Short Single-stranded Region Research Articles

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.

Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System

CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology.

Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction.

The Mechanism of Rolling Circle DNA Replication and the Roles of Initiator Protein RepD

Rolling circle replication is used by plasmids, which carry antibiotic-resistance genes and are transferred between certain bacteria, thereby transmitting the resistance. In this mechanism the two strands are asynchronously copied: one simultaneously with unwinding the parent plasmid, the other strand initially forms a single-stranded circle. As well as polymerase and helicase (PcrA), which are recruited from the host, an initiator protein is also required: RepD in the work described here. This binds specifically to the double-stranded origin of replication and initiates unwinding by nicking one strand and forming a covalent bond with the 5′-end. This nicking opens up a short single-stranded region and so allows the helicase and polymerase to bind and begin unwinding and replication, respecively. After travelling all around the plasmid probably as a single protein complex, the proteins then reach the newly synthesised origin of replication, which provides the signal for termination. RepD then does a series of strand exchanges to close the two plasmid circles. We are using a combination of measurements with whole plasmids and oligonucleotide models to elucidate the series of events at each stage of the replication. In particular, by following individual processes in real time, we are able to describe the order of biochemical steps that enable this process to occur.

In Vivo SELEX of Single-Stranded Domains in the HIV-1 Leader RNA

The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two segments we document the selection of the (nearly) wild-type sequence, thus demonstrating the importance of these primary nucleotide sequences and the power of the in vivo SELEX approach. However, for the third segment a large variety of sequences is compatible with efficient HIV-1 replication. Interestingly, the A-rich sequence of this segment is highly conserved among HIV-1 isolates, which likely reflects the evolutionary tendency of HIV-1 to adopt A-rich sequences.

In vivo SELEX of single-stranded domains in the HIV-1 leader RNA

The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two segments we document the selection of the (nearly) wild-type sequence, thus demonstrating the importance of these primary nucleotide sequences and the power of the in vivo SELEX approach. However, for the third segment a large variety of sequences is compatible with efficient HIV-1 replication. Interestingly, the A-rich sequence of this segment is highly conserved among HIV-1 isolates, which likely reflects the evolutionary tendency of HIV-1 to adopt A-rich sequences.

Interplay between DNA N-glycosylases/AP lyases at multiply damaged sites and biological consequences

Evidence has emerged that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. The first repair event occurring at a multiply damaged site (MDS) is of major importance and will largely contribute to the hazardousness of MDS. Here, using protein extracts from wild type or hOGG1-overexpressing Chinese hamster ovary cells, we investigated the initial incision rate at base damage and the formation of repair intermediates in various complex MDS. These MDS comprise a 1 nt gap and 3–4 base damage, including 8-oxoguanine (oG) and 5-hydroxyuracil (hU). We report a hierarchy in base excision that mainly depends on the nature and the distribution of the damage. We also show that excision at both oG and hU, and consequently DSB formation, can be modulated by hOGG1 overexpression. Anyhow, for all the MDS analyzed, DSB formation is limited, due to impaired base excision. Interestingly, repair intermediates contain a short single-stranded region carrying a potentially mutagenic base damage. This in vitro study provides new insight into the processing of MDS and suggests that repair intermediates resulting from the processing of such MDS are rather mutagenic than toxic.

Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins

Clustered DNA lesions, possibly induced by ionizing radiation, constitute a trial for repair processes. Indeed, recent studies suggest that repair of such lesions may be compromised, potentially leading to the formation of lethal double-strand breaks (DSBs). A complex multiply damaged site (MDS) composed of 8-oxoguanine and 8-oxoadenine on one strand, 5-hydroxyuracil, 5-formyluracil and a 1 nt gap on the other strand, within 17 bp was built and used to challenge several steps of base excision repair (BER) pathway with human whole-cell extracts and purified repair enzymes as well. We show a hierarchy in the processing of lesions within the MDS, in particular at the base excision step. In the present configuration, efficient excision of 5-hydroxyuracil and low cleavage at 8-oxoguanine prevent DSB formation and generate a short single-stranded region carrying the 8-oxoguanine. On the other hand, rejoining of the 1 nt gap occurs by the short-patch BER pathway, but is slightly retarded by the presence of the oxidized bases. Taken together, our results suggest a hierarchy in the processing of the lesions within the MDS, which prevents the formation of DSB, but would dramatically enhance mutagenesis. They also indicate that the mutagenic (or lethal) consequences of a complex MDS will largely depend on the first event in the processing of the MDS.

Type IA DNA topoisomerases: strictly one step at a time.

During DNA replication and transcription, the winding of the DNA helix undergoes dramatic changes that give rise to DNA supercoiling. Additionally, both DNA replication and recombination can leave the DNA in a tangled, catenated, or interwound state. DNA topoisomerases are the enzymes charged with resolving these topological problems in the DNA (for reviews, see refs. 1–3). In addition, in bacteria two or more DNA topoisomerases act in concert to maintain a fixed global level of helical tension in the DNA that is critical for its proper function. Topoisomerases alter the topological state of the DNA by either passing one strand of the helix through the other strand (type I family) or by passing a region of duplex DNA through another region of duplex DNA (type II family). The two families of enzymes carry out this molecular sleight of hand by reversibly cleaving one or both strands of the DNA, respectively. The reversibility of the reaction is ensured by the covalent attachment of the enzyme to a DNA end (or ends) through a phosphodiester linkage involving a tyrosine residue in the enzyme. The type I family is further subdivided into two subfamilies depending on the polarity of attachment of the topoisomerase to the DNA; members of the type IA subfamily become attached to 5′ phosphate termini, whereas members of the type IB subfamily attach to 3′ phosphates. The requirement for a type IA enzyme to bind a short single-stranded region in the substrate further distinguishes the type IA enzymes from the type IB enzymes, which prefer completely duplex DNA. What happens after cleavage to effect the strand passage reaction has long fascinated investigators working on the type I enzymes. The experiments described by Dekker et al. (4) in this issue of PNAS provide an elegant and convincing test of a model …

Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

Masked antisense: a molecular configuration for discriminating similar RNA targets.

Antisense technology has great potential for the control of RNA expression, but there remain few successful applications of the technology. Expressed antisense RNA can effectively down-regulate expression of a gene over long periods, but cannot differentiate partly identical sequences, such as the mRNA of fusion genes or those with point mutants. We have designed a structured form of expressed antisense, which can discriminate between highly similar mRNA molecules. These 'masked' antisense RNAs have most of the antisense sequence sequestered within duplex elements, leaving a short single-stranded region to initiate binding to target RNA. After contacting the correct target, the structured RNA can unravel, releasing the masked antisense region to form a stable duplex with the mRNA. We demonstrate that suitable masked antisense RNA can discriminate between the two forms of BCR-ABL mRNA that result from the Philadelphia chromosomal translocations, as well as discriminating the normal BCR and ABL mRNA.

Inhibition of rev·RRE complexation by triplex tethered oligonucleotide probes

We have described a class of molecules, called tethered oligonucleotide probes (TOPs), that bind RNA on the basis of both sequence and structure. TOPs consist of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. In a triplex TOP, one oligonucleotide recognizes a short single-stranded region in a target RNA through the formation of Watson-Crick base pairs; the other oligonucleotide recognizes a short double-stranded region through the formation of Hoogsteen base pairs. Binding of triplex TOPs to an HIV-1 Rev Response Element RNA variant (RRE AU) was measured by competition electrophoretic mobility shift analysis. Triplex TOP·RRE AU stabilities ranged between −9.6 and −6.1 kcal mol 1 under physiological conditions of pH, salt, and temperature. Although the most stable triplex TOP·RRE AU complex contained 12 contiguous U·AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Triplex TOPs inhibited formation of the RRE·Rev complex with IC 50 values that paralleled the dissociation constants of the analogous triplex TOP·RRE AU complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of Rev·RRE AU complexation by triplex TOPs did not require pre-incubation of RRE AU and a TOP: triplex TOPs competed efficiently with Rev for RRE AU and inhibited RRE AU·Rev complexation at equilibrium.

Renaturase and ribonuclease H: a novel mechanism that influences transcript displacement by RNA polymerase II in vitro.

I have previously reported an activity in HeLa cells which facilitates transcript displacement by purified mammalian RNA polymerase II in vitro. I have shown that this activity copurifies with one of two separable ribonuclease (RNase) H activities in HeLa cells. The RNase H activity in question has characteristics similar to those reported for RNase H2b from calf thymus. RNase H proteins purified from several other sources including Escherichia coli also show renaturase activity. When the renaturase/RNase H protein is present during transcription by purified RNA polymerase II, transcripts are truncated close to the 5' end, and the remainder of the transcript is displaced normally from its template by the polymerase. Since RNA polymerase II dependent transcripts in vivo normally require the presence of the 5'-triphosphate terminus for capping, the in vivo significance of RNase H as a renaturase factor is presently not understood. However, the in vitro action of renaturase/RNase H suggests that the mechanism of this reaction may involve R-loop displacement after formation of a short single-stranded region of DNA on the template strand following hydrolysis of a hybrid transcript oligonucleotide by RNase H.

Locations of methyl groups in 28 S rRNA of Xenopus laevis and man : Clustering in the conserved core of molecule

28 S ribosomal RNA from several vertebrate species contains some 68 to 70 methyl groups. Evidence described in this paper enables some 58 methyl groups to be located in the primary structure of 28 S ribosomal RNA from Xenopus laevis. Most of the locations are unambiguous but a few are currently tentative. In human 28 S ribosomal RNA the great majority of the same sites are methylated as in Xenopus, but there are a few differences between the respective methyl group distributions. The main features of the methyl group distribution are as follows. (1) All of the identified methyl groups are in conserved core regions of 28 S ribosomal RNA. (2) Methylation is much more heavily concentrated in the 3′ region of the molecule than in the 5′ region (in contrast to 18 S ribosomal RNA, in which there is a major cluster of 2′- O-methyl groups in the 5′ region). (3) In addition to the heavily methylated 3′ region, clusters of methyl groups occur elsewhere in 28 S ribosomal RNA in the vicinity of domain boundaries. For domains 3 to 6, the two ends of each domain are united in a helix and are linked to adjacent domains either directly or by short single-stranded regions. It therefore follows that the methyl groups near the boundaries of these domains come together into the same general region of the three-dimensional structure. Within this large-scale pattern of distribution, methyl groups occur in a variety of local environments, examples of which are discussed. The triply methylated sequence Am-Gm-Cm-A occurs in a short single-stranded region which links domain 3 to domain 4. Near the 3′ end of domain 5 there is a cluster of 11 methyl groups including a 2′- O-methyl pseudouridine in a tract of 160 nucleotides whose sequence is totally conserved between Xenopus and man. These methyl groups are variously distributed between single-stranded regions and short or imperfect but conserved helices. A further cluster of methyl groups including the highly conserved Um-Gm-ψ sequence occurs in a region of domain 6 which is implicated in peptidyl transfer. Possible relationships between methylation and other events in ribosome maturation are discussed.

Tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains.

Purification and properties of vaccinia virus DNA-dependent RNA polymerase.

Vaccinia virus DNA-dependent RNA polymerase has been solubilized from purified virions and purified over 2500-fold by chromatography on DEAE-cellulose and phosphocellulose with a 64% yield. RNA synthesis catalyzed by the purified enzyme requires the presence of a DNA template, a divalent cation (Mn2’), and the four ribonucleoside triphosphates. The polymerase activity is inhibited by actinomycin, by monovalent salt concentrations in excess of 0.1 M, by N-ethylmaleimide, and by pyrophosphate, yet is insensitive to rifampicin and to a-amanitin. Single-stranded DNAs are the most effective templates for the vaccinia RNA polymerase. The enzyme is inactive with duplex DNAs, including vaccinia DNA. Superhelical DNAs do serve as template for vaccinia polymerase, yet their template activity is abolished by relaxation of the DNA by DNA topoisomerase. The binding of RNA polymerase to DNA is also most effective with single-stranded DNAs; duplex DNAs are inert. The binding of polymerase to superhelical DNAs may be eliminated by nicking-closing enzyme. The RNA product synthesized in the presence of single-stranded DNA is of large size (23 S), while RNA made in the presence of superhelical DNAs is very small (<4 S), suggesting that the polymerase binds to and transcribes short single-stranded regions present in superhelical DNAs. Although seemingly incapable of displacing and reading through DNA duplex structures, the vaccinia polymerase is capable of transcribing through an RNA:DNA hybrid with concomitant displacement of the RNA strand.

Recognition of duplex DNA containing single-stranded regions by recA protein.

Genetic recombination in Escherichia coli requires recA protein, the product of the recA+ gene. In this paper we show that purified recA protein, which binds strongly to denatured DNA, cooperatively recognizes DNA containing short single-stranded regions. The interaction of varying amounts of recA protein with DNA molecules was investigated by measuring its DNA-dependent ATPase activity. In 3mM Mg2+, the ATPase activity was stimulated by excess single-stranded DNA and was minimal with either intact circular or blunt-ended linear duplexes. Single-strand gaps of about 30 nucleotides were sufficient to increase the ATPase activity to a level almost as great as that observed with single-stranded DNA. Sedimentation studies at neutral pH showed cooperative binding of recA protein to single-stranded DNA or to duplex DNA containing single-stranded regions. In the presence of ATP, an intermediate rate of sedimentation was observed; in contrast, adenosine 5'-gamma-thiotriphosphate (ATP[S]) caused the formation of fast-sedimenting DNA-protein complexes. Gapped plasmid DNA plus recA protein and ATP[S] formed large aggregates containing thousands of molecules. Complex formation and stimulation of the ATPase activity of recA protein with duplex DNA containing single-stranded regions indicates that recA protein may change the conformation of the normally duplex molecules to a conformation prepared for homologous pairing.

CXIII. Total synthesis of the structural gene for an alanine transfer RNA from yeast. Enzymic joining of the chemically synthesized segments to form the DNA duplex corresponding to nucleotide sequence 17 to 50

The DNA duplex (designated [B] and shown in Fig. 1), corresponding to the middle part (nucleotide sequence 17 to 50) of the major yeast alanine transfer RNA has been synthesized. The duplex contained short single-stranded regions at both ends for subsequent joining to the [A](paper CXII) and [C](paper CXIV) parts of the total DNA duplex. The T4 ligase was shown to bring about the joining of d-(5′- 32P)-C-T-A-A-G (segment 9) to the icosanucleotide (segment 6) in the presence of the complementary segment 8 (Fig. 1); similarly, d-(5′- 32P)-A-G-A-G-T-C-T (segment 7) could be joined efficiently to segment 8 in the presence of segment 6. The synthesis of [B] containing the five components (segments 5 to 9) was carried out as follows: the joining of segment 9 to segment 6 and that of segment 7 to segment 8 was carried out in one step at 15 °C. Without isolation of the resulting duplex, 5′- 32P-labeled segment 5 was added and the incubation continued at 25 °C. The joining of segment 7 was complete at this temperature. As required for joining [B] to the two terminal parts, [A] and [C] of the DNA, [B] as obtained above was enzymically phosphorylated at the two 5′-terminal deoxyguanosine units using γ-labeled ATP and polynucleotide kinase and the product was isolated by gel-filtration.

RNA-dependent DNA polymerase activity of RNA tumor viruses. I. Directing influence of DNA in the reaction.

The template requirements and deoxyribonucleic acid (DNA) products of the DNA polymerases isolated from Rauscher leukemia and avian myeloblastosis viruses have been examined. All DNA preparations or synthetic polydeoxynucleotides which are active as primers possess a duplex structure containing single-stranded regions with a 3'-hydroxyl terminus. Native DNA and fully single-stranded DNA are inactive; moreover, their activity is not enhanced by sonic oscillation or treatment with micrococcal nuclease, Neurospora nuclease, or low levels of deoxyribonuclease I. Poor DNA templates are activated by treatment with exonuclease III, large amounts of deoxyribonuclease I, or an endonuclease isolated from Rauscher viral preparations. In reactions primed with deoxyadenylate-deoxythymidylate copolymer, the product formed is covalently attached to primer strands, indicating that no new strands are initiated. DNA polymerase products formed with exonuclease III- or deoxyribonuclase I-treated DNA are duplex structures. Short single-stranded regions are completely filled in, whereas long single-stranded regions are only partly repaired. DNA preparations containing extensive single-stranded regions are poorly utilized as templates.

A symmetrical model for polyoma virus DNA replication

Replicating polyoma virus DNA (DNA II ∗) is known to consist of three-branched circular duplexes containing short single-stranded regions. Under the action of a nuclease from Neurospora crassa conidia, which is highly specific for single-stranded nucleic acids, DNA II ∗ undergoes two successive configurational changes without being fragmented, resulting in marked alterations of its sedimentation properties. Electron microscopic observations performed at intervals on the incubation mixture show that DNA II ∗ is first converted into ring-shaped structubes with one tail attached, while the total number of branched duplexes tends to decrease subsequently. The latter observation suggests that the final product of the reaction might be a non-branched linear duplex. Since the configurational changes described develop concurrently with the disappearance of the single-stranded regions which DNA II ∗ contains, it is proposed that two such regions exist in most replicating molecules. They would be located at the two branch points, but on different replicated branches. On the basis of these observations, it is suggested that each branch point actually represents a growing point where replicating chains are elongated by different mechanisms, in accordance with their opposite polarities.