Replication of X174 DNA: In Vitro Synthesis of X RFI DNA and Circular, Single-stranded DNA

Soluble extracts of adenovirus-infected HeLa cell nuclei support DNA replication on exogenous adenovirus DNA templates. Conditions of synthesis using both wild-type and temperature-sensitive extracts have been defined. Nuclear extracts prepared from cells permissively infected with the adenovirus mutant H5ts125 expressed the temperature-sensitive phenotype and could be inactivated at 370C in vitro. These extracts were completely complemented by the addition of wildtype adenovirus DNA binding protein but not by H5ts125 DNA binding protein. Enhancement by binding protein in the mutant extracts represents replication, as demonstrated by the production of full-sized products and orderly chain elongation originating, as in vivo, at both ends of the linear DNA. Replicative synthesis required the 5'-terminal protein bound covalently to template DNA and could be inhibited by denaturation of this 55,000-dalton protein. Various inhibitors of eukaryotic DNA polymerases, such as aphidicolin and 2',3'-dideoxythymidine triphosphate, inhibited replication of exogenous adenovirus templates in this system as they do in previously reported systems that only elongate endogenous replicating intermediates.have shown that funtional DBP is also required for the elongation reaction (14).This communication describes further studies of the replication reaction on added exogenous viral DNA catalyzed by nuclear extracts from infected cells. E. tracts prepared from H5tsI25-infected cells were thermolabile and could be inactivated by preincubation at 370C. Under these conditions, full activity was reconstituted by the addition of wild-type Ad2 DBP which generated a reaction having all the characteristics of true replication. We have confirmed that replicative DNA synthesis required viral DNA bound to the 5'-terminal protein (2). Denaturation of this protein inactivated the template activity of the DNA-protein complex. Thus, dependence of DNA replication in vitro on specific viral-coded and virus-associated proteins suggests that the system will allow further study of these and other requirements for DNA replication in eukaryotic systems.Challberg and Kelly (1) (3)(4)(5)(6). Although analogous mutants in eukaryotic cells have been difficult to obtain, there are three complementation groups of conditional-lethal DNA-negative Ad mutants (7). The defect of one such AdS mutant, H1tsl25, was identified as the gene coding for a 72,000-dalton polypeptide that binds to singlestranded DNA (8, 9). The mutant is temperature-sensitive for viral DNA replication (10), and the DNA binding protein (DBP), isolated from H5ts125-infected cells at permissive temperature, exhibits a temperature-dependent binding to single-stranded DNA cellulose columns that is different from that of the wild-type protein (9). Studies with whole cells infected with H5ts125 and with nuclei prepared from those cells have suggested that the defect in DNA synthesis is due to a failure of initiation (11,12). Studies with ammonium sulfate nuclear extracts of such cells, which c...

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confidence: 99%

Complementation of the temperature-sensitive defect in H5ts125 adenovirus DNA replication in vitro.

Kaplan¹,

Ariga²,

Hurwitz³

et al. 1979

“…Stage I. and stage II reactions have' been completely resolved and reconstituted in vitro (2)(3)(4)(5)(6). Because In order to elucidate the mechanism of the stage III reaction we have undertaken the task of resolving this process in vitro.…”

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confidence: 99%

Escherichia coli host factor required specifically for the phi X174 stage III reaction: in vitro identification and partial purification.

Wolfson¹,

Eisenberg²

1982

A cell-free extract prepared, from X174-infected Escherichia coli cells sustained in vitro synthesis of viral DNA (stage 11 reaction).when supplemented with fraction II from uninfected cells. The reaction was dependent upon deoxyribonucleoside triphosphate,.ATP, added 4X174 replicative form I DNA template, and the fraction II from uninfected cells. This reaction differed from the stage II reaction (semiconservative replication of duplex replicative form DNA) by the production of stable viral protein-DNA complexes sensitive to anti-+X174 antiserum. Three types ofprotein-DNA complexes were identified, '50S, 92S, and a 114S complex that cobanded in CsCl and cosedimented in neutral sucrose gradients with a 4X174 phage marker. The sensitivity of these complexes to anti-4X174.antiserum and Staphylococcus aureus provided a relatively rapid biochemical assay for direct measurement of the amount of DNA synthesized by -the stage m reaction. With this assay, an E. coli factor .(SI,,) required specifically for the synthesis of viral protein-DNA complexes was identified and purified 200-fold from uninfected E. coli cells.' The partially purified Sm factor was required for the synthesis ofDNA and viral protein-DNA complexes in the +X174-infected cell extracts and could not be replaced by rep The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

“…In contrast, pBR322 RFI DNA was inactive as template over a wide concentration range. As in the case of OX RFI, replication of the recombinant DNAs required the OX A protein and was inhibited by coumermycin, nalidixic acid, and RNase (23).…”

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confidence: 99%

“…Markers for recombinant single-stranded linear [ss(l)] and ss(c) molecules were derived from heat-denatured recombinant RFII molecules prepared as described (21 (23). This synthesis is sensitive to RNase A and to the DNA gyrase inhibitors coumermycin and nalidixic acid (23). Data in Table 1 summarize the template activity of the recombinant DNA preparations containing the OX viral strand origin.…”

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confidence: 99%

See 1 more Smart Citation

In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.

Zipursky¹,

Reinberg²,

Hurwitz³

1980

ABSTRACr The origin of phage OX174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for #X174 RHi DNA as template in XX174-specific reactions in vito. We have shown that the recombinant plasmids: (i) are cleaved by the *X174 A protein; (ii) support net synthesis of unit-length single-stranced circular DNA in the presence of the $X174 A protein and Escherichia cofl rep protein, DNA-binding protein, and DNA polymerase m elongation system; (iii) support replication of duplexes catalyzed by the X174 A protein ana crude extracts of E. coli.The phage 4X174 (OX) A protein initiates RF -RF replication on superhelical OX replicative form I (RFI) by introducing a site-specific endonucleolytic cut in the viral strand (1-4). The phage viral strand (+ strand) is recognized as substrate for endonucleolytic cleavage when in the single-strand configuration or the negatively supercoiled form but not when in the relaxed duplex form (1, 5). The OX A protein cleavage results in a free 3'-OH terminus and an apparent covalent attachment of the protein to the newly generated 5'-phosphate end (6)(7)(8)