In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site.

Wobbe, C R; Dean, Frank B.; Weissbach, Lawrence; Hurwitz, Jerard

doi:10.1073/pnas.82.17.5710

Cited by 323 publications

(293 citation statements)

References 22 publications

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“…3, superhelical viral DNA increased with incubation time and addition of poly(ethyleneglyco1) 6000 during incubation stimulated its synthesis. Replicative forms migrated in a slowmoving region, starting from origin to SV40 (11), consistent with published results [24, 251.…”

Section: Characterization Of the Newly Synthesized Dna In Infectedpersupporting

confidence: 82%

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Sarkar

List

Bánfalvi

1987

European Journal of Biochemistry

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Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV-1 cells, permeabilized with lysolecithin, by incubation with [ u -~~P I~T T P , the other dNTPs and rNTPs and an ATPregenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form 11) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent with the involvement of DNA polymerase a. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.Initiation of a cycle of chromosome replication is not yet clearly understood at the molecular level in eukaryotes. This step is crucial to the regulation of chromosome replication and subsequent cell proliferation. Simian virus 40 (SV40) provides an excellent model system for cellular DNA replication, much like the small DNA bacteriophages in the field of prokaryotes [l]. Initiation of SV40 DNA replication requires the interaction of SV40 T-antigen, permissive cell factors, and ori, a 65-bp sequence essential for viral DNA replication [2, 31. Replication expands bidirectionally from a 2-bp region at the intersection of the strongest T-antigen binding site and the ori sequence [4] and proceeds to a termination region, 180" opposite ori [5, 61. Recent studies on the nucleotide sequences at the 5' ends of RNA-primed nascent DNA chains isolated from SV40-infected cells and on their locations in SV40 ori demonstrated that (a) replication forks within 65 bp of ori initiate Okazaki fragments predominantly on their retrograde arms, (b) that initiation events occur at several sites within ori, but only on the strand that encodes early mRNA, and (c) that the transition from discontinuous synthesis to continuous synthesis oc...

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Section: Characterization Of the Newly Synthesized Dna In Infectedpersupporting

confidence: 82%

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Sarkar

List

Bánfalvi

1987

European Journal of Biochemistry

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“…SV40 DNA was replicated in a large T antigen-dependent manner in HeLa extract (e.g. Stillman and Gluzman, 1985;Li and Kelly, 1984;Wobbe et al, 1985). During replication, the interaction was analysed by immunoprecipitation with anti-Fen1 antibodies followed by immunoblotting with PC10 against PCNA (Figure 7).…”

Section: Evolutionary Conservation Of the Fen1-pcna Interactionmentioning

confidence: 99%

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

et al. 1997

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Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21 Cip1 , which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identi®ed an interaction between PCNA and the structure speci®c nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21 Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is su cient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21 Cip1 . A PCNA binding peptide from p21 Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21 Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

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“…ATPase Activity-MCM proteins were incubated at 37°C for 30 min with 2 Ci of [␥-32 P]ATP (3000 Ci/mmol) in the same solution as that used for measuring the DNA helicase activity, in the absence or the presence of 5 g of single-stranded DNA (heat-denatured), doublestranded DNA (pSV01⌬EP DNA) (27), yeast tRNA, or poly(dT) (Pharmacia). Then, 0.5 l of the reaction was spotted on a poly(ethyleneimine)-cellulose thin layer chromatography plate (Cellulose F, Merck, Darmstadt, Germany).…”

Section: Methodsmentioning

confidence: 99%

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

Ishimi

1997

Journal of Biological Chemistry

506

508

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All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3 to 5 direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.There are at least six MCM 1 proteins that play an essential role in eukaryotic DNA replication as follows: MCM2, -3, -4 (Cdc21), -5, -6 (Mis5), and -7 (CDC47) proteins (see reviews in Refs. 1-3). Genetic evidence suggests that these proteins are required for the initiation of DNA replication in yeast (4, 5). The inactivation of human MCM2 protein with antibody consistently inhibits the initiation of DNA replication (6). Interaction between the MCM proteins has been reported in yeast (4, 7), and the protein complexes containing MCM proteins are detected in Drosophila (8), Xenopus (9), mouse (10, 11), and human cells (12)(13)(14). In human cells, MCM4, -6, and -7 proteins form a stable complex, and MCM2 is loosely associated with this complex (12,15,16). MCM3 and -5 proteins also form a stable complex (10, 13). All six of the MCM proteins contain DNA-dependent ATPase motifs in a central domain that is conserved from yeast to mammals, and these motifs are found in several enzymes that unwind the DNA duplex (17). These findings suggest that MCM protein complexes may be involved in the initiation of DNA replication as an enzyme that unwinds DNA in the origin region. However, DNA helicase activity of MCM proteins has not been reported.MCM proteins bind with chromatin before the initiation of DNA replication and are then released from the chromatin as DNA replication proceeds (18 -20). Our group found that MCM protein complexes containing MCM2, -4, -6, and -7 proteins bound tightly with histone H3 in vitro and were purified to near homogeneity by histone-Sepharose column chromatography (21). In the present study, it is shown that both ATPase and DNA helicase activities are associated with an MCM4, -6, and -7 protein complex. EXPERIMENTAL PROCEDURESPurification of MCM ...

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In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site.

Cited by 323 publications

References 22 publications

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair

A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex

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