RESUMO
The Nucleic Acid Circular Dichroism Database (NACDDB) is a public repository that archives and freely distributes circular dichroism (CD) and synchrotron radiation CD (SRCD) spectral data about nucleic acids, and the associated experimental metadata, structural models, and links to literature. NACDDB covers CD data for various nucleic acid molecules, including DNA, RNA, DNA/RNA hybrids, and various nucleic acid derivatives. The entries are linked to primary sequence and experimental structural data, as well as to the literature. Additionally, for all entries, 3D structure models are provided. All entries undergo expert validation and curation procedures to ensure completeness, consistency, and quality of the data included. The NACDDB is open for submission of the CD data for nucleic acids. NACDDB is available at: https://genesilico.pl/nacddb/.
Assuntos
Bases de Dados de Ácidos Nucleicos , Ácidos Nucleicos , Dicroísmo Circular , Síncrotrons , Ácidos Nucleicos/químicaRESUMO
Based on recently published initial experimental results on the intercalation of a class of broad spectrum antiparasitic compounds, we present a purely theoretical approach for determining if these compounds may preferentially intercalate with guanosine/cytosine (GC)-rich or adenosine/thymidine (TA)-rich regions of DNA. The predictive model presented herein is based upon utilization of density functional theory (DFT) to determine a priori how the best intercalator may energetically and sterically interact with each of the nucleoside base pairs. A potential new method using electrostatic potential maps (EPMs) to visually select the best poses is introduced and compared to the existing brute-force center of mass (COM) approach. The EPM and COM predictions are in agreement with each other, but the EPM method is potentially much more efficient. We report that 4-azatryptantrin, the best intercalator, is predicted to favor π-stacking with GC over that of TA by approximately 2-4 kcal/mol. This represents a significant difference if one takes into account the Boltzmann distribution at physiological temperature. This theoretical method will be utilized to guide future experimental studies on the elucidation of possible mechanism(s) for the action of these antiparasitic compounds at the molecular level.
Assuntos
DNA/química , DNA/metabolismo , Quinazolinas/química , Quinazolinas/metabolismo , Simulação por Computador , Modelos Químicos , Modelos Moleculares , Estrutura MolecularRESUMO
DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.
Assuntos
Canais de Cátion TRPP/metabolismo , Imunoprecipitação da Cromatina , Citosol/metabolismo , DNA/química , DNA/metabolismo , Dano ao DNA , Primers do DNA/genética , Replicação do DNA , Instabilidade Genômica , Células HeLa , Humanos , Íntrons , Conformação de Ácido Nucleico , Fosforilação , Proteínas Proto-Oncogênicas c-myc/metabolismo , Purinas/química , Pirimidinas/química , Canais de Cátion TRPP/genéticaRESUMO
Guanine-rich DNA can fold into highly stable four-stranded DNA structures called G-quadruplexes (G4). Originally identified in sequences from telomeres and oncogene promoters, they can alter DNA metabolism. Indeed, G4-forming sequences represent obstacles for the DNA polymerase, with important consequences for cell life as they may lead to genomic instability. To understand their role in bacterial genomic instability, different G-quadruplex-forming repeats were cloned into an Escherichia coli genetic system that reports frameshifts and complete or partial deletions of the repeat when the G-tract comprises either the leading or lagging template strand during replication. These repeats formed stable G-quadruplexes in single-stranded DNA but not naturally supercoiled double-stranded DNA. Nevertheless, transcription promoted G-quadruplex formation in the resulting R-loop for (G3T)4 and (G3T)8 repeats. Depending on genetic background and sequence propensity for structure formation, mutation rates varied by five orders of magnitude. Furthermore, while in vitro approaches have shown that bacterial helicases can resolve G4, it is still unclear whether G4 unwinding is important in vivo. Here, we show that a mutation in recG decreased mutation rates, while deficiencies in the structure-specific helicases DinG and RecQ increased mutation rates. These results suggest that G-quadruplex formation promotes genetic instability in bacteria and that helicases play an important role in controlling this process in vivo.
Assuntos
Proteínas de Escherichia coli , Quadruplex G , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , DNA/genética , Instabilidade Genômica , Proteínas de Escherichia coli/genéticaRESUMO
Instability of (CTG) x (CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG) x (CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG) x (CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.
Assuntos
Replicação do DNA/fisiologia , DNA/biossíntese , DNA/genética , Instabilidade de Microssatélites , Conformação de Ácido Nucleico , Repetições de Trinucleotídeos/genética , Células Cultivadas , DNA/química , Endonucleases/genética , Endonucleases/metabolismo , Células HeLa , Humanos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Origem de Replicação/genética , Origem de Replicação/fisiologia , Dedos de Zinco/genética , Dedos de Zinco/fisiologiaRESUMO
Spontaneous mutations are stochastic events. The mutation rate, defined as mutations per genome per replication, is generally very low, and it is widely accepted that spontaneous mutations occur at defined, but different, rates in bacteriophage and in bacterial, insect, and mammalian cells. The calculation of mutation rates has proved to be a significant problem. Mutation rates can be calculated by following mutant accumulation during growth or from the distribution of mutants obtained in parallel cultures. As Luria and Delbrück described in 1943, the number of mutants in parallel populations of bacterial cells varies widely depending on when a spontaneous mutation occurs during growth of the culture. Since 1943, many mathematical refinements to estimating rates, called estimators, have been described to facilitate determination of the mutation rate from the distribution or frequency of mutants detected following growth of parallel cultures. We present a rigorous mathematical solution to the mutation rate problem using an unbiased and consistent estimator. Using this estimator we demonstrate experimentally that mutation rates can be easily calculated by determining mutant accumulation, that is, from the number of mutants measured in two successive generations. Moreover, to verify the consistency of our estimator we conduct a series of simulation trials that show a surprisingly rapid convergence to the targeted mutation rate (reached between 25th and 30th generations).
Assuntos
Bactérias/genética , Modelos Genéticos , Taxa de Mutação , Animais , Bactérias/crescimento & desenvolvimento , Meios de Cultura , Plasmídeos/genética , Processos EstocásticosRESUMO
All DNA repeats known to undergo expansion leading to human neurodegenerative disease can form one, or several, alternative conformations, including hairpin, slipped strand, triplex, quadruplex, or unwound DNA structures. These alternative structures may interfere with the normal cellular processes of transcription, DNA repair, replication initiation, or polymerase elongation and thereby contribute to the genetic instability of these repeat tracts. We show that (CCTG) x (CAGG) repeats, in the first intron of the ZNF9 gene associated with myotonic dystrophy type 2, form slipped-strand DNA structures in a length-dependent fashion upon reduplexing. The threshold for structure formation on reduplexing is between 36 and 42 repeats in length. Alternative DNA structures also form in (CCTG)(58) x (CAGG)(58) and larger repeat tracts in plasmids at physiological superhelical densities. This represents an example of a sequence that forms slipped-strand DNA from the energy of DNA supercoiling. Moreover, Z-DNA forms in a (TG) x (CA) tract within the complex repeat sequence 5' of the (CCTG)(n) x (CAGG)(n) repeat in the ZNF9 gene. Upon reduplexing, the presence of the flanking sequence containing the Z-DNA-forming tract reduced the extent of slipped-strand DNA formation by 62% for (CCTG)(57) x (CAGG)(57) compared with 58 pure repeats without the flanking sequence. This finding suggests that the Z-DNA-forming sequence in the DM2 gene locus may have a protective effect of reducing the potential for slipped-strand DNA formation in (CCTG)(n) x (CAGG)(n) repeats.
Assuntos
DNA Forma Z/genética , Distrofia Miotônica/genética , Região 5'-Flanqueadora , Sequência de Bases , Humanos , Dados de Sequência Molecular , Distrofia Miotônica/classificação , Alinhamento de SequênciaRESUMO
G-rich DNA repeats that can form G-quadruplex structures are prevalent in bacterial genomes and are frequently associated with regulatory regions of genes involved in virulence, antigenic variation, and antibiotic resistance. These sequences are also inherently mutagenic and can lead to changes affecting cell survival and adaptation. Transcription of the G-quadruplex-forming repeat (G3T)n in E. coli, when mRNA comprised the G-rich strand, promotes G-quadruplex formation in DNA and increases rates of deletion of G-quadruplex-forming sequences. The genomic instability of G-quadruplex repeats may be a source of genetic variability that can influence alterations and evolution of bacteria. The DNA chaperone Hfq is involved in the genetic instability of these G-quadruplex sequences. Inactivation of the hfq gene decreases the genetic instability of G-quadruplex, demonstrating that the genomic instability of this regulatory element can be influenced by the E. coli highly pleiotropic Hfq protein, which is involved in small noncoding RNA regulation pathways, and DNA organization and packaging. We have shown previously that the protein binds to and stabilizes these sequences, increasing rates of their genomic instability. Here, we extend this analysis to characterize the role of the C-terminal domain of Hfq protein in interaction with G-quadruplex structures. This allows to better understand the function of this specific region of the Hfq protein in genomic instability.
RESUMO
The DNA repeats (CTG).(CAG), (CGG).(CCG), (GAA).(TTC), (ATTCT).(AGAAT), and (CCTG).(CAGG), undergo expansion in humans leading to neurodegenerative disease. A genetic assay for repeat instability has revealed that the activities of RecA and RecB during replication restart are involved in a high rate of deletion of (CTG).(CAG) repeats in E. coli. This assay has been applied to (CCTG).(CAGG) repeats associated with myotonic dystrophy type 2 (DM2) that expand to 11 000 copies and to spinocerebellar ataxia type 10 (SCA10) (ATTCT).(AGAAT) repeats that expand to 4500 copies in affected individuals. DM2 (CCTG).(CAGG) repeats show a moderate rate of instability, less than that observed for the myotonic dystrophy type 1 (CTG).(CAG) repeats, while the SCA10 (ATTCT).(AGAAT) repeats were remarkably stable in E. coli. In contrast to (CTG).(CAG) repeats, deletions of the DM2 and SCA10 repeats were not dependent on RecA and RecB, suggesting that replication restart may not be a predominant mechanism by which these repeats undergo deletion. These results suggest that different molecular mechanisms, or pathways, are responsible for the instability of different disease-associated DNA repeats in E. coli. These pathways involve simple replication slippage and various sister strand exchange events leading to deletions or expansions, often associated with plasmid dimerization. The differences in the mechanisms of repeat deletion may result from the differential propensity of these repeats to form various DNA secondary structures and their differential proclivity for primer-template misalignment during replication.
Assuntos
Instabilidade Genômica , Distrofia Miotônica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Deleção de Sequência/genética , Ataxias Espinocerebelares/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Humanos , Mutação/genética , Plasmídeos/genética , Transdução de SinaisRESUMO
Antigen receptor gene rearrangements are initiated by the RAG1/2 protein complex, which recognizes specific DNA sequences termed RSS (recombination signal sequences). The RAG recombinase can also catalyze transposition: integration of a DNA segment bounded by RSS into an unrelated DNA target. For reasons that remain poorly understood, such events occur readily in vitro, but are rarely detected in vivo. Previous work showed that non-B DNA structures, particularly hairpins, stimulate transposition. Here we show that the sequence of the four nucleotides at a hairpin tip modulates transposition efficiency over a surprisingly wide (>100-fold) range. Some hairpin targets stimulate extraordinarily efficient transposition (up to 15%); one serves as a potent and specific transposition inhibitor, blocking capture of targets and destabilizing preformed target capture complexes. These findings suggest novel regulatory possibilities and may provide insight into the activities of other transposases.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Homeodomínio/genética , Recombinação Genética/genética , Transposases/genética , Animais , Sequência de Bases , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transposases/metabolismoRESUMO
Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.
RESUMO
Slipped strand DNA structures are formed when complementary strands comprising direct repeats pair in a misaligned, or slipped, fashion along the DNA helix axis. Although slipped strand DNA may form in almost any direct repeat, to date, these structures have only been detected in short DNA repeats, termed unstable DNA repeats, in which expansion is associated with many neurodegenerative diseases. This alternative DNA structure, or a similar slipped intermediate DNA that may form during DNA replication or repair, may be a causative factor in the instability of the DNA sequences that can form these structures.
Assuntos
DNA/química , Modelos Genéticos , Reparo do DNA , Enzimas Reparadoras do DNA/fisiologia , Expansão das Repetições de DNA , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Instabilidade Genômica , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido NucleicoRESUMO
Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Elementos de DNA Transponíveis/genética , Marcação de Genes , Recombinação Genética/genética , Animais , Sequência de Bases , Células CHO , Cricetinae , DNA/química , DNA/genética , DNA/metabolismo , DNA Topoisomerases/metabolismo , DNA Recombinante/genética , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico/genética , Especificidade por Substrato , Translocação Genética/genética , VDJ RecombinasesRESUMO
(CTG)n.(CAG)n repeats undergo deletion at a high rate in plasmids in Escherichia coli in a process that involves RecA and RecB. In addition, DNA replication fork progression can be blocked during synthesis of (CTG)n.(CAG)n repeats. Replication forks stalled at (CTG)n.(CAG)n repeats may be rescued by replication restart that involves recombination as well as enzymes involved in replication and DNA repair, and this process may be responsible for the high rate of repeat deletion in E. coli. To test this hypothesis (CAG)n.(CTG)n deletion rates were measured in several E. coli strains carrying mutations involved in replication restart. (CAG)n.(CTG)n deletion rates were decreased, relative to the rates in wild type cells, in strains containing mutations in priA, recG, ruvAB, and recO. Mutations in priB and priC resulted in small reductions in deletion rates. In a recF strain, rates were decreased when (CAG)n comprised the leading template strand, but rates were increased when (CTG)n comprised the leading template. Deletion rates were increased slightly in a recJ strain. The mutational spectra for most mutant strains were altered relative to those in parental strains. In addition, purified PriA and RecG proteins showed unexpected binding to single-stranded, duplex, and forked DNAs containing (CAG)n and/or (CTG)n loop-outs in various positions. The results presented are consistent with an interpretation that the high rates of trinucleotide repeat instability observed in E. coli result from the attempted restart of replication forks stalled at (CAG)n.(CTG)n repeats.
Assuntos
Replicação do DNA/genética , Escherichia coli/genética , Deleção de Sequência/genética , Expansão das Repetições de Trinucleotídeos/genética , Adenosina Trifosfatases/metabolismo , Sequência de Bases , DNA Helicases/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Especificidade por SubstratoRESUMO
Interest to the left-handed DNA conformation has been recently boosted by the findings that a number of proteins contain the Zalpha domain, which has been shown to specifically recognize Z-DNA. The biological function of Zalpha is presently unknown, but it has been suggested that it may specifically direct protein regions of Z-DNA induced by negative supercoiling in actively transcribing genes. Many studies, including a crystal structure in complex with Z-DNA, have focused on the human ADAR1 Zalpha domain in isolation. We have hypothesized that the recognition of a Z-DNA sequence by the Zalpha(ADAR1) domain is context specific, occurring under energetic conditions, which favor Z-DNA formation. To test this hypothesis, we have applied atomic force microscopy to image Zalpha(ADAR1) complexed with supercoiled plasmid DNAs. We have demonstrated that the Zalpha(ADAR1) binds specifically to Z-DNA and preferentially to d(CG)(n) inserts, which require less energy for Z-DNA induction compared to other sequences. A notable finding is that site-specific Zalpha binding to d(GC)(13) or d(GC)(2)C(GC)(10) inserts is observed when DNA supercoiling is insufficient to induce Z-DNA formation. These results indicate that Zalpha(ADAR1) binding facilities the B-to-Z transition and provides additional support to the model that Z-DNA binding proteins may regulate biological processes through structure-specific recognition.
Assuntos
Adenosina Desaminase/química , DNA Super-Helicoidal/ultraestrutura , DNA Forma Z/ultraestrutura , Adenosina Desaminase/metabolismo , Sítios de Ligação , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , DNA Forma Z/química , DNA Forma Z/metabolismo , Humanos , Microscopia de Força Atômica , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/ultraestrutura , Estrutura Terciária de Proteína , Proteínas de Ligação a RNARESUMO
Myotonic dystrophy type 1 (DM1) is caused by the expansion of a (CTG).(CAG) repeat in the DMPK gene on chromosome 19q13.3. At least 17 neurological diseases have similar genetic mutations, the expansion of DNA repeats. In most of these disorders, the disease severity is related to the length of the repeat expansion, and in DM1 the expanded repeat undergoes further elongation in somatic and germline tissues. At present, in this class of diseases, no therapeutic approach exists to prevent or slow the repeat expansion and thereby reduce disease severity or delay disease onset. We present initial results testing the hypothesis that repeat deletion may be mediated by various chemotherapeutic agents. Three lymphoblast cell lines derived from two DM1 patients treated with either ethylmethanesulfonate (EMS), mitomycin C, mitoxantrone or doxorubicin, at therapeutic concentrations, accumulated deletions following treatment. Treatment with EMS frequently prevented the repeat expansion observed during growth in culture. A significant reduction of CTG repeat length by 100-350 (CTG).(CAG) repeats often occurred in the cell population following treatment with these drugs. Potential mechanisms of drug-induced deletion are presented.
Assuntos
Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacos , Alelos , Antineoplásicos Alquilantes/uso terapêutico , Linhagem Celular , Doxorrubicina/uso terapêutico , Metanossulfonato de Etila/uso terapêutico , Humanos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Mitomicina/uso terapêutico , Mitoxantrona/uso terapêuticoRESUMO
DNA supercoiling plays an important role in many genetic processes such as replication, transcription, and recombination. Supercoiling provides energy for helix un-pairing and drives the formation of alternative DNA structural transitions, like cruciforms. Supercoiling also allows distant DNA regions to be brought into close proximity through the formation of inter-wound supercoils. Recently, we showed that the inverted repeat-to-cruciform transition acts as a molecular switch, influencing the global topology of a topological plasmid domain. As alternative DNA structures can affect global topology, a corollary hypothesis might be that the localization of a specific DNA sequence within a topological domain may affect the energetics required for formation of an alternative DNA structure. Here, we test this hypothesis and show that the localization of an inverted repeat to an apical position increases the rate of cruciform formation and reduces the superhelical energy required to drive the transition. For this, we created a series of plasmids containing an inverted repeat and an A-tract bent DNA sequence. The A-tract forms a permanent 180 degrees bend irrespective of DNA topology. The inverted repeat and the bent sequence were placed either at six o'clock or nine o'clock positions with respect to each other. Using 2D agarose gel electrophoresis, we show that the six o'clock construct extrudes the cruciform at a lower superhelical density than a control plasmid without the bend. Atomic force microscopy shows that the nine o'clock construct has the propensity to form branched molecules with the cruciform at the end of one branch. These results demonstrate that the localization of sequences within specific regions of a topological domain can affect the energetics of structural transitions as well as the branching structure of the domain. As structural transitions can be involved in biological processes, localization of alternative conformation-forming sequences to specific locations within a domain provides an additional means for gene regulation.
Assuntos
DNA Super-Helicoidal/química , Conformação de Ácido Nucleico , DNA Super-Helicoidal/ultraestrutura , Microscopia de Força Atômica , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/ultraestruturaRESUMO
A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.
Assuntos
DNA Super-Helicoidal/química , Repetições de Microssatélites , Conformação de Ácido Nucleico , Plasmídeos/genética , Ataxias Espinocerebelares/genética , Composição de Bases , Pareamento de Bases , Replicação do DNA , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/ultraestrutura , Eletroforese em Gel Bidimensional , Células HeLa , Humanos , Microscopia de Força Atômica , Modelos Teóricos , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Plasmídeos/metabolismoRESUMO
The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases, including myotonic dystrophy type 1. However, obtaining defined lengths of long repeats in vitro has been problematic. Strand slippage and/or DNA secondary structure formation may prevent efficient ligation. For example, a purified (CTG)140.(CAG)140 repeat fragment containing 4-bp AGCA/TGCT overhanging ends ligated poorly using T4 or Escherichia coli DNA ligase, although limited repeat ligation occurred using thermostable DNA ligase. Here we describe a general procedure for ligating multimers of DNA repeats. Multimers are efficiently ligated when slippage is prevented or when DNA repeats contain a single G/C overhang. A cloning vector is designed from which pure repeat fragments containing a G/C overhang can be generated for further ligation. (CAG)n.(CTG)n DNA molecules longer than 800 bp were generated using this approach. This approach also worked for (GAA)n.(TTC)n, (CCTG)n-(CAGG)n, and (ATTCT)n.(AGAAT)n tracts associated with Friedreich ataxia, DM2, and spinocerebellar ataxia type 10, respectively.