Complementary strands of CELO virus DNA.

When alkali-denatured DNA from CELO virus (an avian adenovirus) was annealed for 15 min at 37 C in 0.1 M NaCl, 70% of the molecules formed single-stranded circles. This is probably due to base pairing of complementary sequences not more than 110 nucleotides long at the ends of the single strands and implies an inverted terminal repetition in the duplex DNA similar to that reported for the DNA from human adenoviruses. The circular molecules had a uniform length that was approximately the same as that of linear single-stranded molecules. The complementary strands of CELO virus DNA were separated on a preparative scale, and at least 40% of the heavy strands and 56% of the light strands were found to be intact as judged by the formation of single-stranded circles.     

The formation of adjacent triplex-duplex domainsin DNA.

The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix-coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12-20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5'-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a dangling end. The 3'-ends of the 17-20 nt strands could form Watson-Crick pairs to the five base loop of the hairpin or the dangling end of the duplex. Complexes of the hairpin DNA with the single-stranded oligomers showed two step transitions consistent with unwinding of the triplex strand followed by hairpin denaturation. Melting curve and gel competition results indicated that the complex of the hairpin and the 12 nt oligomer was more stable than the complexes involving the extended single strands. In contrast, results indicated that the extended single-stranded oligomers formed Watson-Crick base pairs with the dangling end of the duplex DNA and enhanced the stability of the adjacent triplex region.     

Concerted strand exchange and formation of Holliday structures by E. coli RecA protein

RecA protein makes stable joint molecules from fully duplex DNA and molecules that are partially single-stranded; the latter may be either duplex molecules with an internal gap in one strand or molecules with single-stranded ends. Stable joint molecules form only when the end of at least one strand is in a homologous region. When RecA protein pairs linear duplex molecules and tailed molecules that share the same sequence end to end, the joints, which are located away from the single-stranded tails in most instances, have the electron microscopic appearance associated with the Holliday structure resulting from the reciprocal exchange of strands. The reaction leading to reciprocal strand exchange involves the concerted displacement of a strand from the end of the duplex molecule. These observations support the view that RecA protein makes stable joint molecules only by transferring strands and not by the side-by-side pairing of duplex regions.     

Kinetics of homologous pairing promoted by RecA protein: effects of ends and internal sites in DNA

When recA protein was preincubated with single-stranded DNA in the presence of an ATP-regenerating system prior to the addition of homologous duplex DNA, a slow presynaptic step was eliminated, and the subsequent homologous pairing was revealed as a reaction whose rate exceeds by 1 or 2 orders of magnitude the calculated rate of spontaneous renaturation in 0.15 M NaCl at Tm -25 degrees C. The pairing reaction displayed saturation kinetics with respect to both single-stranded and double-stranded DNA, indicating the existence of a rate-limiting enzyme-substrate complex. The signal observed in the assay of the pairing reaction was due to pairing at free homologous ends of the duplex DNA, as well as pairing in the middle of the duplex molecule, away from a free end. The apparent rate of pairing of circular single strands with linear duplex DNA was equal to the sum of the rates of pairing at sites located at either end of the duplex DNA or at interior sites, but the apparent rates attributable to ends were greater, and nicks also stimulated the apparent rate.     

Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA

The vaccinia virus genome is a single, linear, duplex DNA molecule whose complementary strands are naturally cross-linked. The molecular weight has been determined by contour length measurements from electron micrographs to be 122 ± 2.2 × 10⁶. Denaturation mapping techniques indicate that the nucleotide sequence arrangement of the DNA is unique. Two forms of cross-linked vaccinia DNA were observed in alkaline sucrose gradients. The relative S-values of the two cross-linked species were appropriate for a single-stranded circle and a linear single strand, each with a molecular weight twice that expected for an intact, linear, complementary strand of vaccinia DNA. The fraction of sheared vaccinia DNA able to “snap back” after denaturation suggested a minimum of two crosslinks per molecule. Full-length single-stranded circles were observed in the electron microscope after denaturation of vaccinia DNA. Partial denaturation produced single-stranded loops at the ends of all full-length molecules. Exposure of native vaccinia DNA to a single strand-specific endonuclease isolated from vaccinia virions caused disruption of the cross-links, as assayed by alkaline sedimentation, and produced free single-strand ends when partially denatured DNA was observed in the electron microscope. We conclude that vaccinia DNA contains two cross-links, one at or near (within 50 nucleotides) each end in a region of single-stranded DNA. Two models for the cross-links are presented.     

The specificity of lambda exonuclease. Interactions with single-stranded DNA.

The lambda exonuclease, an enzyme that has been implicated in genetic recombination, rapidly and processively degrades native DNA, starting at the 5' terminus. The enzyme will also degrade the 5'-terminated strand at a single-stranded branch. The experiments reported here reveal various interactions of the enzyme with single-stranded DNA. The rate of digestion is related inversely to the length of single strands. Chains of 100 nucleotides are digested at about 10% the rate of digestion of native DNA. Digestion of the single-stranded ends of lambda DNA does not appear to occur processively. The enzyme binds to circular as well as linear single strands and the affinity for single strands is also related inversely to the chain length. In an equimolar mixture of single- and double-stranded DNA the action of lambda exonuclease on the latteris about half-inhibited. At 20 degrees the initiation of digestion at the 5' terminus of duplex DNA is blocked sterically when such DNA has 3'-terminal single strands that are longer than 100 nucleotides. Information about these properties is important for the practical use of lambda exonuclease as well as for reflections on the role of the enzyme in genetic recombination.     

Underwound loops in self-renatured DNA can be diagnostic of inverted duplications and translocated sequences

DNA that contains inverted duplications separated by non-inverted sequences often can form characteristic “underwound loops” when it is denatured and reannealed. An underwound loop is a partially double-stranded, partially denatured segment between the inverted duplications and is produced as follows. During the early stages of the reannealing, intrastrand stem-loop structures form with first-order kinetics when the inverted duplications pair. In a slower second-order reaction, complementary strands (each with a stem-loop) reanneal. The stem-loop structures produce a cruciform in the hybrid. Because of the unpaired sequences in the loop, the cruciform is unstable. It can isomerize to a linear duplex by double-strand exchange of complementary sequences in the stems. This process requires co-ordinated axial rotation of the stems and the flanking duplexes as well as rotation of the loops. If, however, complementary sequences in the loops start to pair, axial rotation is prevented and the stem-loop structures are trapped in a metastable state. The strands of separate, closed rings cannot interwind when they pair. Consequently, the loops observed by electron microscopy have variable patterns of single-stranded denaturation bubbles and duplex segments with both right-handed and left-handed winding.We have used underwound loops to identify a short inverted duplication flanking the γδ recombination sequence of Escherichia coli F factor (isolated on φ80 d₃ilv⁺ transducing phage) and to study DNA from phages Mu and P1 in which the G segments are flanked by inverted duplications. When deproteinized adenovirus-2 DNA was denatured and reannealed, some underwound circles the length of the entire chromosome were observed by electron microscopy. These resulted from the restricted interaction of complementary single-stranded rings generated when pairing of the short inverted terminal duplications closed the ends of single strands. Another type of underwound loop was seen in heteroduplexes containing complementary insertion loops located at different positions in the hybridized strands, such as occurs with P1 cam DNAs. All these underwound structures are similar in appearance to the hybrids formed when topologically separate, complementary single-stranded circles of Colicin E₁ DNA were allowed to anneal.     

The structure of Tetrahymena pyriformis mitochondrial DNA. II. The complex structure of strain GL mitochondrial DNA.

1. Isolated mtDNA from Tetrahymena pyriformis strain GL is a linear duplex molecule with an average molecular weight of 32.6 - 10(6) and without internal gaps or breaks. Denaturation of this DNA results in single strands with a duplex hairpin at one end. The length of this hairpin varies between 0 and 5 micrometer within one preparation. 2. Uder renaturation conditions the single strands with hairpins are able to circularize in two ways, depending on the length of the hairpin. Circularization is also observed after partial digestion with exonuclease III of native strain GL mtDNA. 3. All these data fit a model (see Fig.2) in which the DNA is heterogeneous in length at both ends. At the left end a 10-micrometer duplication-inversion is present; part of this duplication-inversion is complementary to a region at the right end of the molecule. 4. The analogy between the structural peculiarities of strain GL mtDNA and of some linear viral DNAs is stressed.     

The structure of replicating adenovirus 2 DNA molecules

Adenovirus 2 (Ad2)-infected KB cells were exposed to a 2.5 min pulse of ³H-thymidine at 19 hr after infection. The labeled DNA molecules were separated from cell DNA and mature Ad2 DNA by sucrose gradient sedimentation and CsCI equilibrium centrifugation under conditions designed to minimize branch migration and hybridization of single strands. Electron microscopy-of fractions containing radioactivity revealed two basic types of putative replicating molecules: Ad2 length duplex DNA molecules with one or more single-stranded branches (type I) and Ad2 length linear DNA molecules with a single-stranded region extending a variable distance from one end (type II). Length measurements, partial denaturation studies and 3′ terminal labeling experiments were consistent with the following model for Ad2 DNA replication. Initiation of DNA synthesis occurs at or near an end of the Ad2 duplex. Following initiation, a daughter strand is synthesized in the 5′ to 3′ direction, displacing the parental strand with the same polarity. This results in the formation of a branched replicating molecule (type I). Initiations at the right and left molecular ends are approximately equal in frequency, and multiple initiations on the same replicating molecule are common. At any given displacement fork in a type I molecule, only one of the two parental strands is replicated. Two nonexclusive mechanisms are proposed to account for the replication of the other parental strand. In some cases, before completion of a round of displacement synthesis initiated at one end of the Ad2 duplex, a second initiation will occur at the opposite end. In these doubly initiated molecules, both parental strands serve as templates for displacement synthesis. Two type II molecules are generated when the oppositely moving displacement forks meet. Alternatively, displacement synthesis may proceed to the end of the Ad2 duplex, resulting in the formation of a daughter duplex and a parental single strand. Replication of the displaced parental strand is then initiated at or near its 3′ terminus, producing a type II molecule. Daughter strand synthesis proceeds in the 5′ to 3′ direction in type II molecules generated by either mechanism, and completion of synthesis results in the formation of a daughter duplex.     

Preparation and melting of single strand circular DNA loops.

A method for preparation of single strand DNA circles of almost arbitrary sequence is described. By ligating two sticky ended hairpins together a linear duplex is formed, closed at both ends by single stranded loops. The melting characteristics of such loops are investigated using optical absorbance and NMR. It is shown by comparison with the corresponding linear sequence (closed circle minus the end loops) that the effects of end fraying and the strand concentration dependence of the melting temperature are eliminated in the circular form. Over the concentration range examined (0.5 to 2.0 micromolar strands), the circular DNA has a monophasic melting curve, while the linear duplex is biphasic, probably due to hairpin formation. Since effects of duplex to single strands dissociation do not contribute to melting of the circular molecules (dumbells), these DNAs present a realistic experimental model for examining local thermal stability in DNA.     

Production of triple-stranded recombination intermediates by RecA protein, in vitro

During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state.     

Activation of DNA-dependent protein kinase by single-stranded DNA ends

DNA-dependent protein kinase (DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination. The kinase is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PK(CS). To define the DNA structure required for kinase activation, we synthesized a series of DNA molecules and tested their interactions with purified DNA-PK(CS). The addition of unpaired single strands to blunt DNA ends increased binding and activation of the kinase. When single-stranded loops were added to the DNA ends, binding was preserved, but kinase activation was severely reduced. Obstruction of DNA ends by streptavidin reduced both binding and activation of the kinase. Significantly, short single-stranded oligonucleotides of 3-10 bases were capable of activating DNA-PK(CS). Taken together, these data indicate that kinase activation involves a specific interaction with free single-stranded DNA ends. The structure of DNA-PK(CS) contains an open channel large enough for double-stranded DNA and an adjacent enclosed cavity with the dimensions of single-stranded DNA. The data presented here support a model in which duplex DNA binds to the open channel, and a single-stranded DNA end is inserted into the enclosed cavity to activate the kinase.     

Further characterization of DNA helicase activity of mouse DNA-dependent adenosinetriphosphatase B (DNA helicase B)

The DNA helicase activity of DNA-dependent ATPase B purified from mouse FM3A cells [Seki, M., Enomoto, T., Hanaoka, F., & Yamada, M. (1987) Biochemistry 26, 2924-2928] has been further characterized. The helicase activity was assayed with partially duplex DNA substrates in which oligonucleotides to be released by the enzyme were radiolabeled. Oligonucleotides with or without phosphate at the 5' termini or with a deoxy- or dideoxyribose at the 3'-terminal nucleotides were displaced by this enzyme with essentially the same efficiency and with the same ATP (and dATP) and Mg2+ requirements. Thus, there was no strict structure requirement for both ends of duplex regions of substrates to be unwound by the enzyme. Shorter strands were released more readily than longer strands up to the length of 140 bases. The attachment of the enzyme to a single-stranded DNA region was a prerequisite for the neighboring duplex to be unwound; the enzyme-catalyzed unwinding was inhibited competitively by the coaddition of single-stranded DNAs which act as cofactors of the ATPase activity. Their activities as the inhibitor of helicase were well correlated with those as the cofactor of ATPase. The helicase B was found to migrate along single-stranded DNA in the 5' to 3' direction by the use of single strands with short duplex regions at both 3' and 5' ends as substrate. A possible role of this enzyme in DNA replication in mammalian cells is discussed.     

The homologous recombination system of phage lambda. Pairing activities of beta protein

The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells. These proteins seem to occur in vivo as an equimolar complex. In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000. The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis. beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W. K. (1981) J. Biol. Chem. 256, 12636-12639). To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease. Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands. Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA. beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase. The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions. Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.     

Electron microscopic study of equine herpesvirus type 1 DNA.

Electron microscopic studies of equine herpesvirus DNA revealed that single strands that were allowed to reanneal formed single-stranded loops with double-stranded stems only at one end of the molecule. These observations support restriction enzyme analyses which indicate that the 92-megadalton DNA molecule exists as a long region of unique sequences covalently linked to a short region. The short region is comprised of an internal unique sequence, which forms the loop during reannealing of single strands, and two terminal inverted repeat sequences that bracket the unique sequence and form the double-stranded stem structure observed upon reannealing of single strands. Measurements of the unique sequence and terminal inverted repeat subgenomic sequences indicate a size of 6.4 megadaltons for each and thus fix the size of the short region at approximately 19.2 megadaltons.     

A duplex structure involving two non-complementary DNA strands can be formed and stabilized by M13 phage proteins

M13 phage is a long, thin nucleoprotein filament containing a single-stranded DNA loop. Exposing the filaments to a chloroform/water interface at 20 °C causes them to contract into hollow spherical particles (spheroids), while exposure at low temperatures yields short, thick rods (I-forms). All of the DNA remains within the I-forms while a specific third remains within the spheroids. Here, a photo-crosslinking reagent, psoralen, has been used to probe secondary structure of the DNA in situ in these chloroform-relaxed phage forms. Following photo-crosslinking, the DNA that had been held within the spheroids appeared to be a duplex rod when seen by electron microscopy, while the DNA extruded from the spheroids was an open single-stranded DNA loop. Photo-crosslinking of the DNA in the I-forms yielded linear duplex DNA rods close to the length of M13 phage filaments. Similar observations derived from experiments with deletion and insertion mutant phage showed that the stability of the duplex rods did not depend on the sequence homology between the two opposing strands. These results showthat two non-homologous strands of DNA can exist in an apparently duplex structure and suggest that this is directed by proteins, possibly involving interaction at a membrane.     

Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome

Complementary strands of adeno-associated virus DNA labeled with 32P at the 5' ends were separated and then self-annealed to form single-stranded circles stabilized by hydrogen bonds between the complementary sequences in the inverted terminal repetitions. We have previously shown that there are two distinct sequences in the terminal repetition which represent an inversion of the first 125 nucleotides (E. Lusby et al., J. Virol. 34:402-409, 1980; I. S. Spear et al., Virology 24:627-634, 1977). Base pairing between terminal sequences of the same orientation leads to a normal double helical structure. If sequences of the opposite orientation pair, an aberrant secondary structure is formed. HpaII digestion of the self-annealed, single-stranded circles led to labeled terminal fragments that corresponded both to those generated from termini of a normal double helical structure and those generated from an aberrant terminal secondary structure. Thus, the orientation of the terminal repetition at one end of the genome is not influenced by the orientation at the other end.     

Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands

Human mitochondrial DNA contains two physically separate and distinct origins of DNA replication. The initiation of each strand (heavy and light) occurs at a unique site and elongation proceeds unidirectionally. Animal mitochondrial DNA is novel in that short nascent strands are maintained at one origin (D-loop) in a significant percentage of the molecules. In the case of human mitochondrial DNA, there are three distinct D-loop heavy strands differing in length at the 5' end. We report here the localization of the 5' ends of nascent daughter heavy strands originating from the D-loop region. Analyses of the map positions of 5' ends relative to known restriction endonuclease cleavage sites and 5' end nucleotides indicate that the points of initiation of D-loop synthesis and actual daughter strands are the same. In contrast, the second origin is located two-thirds of the way around the genome where light strand synthesis is presumably initiated on a single-stranded template. Mapping of 5' ends of daughter light strands at this origin relative to known restriction endonuclease cleavage sites reveals two distinct points of initiation separated by 37 nucleotides. This origin is in the same relative genomic position and shows a high degree of DNA sequence homology to that of mouse mitochondrial DNA. In both cases, the DNA region within and immediately flanking the origin of DNA replication contains five tightly clustered tRNA genes. A major portion of the pronounced DNA template secondary structure at this origin includes the known tDNA sequences.