Linear Duplex DNA Molecules Research Articles

A Physical and Functional Interaction between Yeast Pol4 and Dnl4-Lif1 Links DNA Synthesis and Ligation in Nonhomologous End Joining

Genetic studies have implicated the Saccharomyces cerevisiae POL4 gene product in the repair of DNA double-strand breaks by nonhomologous end joining. Here we show that Pol4 preferentially catalyzes DNA synthesis on small gaps formed by the alignment of linear duplex DNA molecules with complementary ends, a DNA substrate specificity that is compatible with its predicted role in the repair of DNA double-strand breaks. Pol4 also interacts directly with the Dnl4 subunit of the Dnl4-Lif1 complex via its N-terminal BRCT domain. This interaction stimulates the DNA synthesis activity of Pol4 and, to a lesser extent, the DNA joining activity of Dnl4-Lif1. Notably, the joining of DNA substrates that require the combined action of Pol4 and Dnl4-Lif1 is much more efficient than the joining of similar DNA substrates that require only ligation. Thus, the physical and functional interactions between Pol4 and Dnl4-Lif1 provide a molecular mechanism for both the recruitment of Pol4 to in vivo DNA double-strand breaks and the coupling of the gap filling DNA synthesis and DNA joining reactions that complete the microhomology-mediated pathway of nonhomologous end joining.

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.

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.

Transcription activates RecA-promoted homologous pairing of nucleosomal DNA.

RecA protein catalyzes the homologous pairing of a single-stranded circular DNA and a linear duplex DNA molecule. When the duplex is packaged into chromatin, formation of homologously paired complexes is blocked. We have established a system for studying the RecA-promoted reaction by using a duplex fragment containing a single-phased nucleosome. Under these conditions there is no reaction leading to formation of joint molecule complexes. However, transcription on the chromatin template activates the formation of complexes. Reaction is dependent on RNA synthesis and DNA sequence homology and proceeds regardless of the direction of transcription.

Transcription activates RecA-promoted homologous pairing of nucleosomal DNA.

RecA protein catalyzes the homologous pairing of a single-stranded circular DNA and a linear duplex DNA molecule. When the duplex is packaged into chromatin, formation of homologously paired complexes is blocked. We have established a system for studying the RecA-promoted reaction by using a duplex fragment containing a single-phased nucleosome. Under these conditions there is no reaction leading to formation of joint molecule complexes. However, transcription on the chromatin template activates the formation of complexes. Reaction is dependent on RNA synthesis and DNA sequence homology and proceeds regardless of the direction of transcription.

Plasmid DNA in a groundwater aquifer microcosm ‐adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23 degrees C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg2+ can be replaced by Na+, K+ or NH4+. The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.

Natural Transformation of Acinetobacter calcoaceticus by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl(2) solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of Acinetobacter calcoaceticus by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl(2) solution). Plasmid DNA adsorbed on sand transformed A. calcoaceticus significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.

Targeting in linear DNA duplexes with two complementary probe strands for hybrid stability

A new in vitro hybridization reaction targets two short complementary RecA protein-coated DNA probes to homologous sequences at any position in a linear duplex DNA molecule. Stable hybrids are obtained after RecA protein removal when both complementary probe strands are present in a four-stranded hybrid, but not when one probe strand is present in a three-stranded hybrid. In four-stranded hybrids with one probe strand biotinylated and the other radiolabelled, the deproteinized hybrids can be isolated and detected by affinity capture on streptavidin-coated magnetic beads. RecA-mediated targeting of complementary biotinylated DNA probe strands allows the affinity capture of 48.5-kilobase duplex lambda genomic DNA. These reactions provide a means of isolating any desired duplex gene or chromosomal DNA fragment.

Cloning and integration of DNA fragments in human cells via the inverted terminal repeats of the adeno-associated virus 2 genome

In current systems for molecular cloning of eukaryotic genes, bacterial cells are routinely utilized as intermediate hosts. We investigated the possibility of using a viral system for cloning DNA fragments independent of bacterial cell usage. In this report, we provide an alternative approach for molecular cloning of DNA fragments in eukaryotic cells by utilizing the inverted terminal repeats (ITRs) of the genome of a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). We constructed a series of chimeric linear duplex DNA molecules, ranging in length from 1.8 to 7.2 kb, containing the cruciform structures of AAV-ITRs at both ends. These ‘no-end’ ( NE) DNA structures, when transfected into adenovirusinfected human cells in the presence of AAV replication proteins (Rep), underwent DNA replication. Furthermore, in the presence of AAV capsid proteins (Cap), all replicated DNA molecules of less than 5.0 kb were packaged into mature, biologically active AAV progeny virions. When a chimeric NE DNA ( NE-neo) containing a gene ( neo) encoding resistance to neomycin was transfected into human cells, neo R clones could be readily isolated in the presence of G418 (Geneticin). Southern-blot analysis of genomic DNA of several independently isolated neo R clones suggested stable integration of the NE-neo DNA into the host chromosomal DNA. AAV-ITRs, therefore, offer an alternative system for molecular cloning, as well as packaging of DNA fragments in mammalian cells independent of bacterial cell usage.

Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus.

Extracts of calf thymus have been fractionated to reveal a nuclease activity that specifically cleaves model Holliday junctions in vitro. The products of cleavage are unbranched linear duplex DNA molecules. Using synthetic four-way junctions, we show that the major sites of cutting are diametrically opposed, at sites one nucleotide from the base of the junction. Other types of four-way junctions, including pseudo-cruciform structures and cruciforms extruded from supercoiled plasmids, are also cleaved by the nuclease. The Mr of the partially purified activity, determined by gel filtration, is approximately 75,000. The calf thymus enzyme provides the first example of an endonuclease from a higher eukaryote that acts specifically on branch points in DNA, and indicates that junction-resolving proteins are normal constituents of somatic cells.

A recombinase from Drosophila melanogaster embryos.

We have partially purified a DNA strand-exchange activity (recombinase) from nuclear extracts of Drosophila melanogaster embryos. The protein fraction forms a joint molecule between a circular single-strand DNA and a homologous linear duplex DNA that is resolved from the substrates by agarose gel electrophoresis. A strand-exchange activity can be obtained from nuclear extracts from embryos as old as 24 hr. The activity is similar to that partially purified from human cells [Hsieh, P., Meyn, S.M. & Camerini-Otero, R.D. (1986) Cell 44, 885-894]. It is homology-dependent, requires Mg2+, appears to be directional in that it prefers to displace the 3' end of the noncomplementary strand, and does not require exogenous ATP. Forty nanograms of protein in the partially purified DNA strand-exchange fraction from D. melanogaster embryos can completely convert 50 ng of substrate single-strand DNA into joint molecules in 10 min. In the electron microscope, joint molecules are seen to consist of a circular single-strand DNA molecule attached to only one end of a linear duplex DNA molecule; a displaced strand is also seen. The region of heteroduplex formation can be as long as 600 base pairs. The demonstration of a strand-exchange activity from wild-type D. melanogaster embryos invites analysis of recombination-defective mutants to explore the role of DNA strand exchange in homologous recombination.

The genome of lipid-containing bacteriophage PRD1, which infects gram-negative bacteria, contains long, inverted terminal repeats.

The bacteriophage PRD1 is a lipid-bearing phage that infects a wide variety of gram-negative bacteria, including Escherichia coli and Salmonella typhimurium when they contain the appropriate plasmid. It contains a linear duplex DNA molecule that is covalently bound by its 5' ends to a terminal protein. We report here that the PRD1 genome contains a 111-base-pair terminal inverted repeat which does not bear homology to that of any known linear duplex DNAs with terminal proteins. We further report that its 3' termini are susceptible to enzymatic digestion by exonuclease III.

Relationship of the physical and enzymic properties of Escherichia coli recA protein to its strand exchange activity

We have shown that performing the recA protein catalyzed strand exchange reaction in the presence of acetate anions, rather than chloride which is commonly used, greatly increases the rate of the reaction. The initial rate of the reaction in an acetate-based buffer is approximately 3-4 times higher in the presence of Escherichia coli single-stranded DNA binding protein (SSB protein) and 2 times higher in its absence than the initial rate in chloride. To determine the enzymatic basis for this stimulatory effect of acetate buffer, we investigated the relationship between a number of physical and enzymatic properties of recA protein and the strand exchange reaction. We have found that although the acetate anion has some effect on the aggregation properties and the single-stranded DNA-dependent ATPase activity of recA protein, these effects cannot explain the enhanced strand exchange activity in an acetate-based buffer. We do find, however, that two aspects of recA protein activity closely parallel the ability of this protein to catalyze strand exchange. The first is the ability of recA protein to displace SSB protein from single-stranded DNA, an event critical to presynaptic complex formation. RecA protein is able to resist displacement by SSB protein at a lower magnesium concentration in acetate than in chloride buffer. The magnesium ion concentration dependence of strand exchange coincides exactly with this behavior. The second activity correlated to strand exchange is the duplex DNA-dependent ATPase activity of recA protein. We find that over a wide variety of sodium chloride and sodium acetate concentrations, this duplex DNA-dependent ATPase activity is linearly related to the amount of product formed in the strand exchange reaction. We postulate that this duplex DNA-dependent ATPase activity is important in the denaturation of the duplex DNA during the branch migration step of strand exchange and have also determined that this reaction is quite efficient, with the number of ATP molecules hydrolyzed per base pair exchanged being 0.75 +/- 0.25. In addition, recA protein catalyzed strand exchange between circular single-strand and linear duplex DNA molecules is shown to be irreversible, and a possible explanation for this irreversibility is presented.

Uncoupling of the DNA breaking and rejoining steps of Escherichia coli type I DNA topoisomerase. Demonstration of an active covalent protein-DNA complex.

DNA topoisomerases have been shown to cleave DNA phosphodiester bond and simultaneously become linked to the DNA at the cleavage site via a phosphotyrosine linkage (Tse, Y.-C., Kirkegaard, K., and Wang, J. C. (1980) J. Biol. Chem. 255, 5560-5565). For prokaryotic DNA topoisomerases, this is observed only when denaturant or protease is added to the topoisomerase-DNA incubation mixture. Previous attempts to reform DNA phosphodiester bonds from the covalent protein-DNA complex have been unsuccessful. Using oligonucleotides as substrates, the cleavage reaction of Escherichia coli DNA topoisomerase I occurs spontaneously (Tse-Dinh, Y.-C., McCarron, B. G. H., Arentzen, R., and Chowdhry, V. (1983) Nucleic Acids Res. 11, 8691-8701). Upon reaction with oligo(dA) labeled with 32P using terminal transferase and [alpha-32P]dATP, the enzyme becomes covalently linked to the 32P-labeled oligonucleotide. This 32P label can then be transferred to the 3'-OH end of a linear or nicked duplex DNA molecule subsequently added to the reaction mixture. This phosphodiester bond rejoining reaction can occur at a recessed, blunt, or protruding 3'-end of double-stranded DNA. It requires magnesium ions. These observations suggest that the covalent protein-DNA complex is a true intermediate during topoisomerization. Implications on the structure of prokaryotic type I DNA topoisomerases as compared to their eukaryotic counterparts are discussed.

Detection of single base-pair mismatches in DNA by chemical modification followed by electrophoresis in 15% polyacrylamide gel.

We have developed a method for distinguishing fragments of DNA that contain single-base mismatches from their perfectly paired homologues. Single-stranded regions within a duplex fragment are accessible to 1-cyclohexyl-3-(2-[4-(4-methyl)morpholinyl]ethyl)carbodiimide, which reacts with unpaired guanidylate and thymidylate residues in DNA. Intact linear duplex DNA molecules do not react with carbodiimide, whereas DNA molecules containing single-base mismatches react quantitatively. After carbodiimide reaction, the DNA molecules are electrophoresed in high-percentage polyacrylamide gels so that modified and unmodified fragments can be resolved. Application of this technique should make it possible to locate and purify DNA fragments that exhibit sequence differences from those that do not; these might be used to signal phenotypic variation as well as to diagnose inherited disease.

A method for unidirectional deletion mutagenesis with application to nucleotide sequencing and preparation of gene fusions

A method is described for the preparation of deletions that extend in one direction from a fixed point. The method is based on the ability of deoxynucleoside [1-thio]triphosphates to be incorporated into DNA by DNA polymerase I, Klenow fragment, and the fact that α-thiophosphate-containing phosphosphodiester bonds are resistant to hydrolysis by the 3′-to-5′ exonucleolytic activity ofphage T4 DNA polymerase. Therefore, linear duplex DNA molecules blocked at one 3′-terminus with a thiophosphate were prepared and then degraded from the other end with the exonuclease. Digestion for different lengths of time followed by treatment with nuclease S1 and ligation allowed the preparation and recovery of a nested set of deletion mutants. Importantly, it was observed that a significant fraction of deletion mutants of recombinant M13 phages carrying the target gene in the same orientation as ‘lacZα’ yielded phage that produced lacZα-complementing activity. Nucleotide (nt) sequencing showed that these phages carried in-frame fusions between the target gene and ‘lacZα’. The deletion mutagenesis procedure is applied to the nt sequencing of a gnd gene from a natural isolate of Escherichia coli.

Avian retrovirus pp32 DNA binding protein. Preferential binding to the promoter region of long terminal repeat DNA

The avian retrovirus pp32 protein possesses DNA endonuclease activity and unique DNA binding properties. An improved purification procedure was developed for pp32, resulting in a severalfold increase in the yield of this virion protein. By use of the nitrocellulose filter binding assay, the protein retains approximately 2-fold more supercoiled (form I) DNA molecules than equivalent linear duplex DNA molecules. Single-stranded DNA is only slightly preferred over double-stranded DNA for pp32 binding. The pp32 DNA binding sites on form I pBR322 DNA which contained an insert of avian retrovirus long terminal repeat (LTR) DNA were determined. A preformed protein-DNA complex was digested with one of several different multicut restriction enzymes and filtered through nitrocellulose filters. Fragments containing viral LTR DNA sequences and plasmid DNA containing promoter sequences for the ampicillin and tetracycline genes, sequences for the "left-end" inverted repeat of transposon 3, and sequences encompassing the carboxyl terminus of the beta-lactamase gene were preferentially retained on the filter by pp32. Partial mapping of pp32 DNA binding sites on LTR DNA was accomplished by generation of deletions in LTR DNA sequences. The pp32 protein preferentially bound viral DNA fragments which contain the viral promoter (TATTTAA) and the adjacent "R" repeat sequences. Computer analysis revealed that three of the four plasmid DNA fragments retained by pp32 contained LTR DNA promoter-like sequences (one mismatch only) which were part of statistically significant and thermodynamically stable hairpin structures.

DNA binding protein from ovaries of the frog, Xenopus laevis which promotes concatenation of linear DNA

A soluble extract of Xenopus laevis ovaries catalyzed ATP-dependent concatenation of linear duplex DNA molecules. DNA ligase and a unique X. laevis DNA binding protein were required for the formation of concatemers. A linear DNA concatenation system was reconstituted using T4 DNA ligase and homogeneous X. laevis DNA binding protein. This system catalyzed inter-molecular ligation of DNA molecules into linear concatemers of up to ten or more times monomer length.

DNA sequence of the US component of the varicella-zoster virus genome.

The linear duplex DNA molecule of varicella-zoster virus is 120 000 bp in size and has the sequence arrangement UL-IRS-US-TRS, where UL and US are unique sequences and IRS and TRS are inverted repeats flanking US. The primary structure of the cloned SstI g DNA fragment containing US (5232 bp) and adjacent portions of IRS and TRS (426 bp of each) was determined, and the following model for genetic expression was derived from an analysis of the sequence. The region specifies four mRNAs encoding primary translation products with mol. wts. of 11, 44, 39 and either 74 or 70 kd. The 39-and 70-kd proteins have primary structures characteristic of membrane proteins. The mRNAs encoding the 11- and 74/70-kd proteins extend from opposite sides of US into IRS/TRS, thus sharing a common 3' terminus. These proteins do not share a common carboxy terminus because the coding region for the 11-kd protein terminates at the junction between US and IRS, whereas that for the 74/70-kd protein extends into TRS. The analysis affirms the hypothesis that the extent of inverted repeats in herpesvirus genomes is primarily a result of constraints imposed by adjacent protein coding sequences.

Purification and properties of the recA protein of Proteus mirabilis. Comparison with Escherichia coli recA protein; specificity of interaction with single strand binding protein.

The product of the cloned recA+ gene of Proteus mirabilis substitutes for a defective recA protein in Escherichia coli recA- mutants and restores recombination, repair, and prophage induction functions to near normal levels (Eitner, G., Adler, B., Lanzov, V. A., and Hofemeister, J. (1982) Mol. Gen. Genet. 185, 481-486). In this paper, we report the purification to near homogeneity of the P. mirabilis recA protein (recApm). The polypeptide has a molecular weight similar to that of E. coli recA protein (recAec) and shows partial identity with recAec when reacted against antibodies specific for the E. coli recA protein. recApm catalyzes the hydrolysis of ATP in the presence of single-stranded but not double-stranded DNA. We have compared the recombination-like activities of recApm with those of recAec and found them to be similar. In the presence of ATP and Mg2+, stoichiometric amounts of recApm promote the complete reciprocal exchange of strands between gapped circular and linear duplex DNA molecules. The enzyme also efficiently promotes the formation of D-loops from circular duplex DNA and homologous single-stranded fragments. However, although recApm and recAec share the above physical and functional similarities, they differ in their ability to interact with the E. coli single strand binding protein to catalyze the transfer of one DNA strand from a linear duplex to a single-stranded circle.