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Biomedical subjects

R R Sinden

Publications and source records attributed to R R Sinden.

At least 37 records · Page 2Linked to original sources
PubMed1998

Stabilization of intramolecular triple/single-strand structure by cationic peptides.

For better comprehension of possible physiological roles of triple-helical DNA structures, it is important to understand if the proteins can stabilize intramolecular triplex (H-DNA). One plausible mode of stabilization is through the neutralization of electrostatic repulsion of negatively charged phosphates in the three DNA strands by positively charged arginine and lysine residues of a bound protein. To gain an insight into interactions between H-DNA and cationic protein domains, we examined the effect of Lys- and Arg-rich oligopeptides on the B-DNA to H-DNA transition. These oligopeptides as well as another type of polycation, spermine, shifted the equilibrium toward H-DNA. These polycations introduced little change in DNA superhelicity, so that an increase in torsional stress was not responsible for facilitated H-DNA formation. Competing influences of polycations and monovalent cations suggest a significant involvement of electrostatic interactions in H-DNA stabilization. The Arg-rich peptides are more effective in H-DNA stabilization than the Lys-rich ones. However, as inferred from experiments on intermolecular complexes, this is not due to a better stabilization of triple helix or destabilization of double helix. It is possible that Arg-rich peptides interact with the unpaired single strand in H-DNA and stabilize its unpaired conformation.

Arginine↗
PubMed1998

Structure and dynamics of supercoil-stabilized DNA cruciforms.

Understanding DNA function requires knowledge of the structure of local, sequence-dependent conformations that can be dramatically different from the B-form helix. One alternative DNA conformation is the cruciform, which has been shown to have a critical role in the initiation of DNA replication and the regulation of transcription in certain systems. In addition, cruciforms provide a model system for structural studies of Holliday junctions, intermediates in homologous DNA recombination. Cruciforms are not thermodynamically stable in linear DNA due to branch point migration, which makes their study using many biophysical techniques problematic. Atomic Force Microscopy (AFM) was applied to visualize cruciforms in negatively supercoiled plasmid DNA. Cruciforms are seen as clear-cut extrusions on the DNA filament with the lengths of the arms consistent with the size of the hairpins expected from a 106 bp inverted repeat. The cruciform exists in two different conformations, an extended one with the angle of ca. 180 degrees between the hairpin arms and a compact, X-type conformation, with acute angles between the hairpin arms and the main DNA strands. The ratio of molecules with the different conformations of cruciforms depends on ionic conditions. In the presence of high salt or Mg cations, a compact, X-type conformation is highly preferable. Remarkably, the X-conformation was highly mobile allowing the cruciform arms to adopt a parallel orientation. The structure observed is consistent with a model of the Holliday junction with a parallel orientation of the exchanging strands.

DNA, Superhelical↗
PubMed1998

Interruptions in the triplet repeats of SCA1 and FRAXA reduce the propensity and complexity of slipped strand DNA (S-DNA) formation.

Models for the disease-associated expansion of trinucleotide repeats involve the participation of alternative DNA structures during replication, repair, or recombination. CAT or AGG interruptions within the (CAG)n or (CGG)n repeats of SCA1 or FRAXA, respectively, confer increased genetic stability to the repeats. In this study, we report the formation of slipped strand structures (S-DNA) using genomic sequences containing pure and interrupted SCA1 and FRAXA repeats having lengths above and below the genetic stability thresholds. S-DNA forms within the repeats during annealing of complementary strands containing equal lengths of repeats. Increased lengths of pure repeats led to an increased propensity for S-DNA formation. CAT or AGG interruptions have both quantitative and qualitative effects upon S-DNA formation: they decrease the total amount of slipped structures as well as limit the specific isomers formed. This demonstrates a unifying inhibitory effect of interruptions in both (CAG)n and (CGG)n tracts. We also present transmission stability data for SCA1 and FRAXA alleles spanning the thresholds and compare these with the ability to form slipped structures. The effect of both the length and purity of the repeat tract on the propensity of slipped structure formation correlates with their effect on genetic instability and disease, suggesting that S-DNA structures may be models for mutagenic intermediates in instability.

Ataxin-1↗
PubMed1998

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.

DNA↗
PubMed1998

CTG repeats associated with human genetic disease are inherently flexible.

The lengthening of tracts of CTG, CGG and GAA triplet repeats during progression of a pedigree has been associated with more than 12 human genetic diseases, including fragile X syndrome, myotonic dystrophy and Friedreich's ataxia. These repetitive sequence elements have the potential to form alternative DNA secondary structures that may contribute to their instability. The alternative DNA secondary structures may mediate errors during DNA replication, repair or recombination of the triplet repeat, leading to expansion. Here we show that DNA composed of pure CTG or CGG repeats exhibits anomalously fast mobility on polyacrylamide gels, confirming a previous observation for DNA containing CTG and CGG triplet repeats flanked by mixed sequence DNA. Moreover, we show that even short tracts of duplex CTG repeats have an unusual helix structure. CTG repeats reduce overall curvature associated with phased A-tract or GGCC curves, but alone they do not introduce curvature into DNA. The reduction in curvature of phased A-tracts by CTG repeats is similar to that afforded by an interspersed flexible region associated with a (TT).(TT) mispair. CTG-containing DNAs exhibit a rapid rate of cyclization, consistent with a flexible helix. These results suggest that tracts of (CTG).(CAG) repeats are inherently flexible. In addition, our results suggest that the unusual rapid electrophoretic mobility of CTG or CGG-containing DNA may be a consequence of an extended flexible DNA chain.

Cytosine↗
PubMed1998

Trinucleotide repeat DNA structures: dynamic mutations from dynamic DNA.

Models for the disease-associated expansion of (CTG)n.(CAG)n, (CGG)n.(CCG)n, and (GAA)n.(TTC)n trinucleotide repeats involve alternative DNA structures formed during DNA replication, repair and recombination. These repeat sequences are inherently flexible and can form a variety of hairpins, intramolecular triplexes, quadruplexes, and slipped-strand structures that may be important intermediates and result in their genetic instability.

Base Composition↗
PubMed1997

Leading strand specific spontaneous mutation corrects a quasipalindrome by an intermolecular strand switch mechanism.

Imperfect inverted repeats or quasipalindromes can undergo spontaneous, often complex mutational events that correct them to perfect palindromes. Two models that depend on the quasipalindrome providing a template for a specific mutational event have been described to explain this mutation: an intramolecular and an intermolecular strand switch model. A 17bp quasipalindrome containing a -1 deletion within the chloramphenicol acetyl transferase (CAT) gene in plasmid pJT7 undergoes a spontaneous +1 frameshift mutation that creates a perfect inverted repeat and a Cm(r) phenotype. By analyzing this mutation frequency in two plasmids that contain the CAT gene in either orientation with respect to the origin of replication, we show that the specific frameshift occurs preferentially in the leading strand during DNA replication. Due to the availability and proximity of the lagging strand template as a single strand during replication of the quasipalindrome in the leading but not lagging strand, we suggest that the specificity for the leading strand correction is due to a leading strand specific intermolecular strand switch rather than an intramolecular strand switch. To test this hypothesis, we have designed a genetic selection to detect a leading strand intermolecular strand switch. This selection utilizes asymmetric quasipalindromes, one of which contains two central stop codons. When cloned into the CAT gene in pJT7, reversion to Cm(r) requires inversion of the stop codons and addition of a +1 frameshift to correct the reading frame. The inversion of the central stop codons, which is predicted by an intermolecular but not an intramolecular strand switch, occurs concomitant with the specific correction of the original 17 bp quasipalindrome. Inversion of an asymmetric center can also be demonstrated when not under selective pressure using a quasipalindrome lacking central stop codons. These results are consistent with the correction of a quasipalindrome occurring predominantly by an intermolecular strand switch during replication of the leading strand.

Base Sequence↗
PubMed1997

Measurement of unrestrained negative supercoiling and topological domain size in living human cells.

Unrestrained DNA supercoiling was measured using a Me3-psoralen photobinding assay within a transcriptionally active hygromycin B phosphotransferase (hph) gene integrated into different chromosomal locations in five transformed human fibrosarcoma cell lines. The level of unrestrained supercoiling in the hph gene varied, from high to low levels, in different chromosomal locations in living human cells. In one cell line, the hph gene contained no unrestrained supercoiling. Consequently, supercoiling was not dictated by the DNA sequence of the active hph gene. The addition of alpha-amanitin, which can inhibit transcription, reduced unrestrained supercoiling by 75% at one chromosomal location, by 50% at two other locations, and had little, if any, effect at two other chromosomal locations. Different levels of supercoiling in separate regions of the chromosome require that the chromosome be organized into independent topological domains in vivo. Evidence for independent topological domains in living cells is presented. From analysis of the relaxation of supercoiling as a function of the number of breaks introduced into the chromosome, the in vivo topological domain size for the human ribosomal RNA genes was estimated between 30,000 and 45,000 kb.

Cell Line, Transformed↗
PubMed1997

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases.

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure-specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.

Binding, Competitive↗
PubMed1996

Relationship between Escherichia coli growth and deletions of CTG.CAG triplet repeats in plasmids.

Instabilities that are intrinsic to natural repetitive DNA sequences produce high frequencies of length changes in vivo. Triplet repeats cloned in plasmids in Escherichia coli undergo expansions and deletions, and this instability is affected by multiple factors. We show that CTG-CAG repeats in plasmids can influence the growth of E. coli, which affects the observed stabilities. At extended growth periods, the observed frequencies of deletions were dramatically increased if the cells passed through stationary phase before subculturing. Deletions were particularly pronounced for a plasmid containing the longest repeat, 525 bp in total, with the CTG sequence as the lagging strand template for replication. Measurements of cell growth showed that the lag phase associated with E. coli growth was increased for cultures containing plasmids with long CTG-CAG repeats, particularly when the CTG-containing strand was the lagging template. High frequencies of deletions were observed because of a growth advantage of cells containing plasmids with deleted triplet repeats. Incubation conditions that reduced the bacterial growth-rate produced a decreased extent of deletions, presumably because they alleviated the growth advantage of cells harboring plasmids with deleted triplet repeats. The experimental observations were simulated by a model in which shorter triplet repeats provided a growth advantage due to a shorter lag phase. We demonstrate that the accumulation of deletions within repeating sequences during growth of E. coli can be prevented, and discuss these findings in relation to the studies of repetitive DNA sequences. These are the first observations to show a direct influence between a plasmid-based DNA sequence or structure and factors controlling bacterial growth.

Base Sequence↗
PubMed1996

Stability of an inverted repeat in a human fibrosarcoma cell.

Deletions and rearrangements of DNA sequences within the genome of human cells result in mutations associated with human disease. We have developed a selection system involving a neo gene containing a DNA sequence inserted into the NcoI site that can be used to quantitatively assay deletion of this sequence from the chromosome. The spontaneous deletion from the neo gene of a 122 bp inverted repeat occurred at a rate of 2.1 x 10(-8) to <3.1 x 10(-9) revertants/cell/generation in three different cell lines. Deletion of the 122 bp inverted repeat occurred between 6 bp flanking direct repeats. Spontaneous deletion of a 122 bp non-palindromic DNA sequence flanked by direct repeats was not observed, indicating a rate of deletion of <3.1 x 10(-9) revertants/cell/generation. This result demonstrates that a 122 bp inverted repeat can exhibit a low level of instability in some locations in the chromosome of a human cell line.

Base Sequence↗
PubMed1996

Formation of a combined H-DNA/open TATA box structure in the promoter sequence of the human Na,K-ATPase alpha2 gene.

Structural variation of DNA within the promoter of the human Na, K-ATPase alpha2 gene, which contains a 35-base pair (bp) homopyrimidine.homopurine (Py.Pu) tract adjacent to a TATA box has been studied. The Py.Pu tract contains a 26-bp quasi-mirror repeat sequence with a potential for intramolecular triplex formation. As analyzed by two-dimensional agarose gel electrophoresis, a plasmid containing 151 bp of the promoter sequence including the 35-bp Py.Pu tract undergoes structural transitions under moderately acidic pH. Chemical probing with chloroacetaldehyde, dimethyl sulfate, and potassium permanganate is consistent with the formation of triplex DNA within the Py.Pu tract at native superhelical density as isolated from Escherichia coli. Chemical probing was used to determine a supercoil dependence for the formation of this combined unwound structure. At the superhelical density sufficient to locally unwind DNA, an H-y3 isomer of intermolecular triplex likely forms. However, at higher superhelical tension an H-y5 structure forms in the Py.Pu tract, and with increasing supercoiling the local DNA unwinding extends into the abutting TATA box. The H-y5/open TATA box combination structure might be favorable at higher superhelical densities since it relaxes more supercoils. The possible involvement of the H-y5/open TATA box structure in transcription is discussed.

Base Sequence↗
PubMed1996

Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci.

Most models proposed to explain the disease-associated expansion of (CTG)n.(CAG)n and (CGG)n.(CCG)n trinucleotide repeats include the formation of slipped strand DNA structures during replication; however, physical evidence for these alternative DNA secondary structures has not been reported. Using cloned fragments from the myotonic dystrophy (DM) and fragile X syndrome (FRAXA) loci containing normal, premutation, and full mutation lengths of repeats, we report the formation of novel alternative DNA secondary structures that map within the repeat tracts during reannealing of complementary strands, containing equal lengths of repeats, into linear duplex DNA molecules. Linear duplex DNA molecules containing these alternative DNA secondary structures are characterized by reduced electrophoretic mobilities in polyacrylamide gels. These alternative secondary structures are stable at physiological ionic strengths and to temperatures up to at least 55 degrees C. Following reduplexing, the CAG strand of the (CTG)n.(CAG)n repeats is preferentially sensitive to mung bean nuclease, suggesting the presence of single-stranded DNA in the alternative DNA structure. For (CTG)17, which is a repeat length found in normal individuals, less than 3% of the DNA molecules formed alternative DNA structures upon reduplexing. DNA molecules containing (CTG)50 or (CTG)255, which represent premutation and full mutation lengths of triplet repeats, respectively, formed a heterogeneous population of alternative DNA structures in >50% of DNA molecules. The complexity of the structures formed increased with the length of the triplet repeat. The relationship between repeat length and the propensity of formation and complexity of the novel structures correlates with the effect of repeat length on genetic instability in human diseases. These are the first results consistent with the existence of slipped strand DNA structures. The potential involvement of these structures, which we term S-DNA, in the gentic instability of triplet repeats is discussed.

Base Sequence↗
PubMed1996

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines.

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)n and (CGG)n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome.

Base Sequence↗
PubMed1995

Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance.

Electron paramagnetic resonance spectroscopy was employed to monitor the dynamics associated with a B-Z transition in both a linear (dG-dC)n and a modified pUC8 plasmid. A spin label consisting of cytidine substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoffel fragment). Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes. The Z conformation was induced by changing the salt concentration from 0.01 to 4.5 M. The EPR line shape changed in response to the switch in DNA conformation. The degree of change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region. A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA. This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA.

Computer Simulation↗
PubMed1995

Anomalous rapid electrophoretic mobility of DNA containing triplet repeats associated with human disease genes.

Eight human genetic diseases have been associated with the expansion of CTG or CGG triplet repeats. The molecular etiology behind expansion is unknown but may involve participation of an unusual DNA structure in replication, repair, or recombination. We show that DNA fragments containing CTG triplet repeats derived from the human myotonic dystrophy gene migrate up to 20% faster than expected in nondenaturing polyacrylamide gels, suggesting the presence of an unusual DNA helix structure within the CTG triplet repeats. The anomalous migration is dependent upon the number of triplet repeats, the length of the flanking DNA, and the percentage and temperature of the polyacrylamide. The effect could be reduced by the addition of actinomycin D. Applying a reptation model for electrophoresis, the results are consistent with a 20% increase in persistence length of the DNA. PCR products containing CTG or CGG repeats from the spinocerebellar ataxia type I gene (SCA1) or the fragile X FMR1 gene, respectively, also showed higher electrophoretic mobility. These are the first sequences of defined length for which a dramatic increase in mobility can be attributed to sequence-dependent structural elements in DNA.

Ataxin-1↗
PubMed1995

Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.

Adenosine Triphosphatases↗