In vitro polyoma DNA synthesis: asymmetry of short DNA chains.

The kinetics of annealing of the separated strands of the polyoma DNA Hpa II restriction fragments 1 and 2 to an excess of purified short DNA chains isolated from in vitro pulse-labeled replicating polyoma DNA were determined. The results indicate that for each growing fork, the DNA strand which must grow discontinuously is represented about 4 times as frequently in the population of short DNA chains as the strand which could replicate continuously. In addition, the absolute concentration of short DNA chains in the two growing forks is approximately the same. The average size of the short DNA chains from the continuous strand was shown to be very similar to that of the short DNA chains from the discontinuous strand. We conclude that polyoma DNA replication in vitro proceeds by a predominantly semi-discontinuous mechanism.     

In vitro polyoma DNA synthesis: discontinuous chain growth

Using an in vitro system for polyoma DNA synthesis from polyoma-infected mouse BALB/3T3 cells, we have shown that short pulses of radioactively labeled deoxynucleoside triphosphates are incorporated into viral replicative intermediates. Upon denaturation, the pulse-labeled replicative intermediates yield two size classes of growing DNA chains, namely a heterogeneous long class with S values up to unit viral DNA length (16 S) and a rather discrete short class of 5 S pieces. We have shown that these short fragments are involved as precursors in viral DNA chain elongation and that they can be chased into mature viral DNA. The fragments are found in replicative intermediates at all stages of replication and are therefore presumably not involved in specialized initiation or termination processes. Kinetic analysis of the appearance of radioactivity in short and long chains shows that initially approximately equal amounts are incorporated at a linear rate into the two classes. Subsequently, the rate of incorporation into long chains approximately doubles, while the amount of radioactivity in short chains reaches a plateau. This not only suggests that short chains are precursors to long chains, but that the synthesis of long chains occurs as a separate event and is not simply a result of joining of short fragments. Under the in vitro labeling conditions the time taken for radioactivity in short chains to reach a constant level can be used as a measure of the average lifetime of a 5 S piece. Our analysis indicates that there may be a considerable lag between the completion of a 5 S piece and its joining into longer DNA. Estimates of the self-annealing of the short chains showed approximately 20% self-complementarity. Thus, polyoma synthesis in vitro appears to proceed predominantly by a semi-discontinuous mechanism in which the nascent DNA on one side of the growing fork is elongated continuously, while on the other side of the fork DNA is synthesized discontinuously as 5 S fragments, which are subsequently joined. Both the short and the long chains are synthesized in the 5′ to 3′ direction.A fraction of the pulse-labeled material is found as free 3 to 5 S single-stranded DNA. These pieces are of both viral and cellular origin. A majority of them appear to be involved as precursors in DNA chain elongation.     

Self-annealing of 4 S strands from replicating simian virus 40 DNA

The nascent short strands (4 S) isolated from replicating Simian, virus 40 DNA hybridize specifically with denatured SV40 DNA and self-anneal extensively (70 to 92%) when incubated at 68 °C in 1 m-NaCl. Since complementary genetic sequences are present in the 4 S strands, both growing chains of SV40 DNA appear to be synthesized discontinuously at each replication fork.     

Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication.

Simian virus 40 replicating DNA was pulse labeled with alpha-32P-dATP using an acellular DNA replication system. Nascent DNA chains of less than 200 nucleotides (Okazaki pieces) were then isolated from the denatured replicating DNA by electrosieving through a polyacrylamide gel column. The purified Okazaki pieces were hybridized to separated strands of Bg1(1)+Hpa1 simian virus 40 DNA restriction fragments immobilized on nitrocellulose filters. Only strands with polarity of the DNA replication fork direction hybridized with Okazaki pieces. Hence, Okazaki pieces in simian virus 40 are synthesized against the DNA replication fork direction.     

In vitro polyoma DNA synthesis: inhibition by 1-beta-d-arabinofuranosyl CTP.

The effects of 1-beta-D-arabinofuranosyl CTP (ara-CTP) on DNA replication were studied in an in vitro system from polyoma-infected BALB/3T3 cells. Ara-CTP concentrations of larger than or equal to 150 muM were found to block in vitro DNA synthesis completely, and concentrations of smaller than or equal to 0.3 muM had no inhibitory effect. Intermediate concentrations resulted in a concentration-dependent reduction of the in vitro synthesis rate. Long-term labeling with [alpha-32-P]ara-CTP demonstrated the incorporation of the analogue into cellular and viral DNA concomitantly with [3-H]TTP. In pulse-labeling experiments, at noninhibitory concentrations of the analogue, ara-CTP was incorporated into short DNA fragments and long growing strands to relatively the same extent as TTP. Partial venom phosphodiesterase digestion liberated the incoporated are-CTP at essentially the same rate as incorporated TTP, excluding a predominantly terminal incorporation, and after total venom phosphodiesterase digestion greater than 80% of the incorporated ara-CTP was recovered as 5'-ara-CMP. Analysis of the long-term in vitro viral DNA product made in the presence of partially inhibiting ara-CTP concentrations demonstrated that none of the steps leading to mature viral DNA were totally inhibited at the ara-CTP concentrations used. Pulse labeling of replicating viral DNA in the presence of ara-CTP revealed two consistent differences in the pattern found in control pulses: (i) predominant labeling of short chains (5S) with reduced amounts of radioactivity in the longer growing viral DNA strands (smaller than or equal to 16S), and (ii) a one-third to one-half reduction in size for short DNA chains labeled in the presence of ara-CTP. Release of the ara-CTP inhibition with excess dCTP resulted in covalent extension of these smaller short chans to approximately the size of regular short chains labeled in the absence of the inhibitor. Isolated short chains synthesized in the presence of ara-CTP exhibited a slightly lower degree of self-complementarity than regular short chains. The predominant labeling of short chains during pulses is, therefore, not a consequence of discontinuous growth on both sides of the replication fork. Similar results were obtained with ara-ATP and N-ethylmaleimide. The experiments indicate that ara-CTP acts primarily on DNA-polymerizing activities, affecting different DNA polymerases to varying degrees. The results are discussed in terms of the possible number and identity of polymerases involved in viral (and cellular) DNA replication.     

DNA replication in Escherichia coli: evidence for two classes of small deoxyribonucleotide chains

We have used [³H]thymidine to pulse label cultures growing at 20 or 37 °C. At these temperatures, thymidine can be used interchangeably with thymine for labeling periods of 0.1 minute or longer. Incorporation does not stop immediately when cultures are poured on to ice-cold medium-KCN mixtures, but can be stopped by pouring on to the same mixture plus pyridine.We have extracted DNA from cells pulse-labeled with [³H]thymidine and measured the number of short deoxynucleotide chains by treating them with bacterial alkaline phosphatase and labeling their 5′ ends with ³²P using [γ-³²P]ATP and polynucleotide kinase. There are at least 40 deoxynucleotide chains per bacterium (20 per replicating chromosome) whose sedimentation coefficient is less than 15 S.The small deoxynucleotide chains labeled by a pulse of [³H]thymidine behave as intermediates in the replication of DNA. They accumulate if ligase is inhibited and they disappear if their synthesis is blocked by inactivating a gene product responsible for their synthesis. In contrast, the ³²P-labeled pieces do not accumulate rapidly or disappear under the same experimental conditions. The data indicate that many of the ³²P-labeled short chains are not located at the replication fork and that they do not behave as intermediates in the replication of DNA.The size distribution of³H pulse-labeled pieces and of the short chains labeled with ³²P are apparently the same when measured by sedimentation through alkaline sucrose. About 25% of the molecules are extremely short. The remainder are distributed in such a way that, roughly, the number of pieces greater than any particular length decreases exponentially as that length increases.     

DNA chain termination by 2',3'-dideoxythymidine in replicating mammalian cells.

The thymidine analog, 2,3-dideoxythymidine (ddT), is rapidly phosphorylated and incorporated terminally at 3-ends of growing DNA chains in replicating mammalian cells. Following some initial loss of ddT incorporated into DNA chains, the major portion is retained for periods equivalent to more than two normal cell generations. Some ddT appears at the termini of oligonucleotides, a portion of which have chromatographic properties suggesting internally complementary sequences. While these oligonucleotides may include degraduation fragments, it is possible that some represent replication initiation sequences.     

Discontinuous or semi‐discontinuous DNA replication in Escherichia coli?

The postulate that a stalled/collapsed replication fork will be generated when the replication complex encounters a UV-induced lesion in the template for leading-strand DNA synthesis is based on the model of semi-discontinuous DNA replication. A review of existing data indicates that the semi-discontinuous DNA replication model is supported by data from in vitro studies, while the discontinuous DNA replication model is supported by in vivo studies in Escherichia coli. Until the question of whether DNA replicates discontinuously in one or both strands is clearly resolved, any model building based on either one of the two DNA replication models should be treated with caution.     

Replication of DNA in mammalian chromosomes

DNA replication has been studied in cells (CHO) synchronized by mitotic selection from roller cultures. A study of the incorporation of ³H supplied as uridine indicates that cells cannot be blocked precisely at the beginning of the S phase, but DNA synthesis can be stopped in early S by treating with F-dU in G₁. After blockage potential initiation sites continue to increase at a linear rate for atleast 13 hours after division. Incorporation of ³H-thymidine begins at most of these sites within seconds after thymidine is supplied in the medium and incorporation continues at a linear rate for 20–24 minutes. There appears to be a pause after this interval before synthesis is resumed at about two times the initial rate. ³H-bromodeoxyuridine can be substituted for thymidine without affecting the kinetic pattern over a similar period. The increased rate is probably an increase in sites of chain growth rather than a change in rate of chain growth. A study of the labeled DNA segments by band sedimentation in a preformed NaClO₄ isokinetic gradient shows that two distinctly different sized segments can be released from the chromosomes by lysis at submelting conditions. One is the previously reported single chain segments averaging about one-half micron in length, but the other is a much larger segment (26S) which is native DNA with perhaps small regions of single chains presumably at the ends. Primarily single chain DNA is released after 1–2 minute pulse labeling, but after 2 minutes the larger segments (26S) contain most of the newly formed DNA except that attached to the chains of the major part of the template DNA which exhibits a discontinuous distribution, sedimenting far faster than either newly replicated segment. A consideration of the kinetics of formation of the 26S component indicates that is may contain the replicating fork. If this proves to be the correct interpretation the template chains would both have non-adjacent nicks preceeding the fork and also in a post-fork site at a mean distance of about 2 microns in both directions. The isolation of the growing points of DNA replication in chromosomes is now possible and the study of properties of the newly replicated regions should be greatly facilitated.     

Polyoma virus DNA replication is semi-discontinuous.

In marked contrast to simian virus 40 (SV40), polyoma virus (PyV) has been reported to replicate discontinuously on both arms of replication forks. In an effort to clarify the relationship between the mechanisms of DNA replication in these closely related viruses, the distribution of RNA-primed DNA chains at replication forks was examined concurrently in PyV and SV40 replicating DNA purified from virus-infected cells. About one third of PyV DNA chains contained 7 to 9 ribonucleotides covalently linked to their 5'-end. A similar fraction of DNA chains from replicating SV40 DNA contained an oligoribonucleotide that was 6 to 9 residues long and began with either (p)ppA or (p)ppG. Greater than 80% of PyV or SV40 RNA-primed DNA chains hybridized specifically to the retrograde template. Moreover, at least 95% of the RNA-primed DNA chains from either PyV or SV40 whose initiation sites could be mapped to unique nucleotide locations originated from the retrograde template. Therefore, PyV and SV40 DNA replication forks are essentially the same; DNA synthesis is discontinuous predominantly, if not exclusively, on the retrograde template.     

DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier

DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells. This translates into an estimated steady-state level of 7–10 DSBs per cell during S-phase. Importantly, breaks detectable in wild-type and sgs1 mutant cells differ from each other in terms of origin and repair. Breaks in wild-type, which were previously reported as DSBs, are likely an artefactual consequence of nicks nearby the rRFB. Sgs1 deficient cells, in which replication fork stability is compromised, reveal a class of DSBs that are detectable only in the presence of functional Dnl4. Under these conditions, Dnl4 also limits the formation of extrachromosomal ribosomal DNA circles. Consistently, dnl4 cells displayed altered fork structures at the replication fork barrier, leading us to propose an as yet unrecognized role for Dnl4 in the maintenance of ribosomal DNA stability.     

Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins

Rolling circle replication has previously been reconstituted in vitro using M13 duplex circles containing preformed forks and the 10 purified T4 bacteriophage replication proteins. Leading and lagging strand synthesis in these reactions is coupled and the size of the Okazaki fragments produced is typical of those generated in T4 infections. In this study the structure of the DNAs and DNA-protein complexes engaged in these in vitro reactions has been examined by electron microscopy. Following deproteinization, circular duplex templates with linear tails as great as 100 kb are observed. The tails are fully duplex except for one to three single-stranded DNA segments close to the fork. This pattern reflects Okazaki fragments stopped at different stages in their synthesis. Examination of the DNA-protein complexes in these reactions reveals M13 duplex circles in which 64% contain a single large protein mass (replication complex) and a linear duplex tail. In 56% of the replicating molecules with a tail there is at least one fully duplex loop at the replication complex resulting from the portion of the lagging strand engaged in Okazaki fragment synthesis folding back to the replisome. The single-stranded DNA segments at the fork bound by gene 32 and 59 proteins are not extended but rather appear organized into highly compact structures ("bobbins"). These bobbins constitute a major portion of the mass of the full replication complex.     

Analysis of the attachment of replicating DNA to a nuclear matrix in mammalian interphase nuclei.

The attachment of replicating DNA to a rapidly sedimenting nuclear structure was investigated by digestion with various nucleases. When DNA was gradually removed by DNase I, pulse label incorporated during either 1 min or during 1 hour in the presence of arabinosylcytosine, remained preferentially attached to the nuclear structure. Single strand specific digestion by nuclease S1 or staphylococcal nuclease at low concentrations caused a release of about 30% of the pulse label, without significantly affecting the attachment of randomly labelled DNA. The released material had a low sedimentation coefficient and contained most of the Okasaki fragments. The remaining pulse label was less accessible to further digestion by double strand specific nuclease activity than the bulk DNA. The results suggest that an attachment of the replication fork to the nuclear structure occurs at sites behind but close to the branch point.     

Two-dimensional gel electrophoretic method for mapping DNA replicons.

We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems.     

Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.     

Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein

Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.     

RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.