Single-stranded Extensions Research Articles

Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase.

Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. Site-specific restriction endonucleases are used to generate linear DNAs with defined termini that can then be joined covalently at their ends via the action of DNA ligase. A novel approach to the synthesis of recombinant DNAs exploits the ability of a single enzyme, vaccinia DNA topoisomerase, to both cleave and rejoin DNA strands with extreme specificity at each step. Placement of the CCCTT cleavage motif for vaccinia topoisomerase near the end of a duplex DNA permits efficient generation of a stable, highly recombinogenic protein-DNA adduct that can religate only to acceptor DNAs that contain complementary single-strand extensions. Linear DNAs containing CCCTT cleavage sites at both ends (bivalent substrates) can be activated by topoisomerase and inserted into a plasmid vector in a simple and rapid in vitro procedure that is especially well suited to the molecular cloning of polymerase chain reaction-amplified DNAs. Activation of polyvalent (e.g. branched) DNA substrates by topoisomerase offers a potentially powerful method for the synthesis of two- and three-dimensional polynucleotide networks.

Replication Intermediates of Rice Tungro Bacilliform Virus DNA Support a Replication Mechanism Involving Reverse Transcription

Rice tungro bacilliform virus (RTBV) replication intermediates have been studied in rice plants infected with the virus. Unencapsidated virus-specific molecules were identified which had open circular, linear, supercoiled (SC), strong-stop, single-stranded, linear double-stranded hairpin, and double-stranded with single-stranded extension DNA forms. The structures of these different DNA forms were consistent with the replication model of cauliflower mosaic virus and support other results that reverse transcription is involved in the replication of RTBV. The existence of nonspecific and defective (+)-strand priming is suggested. The relative amount of SC DNAs differs in various tissues of the same plant and in the same tissue at different ages. This indicates host regulation of the virus replication cycle and a feedback regulatory mechanism in controlling the SC DNA level. There are no obvious differences in the composition of the replication intermediates between insect-infected and agroinoculated rice plants.

Characterization of broken DNA molecules associated with V(D)J recombination.

We previously demonstrated that DNA molecules with double-strand breaks at variable-(diversity)-joining [V(D)J] recombination signal sequences are relatively abundant in mouse thymocytes. This abundance strongly suggests that the mechanism of V(D)J recombination involves double-strand cleavage at recombination signals. As a first step toward understanding the mechanism of cleavage, we used a sensitive PCR assay to characterize the structure of one class of cleavage products, the signal ends, in detail. Here we demonstrate that most of these ends are blunt and terminate in 5' phosphoryl groups. Virtually all of the flush signal ends are full length. A minor subpopulation of broken ends terminates in short single-strand extensions. We have found no evidence for covalent DNA-protein linkages involving the signal ends. These data allow further refinement of the double-strand cleavage model for V(D)J recombination.

Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication.

The mitochondrial genome of Chlamydomonas reinhardtii is a linear double-stranded DNA of 15.8 kb. With the exception of the termini its DNA sequence has been published. Here we describe the unique structure of the two termini determined from cloned fragments or, for the very terminal sequences, by the Maxam and Gilbert method after 5' labeling of uncloned terminal fragments. The 15.8-kb DNA is characterized by terminal inverted repeats of 531 or 532 bp in length including long 3' extensions. The 3' single-stranded extensions of the left and right ends are non-complementary, identical in sequence, and comprise 39 to 41 nucleotides. Remarkably, the linear genome possesses in addition an internal 86-bp repeat of the two outermost sequences. The unusual structure of the 15.8-kb DNA termini is compared with those of other linear mitochondrial DNAs. Possible mechanisms of 15.8-kb DNA replication are discussed.

EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA

We have previously reported the purification and characterization of the transcription factor EBP-80 (Falzon, M., and Kuff, E. L. (1989) J. Biol. Chem. 264, 21915-21922). EBP-80 mediates the DNA methylation effect on transcription from an endogenous proviral long terminal repeat. Here we show that EBP-80 is very similar if not identical to the Ku autoantigen, a heterodimeric nuclear protein first detected by antibodies from autoimmune patients (Mimori, T., Akizuki, M., Yamagata, H., Inada, S., Yoshida, S., and Homma, M. (1981) J. Clin. Invest. 68, 611-620). A number of laboratories have shown that the Ku protein complex binds to free double-stranded DNA ends. In this study, we have examined the binding properties of EBP-80. EBP-80 binds single-stranded DNA with low affinity. Binding to random sequence double-stranded DNA depends on the length of the duplex and is optimal with oligomers of 30 and 32 base pairs; the protein complexes formed with these oligomers have Kd values of 15-20 pM. It binds with comparable high affinities to blunt-ended duplex DNA, to duplex DNA ending in hairpin loops, and to constructs in which an internal segment of duplex DNA is flanked by single-strand extensions. EBP-80 also interacts effectively with circular duplex molecules containing a 30-nucleotide single-stranded region (gap) or a double-stranded segment of nonhomology (bubble), but only weakly with the corresponding closed circular construct made up entirely of duplex DNA. EBP-80 prefers A/T to G/C ends. The binding properties of EBP-80 are consistent with the hypothesis that is recognizes single- to double-strand transitions in DNA. A model is presented for the interaction of EBP-80 with its target sequence in the proviral long terminal repeat.

Discontinuous hairpin DNAs synthesised in vivo following specific and non-specific priming of cauliflower mosaic virus DNA (+) strands

We have analysed, by two-dimensional gel electrophoresis, a population of cauliflower mosaic virus (CaMV) linear DNA forms from infected tissue which contain one or two discontinuities in the plus (+) strand at sites close to the putative priming sequences for (+) strand synthesis (designated G2(+), G3(+)). The proximal ends of the linear DNAs mapped to a region near the origin of DNA (−) strand synthesis at the tRNA primer binding site. The distal end of each linear DNA appeared to comprise heterogeneous single-strand extensions of the (−) strand extending from G2(+) and G3(+) in the direction of reverse transcription. However, strand-specific probes revealed that the ‘single-strand’ extensions contained sequences with both (+) and (−) strand polarity and their single-stranded sizes were twice that expected had they been double-stranded. This suggested that some extensions were hairpin structures which we confirmed by observing their rapid renaturation following transfer to nitrocellulose. We conclude that the linear discontinuous hairpin DNAs arose in vivo by interrupted reverse transcription of RNA templates followed by non-specific self-primed (+) strand synthesis. Generation of ‘full-length’ hairpins was prevented in some molecules by specific priming of (+) strands at G2(+) and G3(+), and in others, extended hairpin formation probably proceeded as a result of loss of specific (+) strand primer RNA fragments at G2(+) and G3(+). The significance of these DNA forms in an understanding of CaMV replication is discussed.

Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction.

Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction.

Although DNA breakage and reunion in nonhomologous recombination are poorly understood, previous work suggests that short sequence homologies may play a role in the end-joining step in mammalian cells. To study the mechanism of end joining in more detail, we inserted a polylinker into the simian virus 40 T-antigen intron, cleaved the polylinker with different pairs of restriction enzymes, and transfected the resulting linear molecules into monkey cells. Analysis of 199 independent junctional sequences from seven constructs with different mismatched ends indicates that single-stranded extensions are relatively stable in monkey cells and that the terminal few nucleotides are critical for cell-mediated end joining. Furthermore, these studies define three mechanisms for end joining: single-strand, template-directed, and postrepair ligations. The latter two mechanisms depend on homologous pairing of one to six complementary bases to position the junction. All three mechanisms operate with similar overall efficiencies. The relevance of this work to targeted integration in mammalian cells is discussed.

Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy.

We describe a method for the construction of large DNA fragment libraries in plasmid vectors, in which complementary, single-stranded extensions are ligated onto both vector and insert DNA using un-phosphorylated adaptor oligonucleotides. Special consideration has been taken of the requirements of expression screening as follows: cDNA synthesis using random oligonucleotide primers is described which maximises the probability of obtaining open reading frame fragments from large mRNA molecules, the adaptors use codons found in high abundance E. coli proteins to minimise problems of premature termination when using strong promoters, and the sequence encoded by the adaptors, when cloned into the bacterial expression vector pEX1, promotes a surface location for the foreign antigenic determinant where it is accessible to antibodies used for screening.

Nucleotide sequence and properties of the cohesive DNA termini from bacteriophage HP1 c1 of Haemophilus influenzae Rd

The termini of the mature DNA of phage HP1 c1 of Haemophilus influenzae Rd have been characterized by DNA ligation, nucleotide sequencing, and deoxynucleotide incorporation experiments. A hybrid plasmid containing the joined phage termini (the cos site) inserted into pBR322 has been constructed. The phage DNA has cohesive termini composed of complementary 5 ' single-stranded extensions which are seven residues long. The left cohesive terminal extension consists only of pyrimidines and the right only of purines. When the ends of the phage are joined, the terminal sequences constitute the central 7 bp of an 11 bp sequence containing only purines on one strand and pyrimidines on the other strand. This oligopyrimidine/oligopurine sequence does not possess rotational symmetry. A 10-bp sequence and its inverted repeat are located approx. 20 bp to the left and right of the fused ends.

Fusion of restriction termini using synthetic adaptor oligonucleotides

A method is proposed for linking a DNA fragment possessing a 5′ single-stranded extension to one carrying a 3′ extension. Synthetic oligonucleotide adaptors can be used to (i) change the site specificity at the termini of a fragment generated by restriction enzyme cleavage and (ii) simultaneously dephosphorylate the extremities of a DNA molecule to prevent recircularisation and allow positive selection for recombinant DNA molecules.

The effect of divalent cations on the mode of action of DNase I. The initial reaction products produced from covalently closed circular DNA.

The effect of different divalent metal ions on the hydrolysis of DNA by DNase I was studied with an assay which distinguishes between cleavage of one or both strands of the DNA substrate during initial encounters between enzyme and DNA. Using covalently closed superhelical SV40(I) DNA as substrate, initial reaction products consisting of relaxed circles or unit-length linears are resolved by electrophoresis of radioactively labeled DNA in agarose gels. Only in the presence of a transition metal ion, such as Mn2+ or Co2+, and only under certain reaction conditions, is DNase I able to cut both DNA strands at or near the same point, generating unit-length linears. This ability to cut both DNA strands is inhibited by such factors as temperature decrease, the addition of a monovalent ion or another divalent cation which is not a transition metal ion, or a reduction in the number of superhelical turns in the DNA substrate. All of these factors lead to a winding of the duplex helix and antagonize the unwinding of the duplex promoted by transition metal ion binding. Transition metal ions may thus convert the DNA substrate locally to a form in which DNase I can introduce breaks into both strands. In the presence of Mg2+, DNase I introduces single strand nicks into SV40(I), generating exclusively the covalently open, relaxed circular SV40(II) as the initial product of the reaction. In the presence of Mn2+, DNase I generates as initial products a mixture of SV40(II) and unit-length SV40 linear DNA molecules, formed by two nicks in opposite strands at or near the same point in the duplex. These circular SV40(II) molecules consist of two types. A minority class is indistinguishable from the nicked SV40(II) produced by DNase I in the presence of Mg2+. The majority class consists of molecules containing a gap in one of the two strands, the mean length of the gap being 11 nucleotides. The SV40(L) molecules produced in the presence of Mn2+ appear to have single strand extensions at one or both ends.

Terminal Repetitions in Herpes Simplex Virus Type 1 DNA

Linear DNA molecules derived from bacteriophage species generally possess some type of repetitious sequence at their termini (Thomas and MacHattie 1967). Basically these repetitious sequences take two forms. The chromosomes of lambdoid phages have short, complementary, single-strand extensions at either end of the DNA; intramolecular association of these regions leads to circularization of the molecule. Other types of phages have longer regions of repetition at each end, and these regions of repetition are fully double stranded. The category of viruses called the herpes-viruses heretofore has not been examined for this property of terminal repetition. We present here evidence that the DNA of herpes simplex virus type 1 (HSV-1) is, in fact, terminally repetitious.

Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli.

We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule.