than 30 bases. The longest repeat has a period of 1267 bases.     

Advances in the application of germline tandem repeat instability for in situ monitoring

Alterations in tandem repetitive DNA sequences such as minisatellite DNA and expanded simple tandem repeats (ESTRs) may provide useful biomarkers of induced germline effects. In this review, I describe the differences between ESTRs and minisatellites with respect to their structure and mutational mechanisms, and discuss field applications measuring induced germline instability. It is evident that both types of loci have high rates of mutation that facilitate the measurement of induced mutation measured in relatively small numbers of samples following environmentally relevant exposures. Several research groups have used these loci to demonstrate a significant increase in germline mutation in humans and animals exposed to radioactive or chemical pollutants in their natural environment. Mutations are manifested as gains or losses in repeat units and are detected either by pedigree screening or by PCR amplification of sperm DNA. Mutations at both ESTRs and minisatellites appear to arise via indirect mechanisms rather than by direct damage to the repeat locus itself. Most interestingly, ESTR instability following radiation has been shown to be heritable and transmitted to subsequent generations. An understanding of the mechanisms involved in induced instability is required in order to begin to decipher the potential biological implications of increased germline tandem repeat mutation. Furthermore, relatively few studies have investigated the ability of different genotoxins to induce tandem repeat instability. Such laboratory-based experiments will be crucial in clarifying the particular environmental or occupational exposures that should be targeted for future studies and for isolating and subsequently identifying the putative mutagens in complex environmental matrices.     

Repeat instability during DNA repair: Insights from model systems

Abstract

The expansion of repeated sequences is the cause of over 30 inherited genetic diseases, including Huntington disease, myotonic dystrophy (types 1 and 2), fragile X syndrome, many spinocerebellar ataxias, and some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions are dynamic, and disease inheritance and progression are influenced by the size and the rate of expansion. Thus, an understanding of the various cellular mechanisms that cooperate to control or promote repeat expansions is of interest to human health. In addition, the study of repeat expansion and contraction mechanisms has provided insight into how repair pathways operate in the context of structure-forming DNA, as well as insights into non-canonical roles for repair proteins. Here we review the mechanisms of repeat instability, with a special emphasis on the knowledge gained from the various model systems that have been developed to study this topic. We cover the repair pathways and proteins that operate to maintain genome stability, or in some cases cause instability, and the cross-talk and interactions between them.     

Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences

The influence of negative superhelical density on the genetic instabilities of long GAA.TTC, CGG.CCG, and CTG.CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (-sigma). All three strategies revealed that higher -sigma in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of -sigma stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slippage of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the -sigma of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases.     

Non-B DNA structure-induced genetic instability

Repetitive DNA sequences are abundant in eukaryotic genomes, and many of these sequences have the potential to adopt non-B DNA conformations. Genes harboring non-B DNA structure-forming sequences increase the risk of genetic instability and thus are associated with human diseases. In this review, we discuss putative mechanisms responsible for genetic instability events occurring at these non-B DNA structures, with a focus on hairpins, left-handed Z-DNA, and intramolecular triplexes or H-DNA. Slippage and misalignment are the most common events leading to DNA structure-induced mutagenesis. However, a number of other mechanisms of genetic instability have been proposed based on the finding that these structures not only induce expansions and deletions, but can also induce DNA strand breaks and rearrangements. The available data implicate a variety of proteins, such as mismatch repair proteins, nucleotide excision repair proteins, topoisomerases, and structure specific-nucleases in the processing of these mutagenic DNA structures. The potential mechanisms of genetic instability induced by these structures and their contribution to human diseases are discussed.     

Repeat instability: mechanisms of dynamic mutations

Disease-causing repeat instability is an important and unique form of mutation that is linked to more than 40 neurological, neurodegenerative and neuromuscular disorders. DNA repeat expansion mutations are dynamic and ongoing within tissues and across generations. The patterns of inherited and tissue-specific instability are determined by both gene-specific cis-elements and trans-acting DNA metabolic proteins. Repeat instability probably involves the formation of unusual DNA structures during DNA replication, repair and recombination. Experimental advances towards explaining the mechanisms of repeat instability have broadened our understanding of this mutational process. They have revealed surprising ways in which metabolic pathways can drive or protect from repeat instability.     

Structure and heterogeneity of the a sequences of human herpesvirus 6 strain variants U1102 and Z29 and identification of human telomeric repeat sequences at the genomic termini.

The unit-length genome of human herpesvirus 6 (HHV-6) consists of a single unique component (U) bounded by direct repeats DRL and DRR and forms head-to-tail concatemers during productive infection. cis-elements which mediate cleavage and packaging of progeny virions (a sequences) are found at the termini of all herpesvirus genomes. In HHV-6, DRL and DRR are identical and a sequences may therefore also occur at the U-DR junctions to give the arrangement aDRLa-U-aDRRa. We have sequenced the genomic termini, the U-DRR junction, and the DRR.DRL junction of HHV-6 strain variants U1102 and Z29. A (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was found adjacent to, but did not form, the termini of both strain variants. The DRL terminus and U-DRR junction contained sequences closely related to that of the well-conserved herpesvirus packaging signal Cn-Gn-Nn-Gn (pac-1), followed by tandem arrays of TRSs separated by single copies of a hexanucleotide repeat. HHV-6 strain U1102 contained repeat sequences not found in HHV-6 Z29. In contrast, the DRR terminus of both variants contained a simple tandem array of TRSs and a close homolog of a herpesvirus pac-2 signal (GCn-Tn-GCn). The DRR.DRL junction was formed by simple head-to-tail linkage of the termini, yielding an intact cleavage signal, pac-2.x.pac-1, where x is the putative cleavage site. The left end of DR was the site of intrastrain size heterogeneity which mapped to the putative a sequences. These findings suggest that TRSs form part of the a sequence of HHV-6 and that the arrangement of a sequences in the genome can be represented as aDRLa-U-a-DRRa.     

Escherichia coli thioredoxin-like protein YbbN contains an atypical tetratricopeptide repeat motif and is a negative regulator of GroEL

Many proteins contain a thioredoxin (Trx)-like domain fused with one or more partner domains that diversify protein function by the modular construction of new molecules. The Escherichia coli protein YbbN is a Trx-like protein that contains a C-terminal domain with low homology to tetratricopeptide repeat motifs. YbbN has been proposed to act as a chaperone or co-chaperone that aids in heat stress response and DNA synthesis. We report the crystal structure of YbbN, which is an elongated molecule with a mobile Trx domain and four atypical tetratricopeptide repeat motifs. The Trx domain lacks a canonical CXXC active site architecture and is not a functional oxidoreductase. A variety of proteins in E. coli interact with YbbN, including multiple ribosomal protein subunits and a strong interaction with GroEL. YbbN acts as a mild inhibitor of GroESL chaperonin function and ATPase activity, suggesting that it is a negative regulator of the GroESL system. Combined with previous observations that YbbN enhances the DnaK-DnaJ-GrpE chaperone system, we propose that YbbN coordinately regulates the activities of these two prokaryotic chaperones, thereby helping to direct client protein traffic initially to DnaK. Therefore, YbbN may play a role in integrating the activities of different chaperone pathways in E. coli and related bacteria.     

DNA tertiary structures formed in vitro by misaligned hybridization of multiple tandem repeat sequences.

DNA tertiary structures are shown to be formed by denaturation and reannealing in vitro of molecularly-cloned DNA containing multiple tandem repeat sequences. Electron microscopy of homoduplex DNA molecules containing the human c-Harvey-ras gene revealed knot-like structures which mapped to the position of the 812 bp variable tandem repeat (VTR) sequence. We propose that the structures result from slipped-strand mispairing within the VTR and hybridisation of homologous repetitive sequences in the single-stranded loops so produced. Similar structures were also found in freshly-linearized supercoiled plasmids. More complex knot-like structures were found in homoduplexes of a 4 kb tandem array from the hypervariable region 3' to the human alpha-globin locus. Formation of such DNA tertiary structures in vitro also provides a practical method for identifying and mapping direct tandem repeat arrays that are at least 800 bp long.     

Evidence for active maintenance of inverted repeat structures identified by a comparative genomic approach

Inverted repeats have been found to occur in both prokaryotic and eukaryotic genomes. Usually they are short and some have important functions in various biological processes. However, long inverted repeats are rare and can cause genome instability. Analyses of C. elegans genome identified long, nearly-perfect inverted repeat sequences involving both divergently and convergently oriented homologous gene pairs and complete intergenic sequences. Comparisons with the orthologous regions from the genomes of C. briggsae and C. remanei show that the inverted repeat structures are often far more conserved than the sequences. This observation implies that there is an active mechanism for maintaining the inverted repeat nature of the sequences.     

Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis-elements unrelated to canonical translation initiation signals

In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5' neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis-elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome.     

Hypermutable minisatellites,a human affair?

Minisatellites are a class of highly polymorphic GC-rich tandem repeats. They include some of the most variable loci in the human genome, with mutation rates ranging from 0.5% to >20% per generation. Structurally, they consist of 10- to 100-bp intermingled variant repeats, making them ideal tools for dissecting mechanisms of instability at tandem repeats. Distinct mutation processes generate rare intra-allelic somatic events and frequent complex conversion-like germline mutations in these repeats. Furthermore, turnover of repeats at human minisatellites is controlled by intense recombinational activity in DNA flanking the repeat array. Surprisingly, whereas other mammalian genomes possess minisatellite-like sequences, hypermutable loci have not been identified that suggest human-specific turnover processes at minisatellite arrays. Attempts to transfer minisatellite germline instability to the mouse have failed. However, yeast models are now revealing valuable information regarding the mechanisms regulating instability at these tandem repeats. Finally, minisatellites and tandem repeats provide exquisitely sensitive molecular tools to detect genomic insults such as ionizing radiation exposure. Surprisingly, by a mechanism that remains elusive, there are transgenerational increases in minisatellite instability.     

Generating DNA sequences encoding tandem peptide repeats suitable for expression and immunological application

Tandem repeats of single short peptide sequences are useful for many purposes. Here we describe a method called ligation-PCR to construct DNA sequences encoding numerous tandem peptide repeats that can stably produce such repeats in both prokaryotic and eukaryotic cells. The method employs double-strand target monomers consisting of a short peptide coding sequences. These sequences contain 3-bp cohesive overhangs to ensure correct repeat orientation and reading frame during ligation. The ligation products are PCR amplified and directly cloned into a new TA-cloning vector, pZeroT. Constructs containing tandem 10-amino-acid myc-tag peptide coding sequence repeats that ranged from approximately 0.45–1.2 kb, representing 15–40 copies of the corresponding peptide, were successfully obtained by this method. When one of the constructs was subcloned into prokaryotic vector pET-28 c (+) and eukaryotic vector rGHpcDNA3.0, and introduced into E. Coli and COS-7 cells, respectively, proteins containing tandem myc-tag peptide repeats were expressed with expected molecular weights. Purified proteins from E. Coli could successfully stimulate a peptide specific immune response. This method provides a means to manipulate peptides at the nucleic acid level, and can serve as the basis for biological peptide synthesis, epitope-specific antibody production, and epitope-based DNA vaccine construction.     

化学药物导致的与Friedreich共济失调相关的GAA重复序列不稳定性的研究

Friedreich共济失调是由位于FRDA基因上的GAA重复序列动态突变扩增所引起的,目前其不稳定性机制还不清楚。为了探索化学药物对GAA重复序列的作用,本实验构建了含有(GAA)42的重组质粒,转入大肠杆菌中,分别经过丝裂霉素C和甲基磺酸乙酯连续多次处理后,检测其长度变化发现,两种化学药物在不同程度上都可以促使(GAA)42发生删除,且删除后长度多数处于体内的正常范围。试验结果表明,化学药物可以促进与Friedreich共济失调相关的GAA重复序列发生删除。本工作为研究Friedreich共济失调的致病机理及治疗方案奠定了一定的基础。