Discontinuous DNA replication: Accumulation of simian virus 40 DNA at specific stages in its replication

Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.Topoisomerases are enzymes that change the superhelical state of DNA by transiently breaking one or both strands of the duplex. Type II topoisomerases make a staggered double-strand break, whereas the type I enzymes introduce a single-strand break. The known eucaryotic topoisomerases are capable only of relaxing supercoiled DNA, whereas some of the bacterial enzymes are able to introduce supercoils (e.g., DNA gyrase in Escherichia coli) (for recent reviews, see references 39 and 62 to 64). The eucaryotic type

Section: Resultsmentioning

confidence: 99%

Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules.

Porter¹,

Champoux²

1989

“…1, lane 0) appears to indicate more extensive replication. Together, these data suggest that RI becomes topologically relaxed during incubation in vitro, limiting the fractionation range during gel electrophoresis from the position of form II (0% replication) to RI* (85 to 95% replication) (32,33). As replication continued in vitro for up to 3 h, the amount of DNA synthesis in the termination region increased in the manner expected for bidirectional replication of SV40 (data not shown).…”

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confidence: 84%

“…The highmolecular-weight products at the top of the gel are quantitatively converted to form III by cleavage with a single-site endonuclease (40) and may consist of multiple interlocked catenates, the result of inefficient separation of sibling DNA molecules (38). In addition, nonspecific DNA aggregation can occur that traps a random population of molecules (32). Problems with completion of circular plasmid DNA replication in vitro have been observed in other systems as well (27,39).…”

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confidence: 99%

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

Decker¹,

Yamaguchi²,

Possenti³

et al. 1986

Aphidicolin, a specific inhibitor of DNA polymerase a, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (on) and continuation of replication beyond on. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600-to 700-base-pair region centered at on. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 on-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the on region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of on, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase a. Synthesis of pppA/G(pN)64(pdN)21_23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at on first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.Simian virus 40 (SV40) is a small (5.2 kilobases [kb]) DNA virus that replicates in the nuclei of permissive monkey or human cells as a circular chromosome whose nucleosome striicture and histone composition are indistinguishable from that of its host (reviewed in reference 5 and in M. L. DePamphilis and M. K. Bradley, in N. P. Salzman, ed., The Viruses: Polyoma Viruses, in press). With the exception of initiation of viral DNA replication, all steps in the replication and assembly of viral chromosomes are carried out exclusively by cellular components. Initiation of SV40 DNA replication requires a cis-acting sequence that functions as the origin of replication (ori), SV40 large tumor antigen (T-ag), and one or more permissive cell factors. Bidirectional replication then begins at the junction between the strongest DNA-binding site for T-ag and the ori core. Analysis of initiation sites for RNA-primed DNA synthesis in SV40 replicating intermediates led to the hypothesis that initiation of DNA synthesis at ori involves the same mechanism used to synthesize Okazaki...

“…We have used replicating DNA of SV40 to test our procedure and to optimize experimental conditions. SV40 DNA contains a single, specific replication origin whose location is well known (9,17,48), and replicating SV40 DNA can easily be prepared in quantities sufficient for these studies (40).…”

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confidence: 99%

“…The origin location procedure requires that diagonals on the autoradiograms (as diagrammed in Fig. 1 (40) observed that unrestricted circular SV40 DNA molecules can be displayed as a linear function of extent of replication by agarose gel electrophoresis. The procedure described here requires that the ordering of replication intermediates achieved by the first-dimension agarose gel be maintained in the second-dimension analysis.…”

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confidence: 99%

Method of mapping DNA replication origins.

Spotila¹,

Huberman²

1985

We have developed a method which allows determination of the direction in which replication forks move through segments of chromosomal DNA for which cloned probes are available. The method is based on the facts that DNA restriction fragments containing replication forks migrate more slowly through agarose gels than do non-fork-containing fragments and that the extent of retardation of the fork-containing fragments is a function of the extent of replication. The procedure allows the identification of DNA replication origins as sites from which replication forks diverge. In this paper we demonstrate the feasibility of this procedure, with simian virus 40 DNA as a model, and we discuss its applicability to other systems.During the eucaryotic S phase, DNA replication initiates at multiple discrete sites spaced irregularly along chromosomal DNA (23). These sites are termed origins because replication proceeds bidirectionally away from them (23). The question of whether chromosomal replication origins are located at specific nucleotide sequences or are located randomly has been intensively