Chromatin assembly during SV40 DNA replication in vitro

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.     

Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex

The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled.     

E. coli DNA binding protein HU forms nucleosomelike structure with circular double-stranded DNA.

The incubation of the E coli DNA binding protein HU with relaxed circular SV40 DNA in the presence of pure nicking-closing enzyme introduces up to 18 negative superhelical turns in the DNA molecules as measured by agarose gel electrophoresis. The maximal density of supercoiling is obtained at a HU-DNA mass ratio of 1. Reconstituted DNA-HU complexes prefixed with glutaraldehyde appear as condensed circular structures having an average of 14 "beads" per circular SV40 DNA molecule, with a "bead" diameter of 180 +/- 23 A. The circular SV40 DNA is condensed by a ratio of 2.0-2.5 relative to naked DNA. This is similar to the ratio (2.4) measured for chromatin formed by reassociation of relaxed SV40 DNA with the four core histones.     

Supercoiling unwinds two-micrometer plasmid yeast DNA at the origin of replication

All studied origins of replication of DNA in Saccharomyces cerevisiae contain DNA unwinding elements. The introduction of unrestrained negative supercoiling leads to melting of the two DNA strands in DNA unwinding elements. To understand the mechanism of DNA replication it is important to know whether the most unstable region of DNA coincides with the origin of replication. Two-micrometer plasmid DNA from S. cerevisiae inserted in pBR322 was investigated by cleaving with snake venom phosphodiesterase. Its single-strand endonucleolytic activity allows cutting of negatively supercoiled DNA in the DNA unwinding elements. The sites of the venom phosphodiesterase hydrolysis were mapped by restriction enzymes. This study shows that the unwinding of the two-micrometers plasmid DNA of S. cerevisiae takes place only in the origin of replication as a result of unrestrained negative supercoiling.     

The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication

Human single-stranded DNA binding protein (human SSB) is a multisubunit protein containing polypeptides of 70, 34, and 11 kDa that is required for SV40 DNA replication in vitro. In this report we identify the functions of the SSB and its individual subunits in SV40 DNA replication. The 70 kDa subunit was found to bind to single-stranded DNA, whereas the other subunits did not. Four monoclonal antibodies against human SSB were isolated which inhibited SV40 DNA replication in vitro. The antibodies have been designated alpha SSB70A, alpha SSB70B, alpha SSB70C, and alpha SSB34A to indicate which subunits are recognized. Immunolocalization experiments indicated that human SSB is a nuclear protein. Human SSB is required for the SV40 large tumor antigen-catalyzed unwinding of SV40 DNA and stimulates DNA polymerases (pol) alpha and delta. The DNA unwinding reaction and stimulation of pol delta were blocked by alpha SSB70C, whereas the stimulation of pol alpha by human SSB was unaffected by this antibody. Conversely, alpha SSB70A, -70B, and -34A inhibited the stimulation of pol alpha, but they had no effect on DNA unwinding and pol delta stimulation. None of the antibodies inhibited the binding of SSB to single-stranded DNA. These results suggest that DNA unwinding and stimulation of pol alpha and pol delta are required functions of human SSB in SV40 DNA replication. The human SSB 70-kDa subunit appears to be required for DNA unwinding and pol delta stimulation, whereas both the 70- and 34-kDa subunits may be involved in the stimulation of pol alpha.     

DNA helicase and duplex DNA fragment unwinding activities of polyoma and simian virus 40 large T antigen display similarities and differences.

We have characterized the biochemical activities of purified polyoma (Py) large T antigen (T Ag) that was capable of mediating the replication of a plasmid containing the Py origin (ori(+) DNA) in mouse cell extracts. We report here that like the T Ag encoded by simian virus 40 (SV40), Py T Ag has DNA helicase and double-stranded DNA fragment unwinding activities. Py T Ag displaced DNA fragments greater than 1,600 nucleotides which were annealed to complementary sequences in single-stranded M13 by translocating in the 3' to 5' direction. Both helicase and double-stranded DNA fragment unwinding reactions were completely dependent upon NTP hydrolysis, displaying a strong preference for ATP and dATP. At low T Ag concentrations, significantly more Py ori(+) DNA fragment was unwound compared with a fragment lacking the replication origin. However, at higher ratios of Py T Ag to DNA, equivalent to those used in replication reactions, unwinding of both ori-containing and -lacking fragments was equally efficient. This is in contrast to SV40 T Ag which exhibited a more stringent requirement for SV40 origin sequences under similar conditions. Furthermore, some of the nucleotides that supported the helicase and unwinding activities of Py T Ag were different from those for the same SV40 T Ag reactions. We have also observed that in contrast to the very poor replication of linear SV40 ori(+) DNA by SV40 T Ag in human cell extracts, linear Py ori(+) DNA was replicated efficiently in mouse cell extracts by Py T Ag. However, despite the fact that linear Py ori(+), SV40 ori(+), and ori(-) DNA fragments could be unwound with comparable efficiency by Py T Ag, only fragments containing the Py replication origin were replicated in vitro. These results suggest that the initiation of DNA synthesis at the Py origin of replication requires features in addition to unwinding of the template.     

Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro.

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.     

The unwinding of duplex regions in DNA by the simian virus 40 large tumor antigen-associated DNA helicase activity

The DNA helicase activity associated with purified simian virus 40 (SV40) large tumor (T) antigen has been examined. A variety of DNA substrates were used to characterize this ATP-dependent activity. Linear single-stranded M13 DNA containing short duplex regions at both ends was used to show that SV40 T antigen helicase displaced the short, annealed fragment by unwinding in a 3' to 5' direction. Three different partial duplex structures consisting of 71-, 343-, and 851-nucleotide long fragments annealed to M13 single-stranded circular DNA were used to show that SV40 T antigen can readily unwind short and long duplex regions with almost equal facility. ATP and MgCl2 were required for this reaction. With the exception of GTP, dGTP, and CTP, the other common nucleoside triphosphates substituted for ATP with varied efficiency, while adenosine 5'-O-(thiotriphosphate) was inactive. The T antigen helicase activity was also examined using completely duplex DNA fragments (approximately 300 base pairs) with or without the SV40 origin sequence as substrates. In reactions containing small amounts (0.6 ng) of DNA, the ATP-dependent unwinding of duplex DNA fragments occurred with no dependence on the origin sequence. This reaction was stimulated 5- to 6-fold by the addition of the Escherichia coli single-stranded DNA-binding protein. When competitor DNA was added so that the ratio of SV40 T antigen to DNA was reduced 1000-fold, only DNA fragments containing a functional SV40 origin of replication were unwound. This reaction was dependent on ATP, MgCl2, and a DNA-binding protein, and was stimulated by inorganic phosphate or creatine phosphate. The origin sequence requirements for the unwinding reaction were the same as those for replication (the 64-base pair sequence present at T antigen binding site 2). Thus, under specified conditions, only duplex DNA fragments containing an intact SV40 core origin were unwound. In contrast, unwinding of partially duplex segments of DNA flanked by single-stranded regions can occur with no sequence specificity.     

Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling

In the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus-mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene (topA869), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxyl-terminal domain of topoisomerase I, topA10 and topA66, also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus-Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting Tus-Ter interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC, indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity. We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus-DnaB interaction and leading to decreased Tus activity.     

Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

Summary The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 μM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 μM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.     

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.     

Histone H1 preferentially binds to superhelical DNA molecules of higher compaction.

In chromatin, the physiological amount of H1 is one molecule per nucleosome or, roughly, one molecule per 200 bp of DNA. We observed that at such a stoichiometry, H1 selectively binds to supercoiled DNA with magnitude of sigma > or = 0.012 (both negative and positive), leaving relaxed, linear, or nicked DNA molecules unbound. When negative and positive DNA topoisomers of varying superhelicity are simultaneously present in the binding mixture, H1 selectively binds to the molecules with highest superhelicity; less supercoiled forms are gradually involved in binding upon increasing the amount of input protein. We explain this topological preference of H1 as the consequence of an increased probability for more than one H1-DNA contact provided by the supercoiling. The existence of simultaneous contacts of H1 with both intertwined DNA strands in the supercoiled DNA molecules is also inferred by topoisomerase relaxation of H1-DNA complexes that had been prefixed with glutaraldehyde.     

Transfer of nucleosomes from parental to replicated chromatin.

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.     

Molecular interactions between DNA, poly(ADP-ribose) polymerase, and histones

Molecular interactions between purified poly(ADP-ribose) polymerase, whole thymus histones, histone H1, rat fibroblast genomic DNA, and closed circular and linearized SV40 DNA were determined by the nitrocellulose filter binding technique. Binding of the polymerase protein or histones to DNA was augmented greatly when both the enzyme protein and histones were present simultaneously. The polymerase protein also associated with histones in the absence of DNA. The cooperative or promoted binding of histones and the enzyme to relaxed covalently closed circular SV40 DNA was greater than the binding to the linearized form. Binding of the polymerase to SV40 DNA fragments in the presence of increasing concentrations of NaCl indicated a preferential binding to two restriction fragments as compared to the others. Polymerase binding to covalently closed relaxed SV40 DNA resulted in the induction of superhelicity. The simultaneous influence of the polymerase and histones on DNA topology were more than additive. Topological constraints on DNA induced by poly(ADP-ribose) polymerase were abolished by auto ADP-ribosylation of the enzyme. Benzamide, by inhibiting poly(ADP-ribosylation), reestablished the effect of the polymerase protein on DNA topology. Polymerase binding to in vitro-assembled core particle-like nucleosomes was also demonstrated.     

Dye titrations of covalently closed supercoiled DNA analyzed by agarose gel electrophoresis.

We have developed a rapid electrophoretic technique for performing ethidium bromide dye titrations in cylindrical 0.7% agarose gels. The technique was used to analyze the extent of supercoiling in circular covalently closed SV40, Co1E1, and pSC101 DNA. We have estimated the superhelical densities of SV40, Co1E1, and pSC101 DNA to be ?0.050, ?0.078, and ?0.085 respectively. The results obtained for native SV40 DNA correlate well with previously published values for the superhelical density of this DNA when these values are corrected to reflect a 26° duplex unwinding angle for ethidium bromide. Ethidium bromide concentrations sufficient to partially relax a supercoiled DNA allow the DNA to be resolved into a series of discrete bands in agarose gels. The distribution of bands represents a natural heterogeneity in the superhelical densities of the DNA molecules in the population.