Method of mapping DNA replication origins.

Spotila, Loretta D.; Huberman, Joel A.

doi:10.1128/mcb.5.1.85

Cited by 13 publications

(8 citation statements)

References 48 publications

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“…All of the DNA fragments in the 22 puM aphidicolin sample were equally faint, consistent with a low level of nonspecific DNA damage and repair that obscured any replication from ori. In addition, replication forks that remained in fragments G, I, and N would prevent normal migration of these DNA fragments during gel electrophoresis (4,26). These results were confirmed by extracting DNA from the early RI region of each gel as described below and then digesting it with a restriction endonuclease (data not shown).…”

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confidence: 77%

“…4. Replication forks must traverse the entire length of a restriction fragment for that fragment to be identified by gel electrophoresis, because restriction fragments containing replication forks migrate more slowly than those that are completely replicated, and the rate of migration varies with the number of forks and extent of replication (4,26). This complication means that the amount of a [32P]DNA fragment will be inversely proportional to its length as well as to its distance from the origin of DNA synthesis.…”

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confidence: 99%

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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

Mol. Cell. Biol.

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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...

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confidence: 77%

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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

Mol. Cell. Biol.

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“…Therefore, the total amount of form III 32P-labeled plasmid DNA observed was a measurement of total in vitro DNA synthesis (i.e., replication plus repair), while the amount of Dpn I resistant form III 32P-labeled plasmid DNA measured only DNA replication. When cut with EcoRI, replicating DNA intermediates (RI) produced molecules with branches of various lengths, depending on the extent of their replication, that migrated more slowly than form III DNA during gel electrophoresis and appeared as a smear of radioactivity (30,31;Fig. 2).…”

Section: Methodsmentioning

confidence: 99%

DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

Yamaguchi¹,

DePamphilis²

1986

Proc. Natl. Acad. Sci. U.S.A.

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Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J. J. Li and T. J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, [6973][6974][6975][6976][6977].Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (on) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing on and plasmids carrying deletions in and about on served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 on sequence that is required for initiation of replication (oni-core) plus the contiguous sequence on the late gene side of on-core containing G+C-rich repeats that facilitates initiation (oni-auxiliary). This initiation factor binding site was specific for the simian virus 40 on region, even though it excluded the high-affmlity large tumor antigen DNA binding sites.With the exception of initiation of DNA replication, all subsequent steps in the replication and assembly of simian virus 40 (SV40) chromosomes utilize host-cell components exclusively (1, 2). Initiation ofviral DNA replication requires the cis-acting 63-base-pair (bp) ori-core sequence, SV40 large tumor antigen (T-Ag), and permissive cell factors from monkey or human cells. Bidirectional replication then originates at the junction between the strongest DNA binding site for T-Ag and ori-core (Fig. 1). The 40-45 bp flanking either side of ori-core (ori-auxiliary; Fig. 1) are not required for replication, although they do stimulate replication 3-to 5-fold.Purified SV40 T-Ag binds most strongly to the early gene-side auxiliary sequence (site I), less strongly to the ori-core sequence (site II), and marginally to the late geneside auxiliary sequence and beyond (site III; Fig. 1). Mutations within sites I and II alter the affinity of T-Ag for DNA (6)(7)(8)19). These sites are biologically significant because mutations within ori that reduce or prevent replication can be suppressed by mutations in T-Ag (20), and mutations within site I that reduce the ability of T-Ag to regulate its own synthesis are overcome by mutations in T-Ag (7). Since most of binding sites I and III can be deleted with only a marginal effect on replication, initiation of DNA replication must involve binding of T-Ag to site II (ori-core), perhaps aided by the strong affinity of T-Ag to site I. Thus, one might expect ori-core and perhaps T-Ag binding site I to be the primary binding site for the protein complex responsible for initiating SV40 DNA replication.With the advent of soluble systems capable of initiating SV40 DNA replication in vitro (21-24), it was possible to determine which DNA sequence exhibited the strongest affinity for replication initiation factors by its ability to inhibit replication of a s...

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“…We are grateful to Loretta Spotila for paving the way with her earlier work (10). We thank the following people for helpful discussions: Michael McCleland, Robert Benezra, Randall Morse, Les Davis, Robert Givens, Ravindra Hajela, and Jiguang Zhu.…”

Section: Acknowledgmentsmentioning

confidence: 95%

“…We have found that digestions shorter than 4 h at 37°C show no evidence of significant branch migration. For the experiments reported here, replicating SV40 DNA molecules were enriched by adsorption to BND-cellulose by a previously described column procedure (10). However, we have found that our recently described batch adsorption method (7) provides superior enrichment of replicating molecules (20-to 50-fold) with considerably less effort.…”

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Two-dimensional gel electrophoretic method for mapping DNA replicons.

1988

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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.Although considerable indirect evidence suggests that initiation of eucaryotic DNA replication occurs at specific nucleotide sequences, or origins (2), only recently have direct tests for initiation sites become available. Both Huberman et al. (7) and Brewer and Fangman (1) recently reported the use of two-dimensional (2D) gel electrophoretic methods which allowed localization of the replication origins and termini (sites where replication forks stop) used in vivo by Saccharomyces cerevisiae plasmids. Both methods show promise for application to eucaryotic chromosomal DNA. This report provides additional details of the method of Huberman et al. (7) and a current assessment of its applicability to higher eucaryotic organisms.A hypothetical stretch of chromosomal DNA containing a replication origin and two sites for a restriction endonuclease (R) is shown in Fig. 1. The short segments labeled 1, 2, and 3 are assumed to be available for use as hybridization probes, and the entire restriction fragment is assumed to be replicated by forks traveling from left to right. DNA is isolated from cells (which must be growing but need not be synchronized), cut with restriction enzyme R, and enriched for fork-containing molecules by chromatography through benzoylated, naphthoylated DEAE-cellulose (BND-cellulose). The planar, aromatic benzoyl and naphthoyl groups can interact (in a manner similar to base stacking in native DNA) with the unpaired bases present in single-stranded DNA. Replicating molecules contain small single-stranded regions at their forks. These and other molecules with single-stranded regions are eluted from BND-cellulose with caffeine, which competes with the unpaired bases for the aromatic groups on BND-cellulose.The replication-fork-enriched DNA molecules are then electrophoresed through a neutral agarose gel (Fig. 1D). Nonreplicating molecules form a sharp band (closed vertical bar), whereas the replicating molecules form a trailing smear. The position of a replicating molecule in the smear is determined by its extent of replication: molecules beginning replication migrate just behind the nonreplicating molecules, whereas almost fully replicated molecules are ...

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Method of mapping DNA replication origins.

Cited by 13 publications

References 48 publications

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.

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.

DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

Two-dimensional gel electrophoretic method for mapping DNA replicons.

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