Duplex DNA Molecules Research Articles

Stoichiometric analysis of protein- and nucleic acid-based structures in the cell nucleus

We describe a method to image selectively the protein-based architecture in the eukaryotic cell nucleus using nitrogen and phosphorus mapping. In addition, we describe a method to determine total mass as well as stoichiometric relationships between protein and RNA. This method is illustrated using particulate structures in the nucleus called interchromatin granules. In so doing, we demonstrate that these granules contain heterogeneous nuclear RNA, and have an average protein and RNA content of 3.094 and 1.672 MDa, respectively. We also tested the sensitivity of phosphorus detection by exogenously applying purified duplex DNA to the surfaces of thin sections, and have shown that structures as small as single molecules of duplex DNA can be detected in situ using these electron spectroscopic imaging techniques.

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4′,6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPγS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.

Substrate specificity of ultraviolet DNA endonuclease (UVDE/Uve1p) from Schizosaccharomyces pombe.

Schizosaccharomyces pombe ultraviolet DNA endonuclease (UVDE or Uve1p) has been shown to cleave 5' to UV light-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). This endonuclease is believed to function in the initial step in an alternative excision repair pathway for the removal of DNA damage caused by exposure to UV light. An active truncated form of this protein, Delta228-Uve1p, has been successfully overexpressed, affinity purified and partially characterized. In the present study we present data from a detailed substrate specificity trial. We have determined that the substrate range of Uve1p is much greater than was originally believed. We demonstrate that this DNA damage repair protein is capable of recognizing an array of UV-induced DNA photoproducts (cis-syn-, trans-syn I- and trans-syn II CPDs, 6-4PP and Dewar isomers) that cause varying degrees of distortion in a duplex DNA molecule. We also demonstrate that Uve1p recognizes non-UV-induced DNA damage, such as platinum-DNA GG diadducts, uracil, dihydrouracil and abasic sites. This is the first time that a single DNA repair endonuclease with the ability to recognize such a diverse range of lesions has been described. This study suggests that Uve1p and the alternative excision repair pathway may participate broadly in the repair of DNA damage.

The Utility of Capped Duplex DNA (σ-DNA) in Triplex Interaction Studies

Two optimally capped duplex DNA molecules (σ-DNA; see 1 and 4) were synthesized and their utility demonstrated for triplex investigations with their corresponding homopyrimidine DNA and RNA single strands in the D⋅(D⋅D) and R⋅(D⋅D) Hoogsteen mode. Furthermore, it was established that σ-DNA is an ideal tool to study the pH dependency of triplex formation.

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.

On the Mechanism of RecA-Mediated Repair of Double-Strand Breaks: No Role for Four-Strand DNA Pairing Intermediates

RecA protein will bind to a gapped duplex DNA molecule and promote a DNA strand exchange with a second homologous linear duplex. A double-strand break in the second duplex is efficiently bypassed in the course of these reactions. We demonstrate that the bypass of double-strand breaks is not explained by a mechanism involving homologous interactions between two duplex DNA molecules, but instead requires the ATP-mediated generation of DNA torsional stress brought about by the action of RecA. The results suggest new pathways for the repair of double-strand breaks and underline the need for new paradigms to explain the alignment of homologous DNAs during genetic recombination.

Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum.

The homing endonuclease I-PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26S rRNA gene. This endonuclease promotes the lateral transfer or "homing" of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26S rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I-PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombinant I-PpoI has been co-crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3(1)21, with unit cell dimensions a = b = 114 A, c = 89 A. The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A3/dalton. These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.

The DNA replication protein PriA and the recombination protein RecG bind D-loops

The PriA protein of Escherichia coli provides a vital link between recombination and DNA replication. To establish the molecular basis for this link, we investigated the ability of PriA to target DNA substrates modelled on D-loops, the intermediates formed during the early stages of RecA-mediated recombination. We show that PriA binds D-loops and unwinds the DNA in reactions that rely on its ability to function as a helicase. The minimal structure that binds PriA is a duplex DNA molecule with unpaired single strands at one end, an arrangement likely to occur at a D-loop. It resembles features of the stem-loop formed by primosome assembly site (PAS) sequences in the DNA of bacteriophage φX174 and plasmid ColE1, and which enable PriA to assemble active primosomes for the initiation of lagging strand synthesis. We suggest that PAS sequences may have evolved to mimic the natural D-loop target for PriA formed in the chromosome of E. coli during recombination and DNA repair. Genetic studies have revealed an interaction between PriA and RecG, a DNA helicase that drives branch migration of recombination intermediates. We therefore compared PriA and RecG for their ability to bind and unwind DNA. RecG, like PriA, binds D-loops and unwinds the DNA. However, it prefers branched structures with at least two duplex components. The possibility that it competes with PriA for binding recombination intermediates is discussed.

Recombination-dependent repair of DNA double-strand breaks with purified proteins from Escherichia coli.

We have developed an in vitro system in which repair of DNA double-strand breaks is performed by purified proteins of Escherichia coli. A segment was deleted from a circular duplex DNA molecule by restriction at two sites. 3' single-stranded overhangs were introduced at both ends of the remaining linear fragment. In a first step, RecA protein catalyzed the formation of a D-loop between one single-stranded tail and a homologous undeleted supercoiled DNA molecule. In a second step, E. coli DNA polymerase II or III used the 3' end in the D-loop as a primer to copy the missing sequences of the linear substrate on one strand of the supercoiled template. Under proper conditions, the integrity of the deleted substrate was restored, as shown by analysis of the products by electrophoresis, restriction, and transformation. In this reaction, DNA synthesis is strictly dependent on recombination, and repair is achieved without formation of a Holliday junction.

Development and use of an in vitro HSV-tk forward mutation assay to study eukaryotic DNA polymerase processing of DNA alkyl lesions.

We have developed an in vitro DNA polymerase forward mutation assay using damaged DNA templates that contain the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. The quantitative method uses complementary strand hybridization to gapped duplex DNA molecules and chloramphenicol selection. This design ensures exclusive analysis of mutations derived from the DNA strand produced during in vitro synthesis. We have examined the accuracy of DNA synthesis catalyzed by calf thymus polymerase alpha-primase, polymerase beta and exonuclease-deficient Klenow polymerase. Using unmodified DNA templates, polymerase beta displays a unique specificity for the loss of two bases in a dinucleotide repeat sequence within the HSV-tk locus. Treatment of the DNA template with N-ethyl-N-nitrosourea resulted in a dose-dependent inhibition of DNA synthesis concomitant with an increased mutation frequency. Similar dose-response curves were measured for the three polymerases examined; thus the identity of the DNA polymerase does not appear to affect the mutagenic potency of ethyl lesions. The HSV-tk system is unique in that damage-induced mutagenesis can be analyzed both quantitatively and qualitatively in human cells, in bacterial cells and in in vitro DNA synthesis reactions at a single target sequence.

Saccharomyces cerevisiae MSH2, a mispaired base recognition protein, also recognizes Holliday junctions in DNA

Genetic and biochemical studies have suggested that mismatch repair proteins interact with recombination intermediates to prevent recombina tion, or to limit the extent of formation of heteroduplex DNA during recombination between divergent DNA sequences. To test the idea that mismatch repair proteins regulate recombination by interacting with recombination intermediates, we investigated whether the Saccharomyces cerevisiae MutS homolog MSH2 could interact with Holliday junctions. Both filter-binding and electron-microscopic analysis showed that MSH2 bound to duplex DNA molecules containing Holliday junctions with a higher affinity than to control duplex DNA, single-stranded DNA or a control duplex DNA containing a mispaired base. The MSH2 - Holliday junction complexes were also more stable than MSH2 - duplex DNA complexes. This observation suggests that MSH2 protein could directly coordinate the interaction between mismatch repair and genetic recombination observed in genetic studies.

Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes

Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.

The directionality of RuvAB-mediated branch migration: in vitro studies with three-armed junctions.

The Escherichia coli RuvA and RuvB proteins promote the branch migration of 4-way (Holliday) junctions during genetic recombination. The active complex is a tripartite structure in which RuvA protein is bound to the crossover and is sandwiched between two hexameric rings of RuvB. Branch migration requires ATP hydrolysis and occurs as the DNA passes through each RuvB ring. In this work, we have investigated the mechanism by which RuvAB catalyses the branch migration of a three-armed (Y) junction. Using synthetic DNA structures, we observed the formation of DNA products, a partial duplex DNA molecule and a single-stranded oligonucleotide, indicative of a branch migration reaction that occurred with unique polarity. Analysis of the RuvAB-junction complex by DNase footprinting showed that RuvA bound asymmetrically to the junction and targeted a single hexameric RuvB ring to one arm of DNA. Branch migration of a three-armed junction occurs in a unidirectional manner that is determined by the assembly of a single RuvB ring onto one arm of the DNA. The asymmetry of the complex and observed directionality of branch migration indicate that strand passage occurs as the DNA is pulled into the RuvB ring structure, a reaction likely to be coupled with DNA unwinding.

DNA: An Extensible Molecule

The force-displacement response of a single duplex DNA molecule was measured. The force saturates at a plateau around 70 piconewtons, which ends when the DNA has been stretched about 1.7 times its contour length. This behavior reveals a highly cooperative transition to a state here termed S-DNA. Addition of an intercalator suppresses this transition. Molecular modeling of the process also yields a force plateau and suggests a structure for the extended form. These results may shed light on biological processes involving DNA extension and open the route for mechanical studies on individual molecules in a previously unexplored range.

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.

Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circle replication of circular duplex DNA molecules. The products of the reaction are longer than monomer unit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance to DpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on the gradient consistent with those of molecules composed mainly of one parental DNA strand and one newly synthesized DNA strand, and (iii) the appearance in the electron microscope of molecules consisting of duplex circles with multiunit linear appendages, a characteristic of a rolling circle mode of DNA replication. The reaction requires ATP and is dependent on herpes simplex virus type 1-encoded DNA polymerase.

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.

Condensation and cohesion of λ DNA in cell extracts and other media: Implications for the structure and function of DNA in prokaryotes

DNA added to concentrated extracts of Escherichia coli undergoes a reversible transition to a readily-sedimentable (‘condensed’) form. The transition occurs over a relatively small increment in extract concentration. The extract appears to play two roles in this transition, supplying both DNA-binding protein(s) and a crowded environment that increases protein binding and favors compact DNA conformations. The two roles of the extract are suggested by properties of fractions prepared by absorption of extracts with DNA-cellulose. The DNA-binding fraction and the DNA-nonbinding fractions from these columns are separately poorer condensing agents than the original extract, but when rejoined are similar to the original extract in the amount required for condensation. The dual role for the extract is supported by model studies of condensation with combinations of purified DNA-binding materials (protein HU or spermidine) and concentrated solutions of crowding agents (albumin or polyethylene glycol 8000); in each case, crowding agents and DNA-binding materials jointly reduce the amounts of each other required for condensation. The condensation reaction as studied in extracts or in the purified systems may be a useful approach to the forces which stabilize the compact form of DNA within the bacterial nucleoid. The effect of condensation on the reactivity of the DNA was measured by changes in the rate of cohesion between duplex DNA molecules bearing the complementary single-strand termini of λ DNA. Condensation caused large increases in the rates of cohesion of both λ DNA and of restriction fragments of λ DNA bearing the cohesive termini. Cohesion products of λ DNA made in vitro are a mixture of linear and circular aggregates, whereas those made in vivo are cyclic monomers. We suggest a simple mechanism based upon condensation at the site of viral injection which may explain this discrepancy.

The search for DNA homology does not limit stable homologous pairing promoted by RecA protein

The basic molecular mechanisms that govern the search for DNA homology and subsequent homologous pairing during genetic recombination are not understood. RecA is the central homologous recombination protein of Escherichia coli; because several RecA homologues have been identified in eukaryotic cells, it is likely that the mechanisms employed by RecA are conserved throughout evolution. Analysis of the kinetics of the homologous search and pairing reactions catalyzed by RecA should therefore provide insights of general relevance into the mechanisms by which macromolecules locate, and interact with, specific DNA targets. RecA forms three-stranded synaptic complexes with a single-stranded oligonucleotide and a homologous region in duplex DNA. The kinetics of this initial pairing reaction were characterized using duplex DNA molecules of various concentrations and complexities containing a single target site, as well as various concentrations of homologous single-stranded oligonucleotides. The formation of the synaptic complex follows apparent second-order reaction kinetics with a rate proportional to the concentrations of both the homologous single-stranded oligonucleotide and the target sites within the duplex DNA. The reaction rate is independent of the complexity of duplex DNA in the reaction. We propose a kinetic scheme in which the RecA-single-stranded DNA filament interacts with duplex DNA and locates its target in a relatively fast reaction. We also suggest that complex conformational changes occur during the subsequent rate-limiting step. We conclude that, during the formation of synaptic complexes by RecA, the search for homology is not rate-limiting, and that the iteration frequency of the search is around 10(2)-10(3) s-1. This value agrees well with what has been calculated as the minimum number for such a frequency in genome-wide searches, and limits the possible structures involved in the search for homology to those involving very soft (low energy) interactions. Furthermore, from the order of the reaction at the DNA concentrations found in eukaryotic nuclei, and the rate constant of the overall reaction, we predict that the search for homology is also not the rate-limiting step in the genome-wide searches implicated in meiosis and in gene targeting.

Uptake and processing of duplex DNA by RecA nucleoprotein filaments: insights provided by a mixed population of dynamic and static intermediates.

In the polarized strand exchange that is promoted by Escherichia coli RecA protein, when the initiating end of a duplex DNA molecule is blocked by heterology, the homologous distal end nonetheless forms a joint with single-stranded DNA, but strand exchange in that joint cannot be completed because the strand that would otherwise be displaced lacks a free 5' end. Instead, 2/3 to 3/4 of such distal joints cyclically form and dissociate. Dissociation requires the hydrolysis of ATP (Burnett et al., 1994). Observations on DNase protection revealed that consistent with their dynamic nature, these joints were heterogeneous in length, extending from the labeled distal end of the duplex up to 600 base pairs within the homologous region. Switching of base pairs was undetectable in this fraction of distal joints. However, the other 1/3 to 1/4 of distal joints, which did not cycle, were as long as the entire homologous region (6 kb), and underwent complete switching of base pairs. The formation of these static joints occurred at a rate in excess of 100 bp per second, without requiring hydrolysis of ATP. These and earlier observations suggest that the RecA filament containing single-stranded DNA rapidly incorporates duplex DNA into a coaxial three-stranded helix by a passive process, whereas additional energy is required to convert the three-stranded intermediate into products or back into substrates, both of which involve the unwinding of many turns of three-stranded helix.