c-myc protein and DNA replication: separation of c-myc antibodies from an inhibitor of DNA synthesis

Antibodies against human c-myc protein have been reported to inhibit DNA polymerase activity and endogenous DNA synthesis in isolated nuclei, suggesting a role for c-myc in DNA replication. Using the same antibody preparations, we observed equivalent inhibition of simian virus 40 DNA replication and DNA polymerase alpha and delta activities in vitro, as well as inhibition of DNA synthesis in isolated nuclei. However, the c-myc antibodies could be completely separated from the DNA synthesis inhibition activity. c-myc antibodies prepared in other laboratories also did not interfere with initiation of simian virus 40 DNA replication, DNA synthesis at replication forks, or DNA polymerase alpha or delta activity. Therefore, the previously reported inhibition of DNA synthesis by some antibody preparations resulted from the presence of an unidentified inhibitor of DNA polymerases alpha and delta and not from the action of c-myc antibodies.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3815 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3815-3825 ◽

Cited By ~ 41

Author(s):

R S Decker ◽

M Yamaguchi ◽

R Possenti ◽

M L DePamphilis

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Early Gene ◽

Initial Direction ◽

Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. 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 ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-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 ori 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 ori, 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 alpha. Synthesis of pppA/G(pN)6-8(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 ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

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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

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3815-3825.1986 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3815-3825

Author(s):

R S Decker ◽

M Yamaguchi ◽

R Possenti ◽

M L DePamphilis

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Early Gene ◽

Initial Direction ◽

Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. 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 ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-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 ori 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 ori, 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 alpha. Synthesis of pppA/G(pN)6-8(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 ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

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Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.94 ◽

1996 ◽

Vol 16 (1) ◽

pp. 94-104 ◽

Cited By ~ 27

Author(s):

F Stadlbauer ◽

C Voitenleitner ◽

A Brückner ◽

E Fanning ◽

H P Nasheuer

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Polymerase Alpha ◽

Cell Extracts ◽

Human Dna ◽

Sv40 Dna ◽

Hybrid Complexes

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.

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T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.4077-4087.1986 ◽

1986 ◽

Vol 6 (11) ◽

pp. 4077-4087

Author(s):

S T Smale ◽

R Tjian

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Affinity Column ◽

Specific Domain ◽

Dna Polymerase Alpha ◽

Sv40 Dna

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.

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Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.83.9.2869 ◽

1986 ◽

Vol 83 (9) ◽

pp. 2869-2873 ◽

Cited By ~ 85

Author(s):

Y. Murakami ◽

C. R. Wobbe ◽

L. Weissbach ◽

F. B. Dean ◽

J. Hurwitz

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Polymerase Alpha ◽

Dna Primase

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T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.4077 ◽

1986 ◽

Vol 6 (11) ◽

pp. 4077-4087 ◽

Cited By ~ 85

Author(s):

S T Smale ◽

R Tjian

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Affinity Column ◽

Specific Domain ◽

Dna Polymerase Alpha ◽

Sv40 Dna

We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication.

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Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1476-1482.1984 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1476-1482

Author(s):

H Ariga

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Rna Polymerase Iii ◽

Simian Virus 40 ◽

Cell Extract ◽

Simian Virus ◽

T Antigen ◽

Sv40 T Antigen ◽

Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

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Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases

Biochemical Journal ◽

10.1042/bj1780621 ◽

1979 ◽

Vol 178 (3) ◽

pp. 621-626 ◽

Cited By ~ 15

Author(s):

J F Burke ◽

P M Duff ◽

C K Pearson

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Deoxyribonucleic Acid ◽

Acid Synthesis ◽

Cell Nuclei ◽

Isolated Nuclei ◽

Dna Polymerase Alpha ◽

Polymerase Beta ◽

Deoxyribonucleic Acid Synthesis

In order to ascertain the identity of the DNA-dependent DNA polymerase responsible for the observed DNA synthesis in nuclei isolated from baby-hamster kidney (BHK-21/C13) cells a comparative study was carried out on the effects of some drugs, reported to influence DNA synthesis, on DNA synthesis catalysed by these nuclei and by partially purified DNA polymerase-alpha and -beta. In all cases DNA synthesis by isolated nuclei and polymerase-alpha was inhibited to similar extents by N-ethylmaleimide, p-hydroxymercuribenzoate, novobiocin, heparin and phosphonoacetic acid; polymerase-beta was much less affected by these compounds. Ethidium bromide inhibited all DNA synthesis to similar extents, although at low concentrations (about 2 microgram/ml) synthesis in isolated nuclei was stimulated. The results are discussed in relation to the proposal that DNA polymerase-alpha catalyses the covalent extension of Okazaki fragments that these nuclei carry out in vitro.

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Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

Journal of Virology ◽

10.1128/jvi.75.18.8569-8578.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8569-8578 ◽

Cited By ~ 6

Author(s):

Armin R. Kautz ◽

Klaus Weisshart ◽

Annerose Schneider ◽

Frank Grosse ◽

Heinz-Peter Nasheuer

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Protein Complexes ◽

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Specific Factor ◽

Dna Polymerase Α

ABSTRACT Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

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