The In Vitro Replication of Adenovirus DNA

Friefeld, Beth R.; Lichy, Jack H.; Field, Jeffrey; Gronostajski, Richard M.; Guggenheimer, R A; Krevolin, Mark D.; Nagata, Kyosuke; Hurwitz, Jerard; Horwitz, Marshall S.

doi:10.1007/978-3-642-46494-2_8

Cited by 41 publications

(28 citation statements)

References 68 publications

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“…The strongest evidence that the TGG(N)67GCCAA sequence is not the only requirement for the binding of nuclear aggtcTGGctt-tggGCCAAgagcc 4) tcctcTOGatt-tgtGCCAAatgtc 5) atgccTGGaag-gcaGCCAAatttt 6) ctgggTOGaag-gt&TCCAAtccag 7)…”

Section: Discussionmentioning

confidence: 99%

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Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Gronostajski¹,

Adhya²,

Nagata³

et al. 1985

Mol. Cell. Biol.

130

120

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Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)67GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is importadt for the binding of nuclear factor I. Nuclear factor I protects a 25-to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)67GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.Considerable progress has been made in the purification and characterization of procaryotic DNA replication proteins. (12). However, the lack of both genetic analysis and in vitro replication systems has hampered similar studies on DNA synthesis in eucaryotes. Adenovirus (Ad) DNA replication is the only system in eucaryotes that is amenable to genetic manipulation (5, 22, 30) and for which the initiation and elongation of DNA chains have been reconstituted with purified proteins (20; for reviews see references 3, 9, and 28). We have fractionated and purified the viral and cellular proteins required for the in vitro replication of Ad DNA with the goal of isolating proteins involved in host DNA synthesis (7,9,16). A protein of particular interest is nuclear factor I, a cellular site-specific DNA-binding protein that is required for the efficient in vitro initiation of Ad DNA replication (19,21,23).Nuclear factor I was purified from nuclear extracts of uninfected HeLa cells by its ability to support the replication of Ad DNA in vitro (19). The synthesis of full-length Ad DNA in vitro requires five purified proteins (4,20). Three of these proteins are viral coded, the 80,000-dalton precursor to the 55,000-dalton terminal protein found at the 5' end of the Ad genome (pTP), the Ad DNA polymerase, and the Ad DNA-binding protein. The two remaining proteins, nuclear factors I and II, are host coded and have been purified from nuclear extracts of uninfected HeLa cells. The initiation of Ad DNA synthesis occurs by the covalent attachment of dCMP, the 5'-terminal deoxynucleotide of Ad DNA, to the pTP (2, 15). This initiation reaction is catalyzed by the Ad DNA polymerase and, in the presence of the Ad DNA-binding protein, is completely dependent on nuclear factor 1 (19). Nuclear factor I has been shown to specifically bind, and protect from DNase I digestion, a 32-base-pair (bp) DNA sequence located in the replication origin present at the termini of the 36,000-bp Ad genome (21. 23). The ability of nuclear fact...

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Section: Discussionmentioning

confidence: 99%

“…The synthesis of full-length Ad DNA in vitro requires five purified proteins (4,20). Three of these proteins are viral coded, the 80,000-dalton precursor to the 55,000-dalton terminal protein found at the 5' end of the Ad genome (pTP), the Ad DNA polymerase, and the Ad DNA-binding protein.…”

mentioning

confidence: 99%

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Gronostajski¹,

Adhya²,

Nagata³

et al. 1985

Mol. Cell. Biol.

130

120

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“…The initiation reaction for DNA synthesis was performed essentially as described (3,22). Reaction mixtures (50 ,ul) contained various extracts of Ad Pol mutants, Ad type 35 DNA covalently linked to TP at each 5' end (Ad35 DNA-pro, 0.15 ,ug), the DEAE-cellulose fraction of nuclear extract from uninfected HeLa cells, wild-type (wt) pTP extract from transfected CMT4 cells (J-pTP), 10%o (vol/vol) glycerol, 20 mM NaCl, 15 ,Ci of [a-32P]dCTP (3000 Ci/mmol; 1 Ci = 37 GBq), 5 mM MgCl2, 25 mM Hepes (pH 7.4), 3 mM ATP, 0.5 mM dithiothreitol, and 0.1 mM aphidicolin. After a 1-hr incubation at 30°C, the reaction was terminated by the addition of 50 1g of 1% SDS/10 mM Tris-HCI, pH 8/1 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis.

Chen

Horwitz

1989

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

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Linker-insertion mutations were introduced into the cloned adenovirus DNA polymerase gene and the functional effects on the initiation and elongation of DNA in vitro were measured. Essential regions of the polymerase appear to be scattered in patches across the entire molecule and are not limited to the five regions of homology shared with a variety of other replicating polymerases. Thus, the adenovirus DNA polymerase presumably contains active sites that must be formed by distant interactions across the polymerase molecule.Adenovirus (Ad) type 2 contains a linear, duplex, 36-kilobase-pair (kb) DNA with inverted terminal repeats and a 55-kDa terminal protein (TP) covalently bound to each 5' end (1, 2). Initiation of DNA replication occurs by a proteinpriming mechanism in which the first deoxynucleotide, dCMP, is covalently attached to an 80-kDa precursor of the TP (pTP) (2, 3). The replication of Ad DNA in vitro requires six proteins of which three, the 80-kDa pTP, the 140-kDa Ad DNA polymerase (Ad Pol), and the 59-kDa Ad DNA-binding protein (DBP), are virus-encoded (4-7). Three host proteins, nuclear factors (NF) I-III, have been purified from uninfected HeLa cells. NFI and NFIII specifically recognize the Ad origin of replication at each end of the template and stimulate replication (8)(9)(10). NFII is a type I DNA topoisomerase (11).Ad Pol is an essential enzyme for both the initiation and elongation steps of DNA replication; it has been characterized biochemically and genetically by using the AdS temperature-sensitive, DNA-negative mutants ts149 and ts36 (7,12,13). Ad Pol is isolated as an equimolar complex with pTP from Ad-infected cytoplasmic extracts but can be separated from pTP by glycerol gradient sedimentation in the presence of 1.7 M urea (14). Ad Pol is resistant to aphidicolin, a specific inhibitor of the eukaryotic DNA polymerases a and 8, and is a multifunctional enzyme for which the following activities have been characterized: (i) an aphidicolin-resistant DNA polymerase reaction that adds deoxynucleosides to 3' ends of gapped DNA (14,15); (ii) a de novo chain-initiation activity measured as covalent complex formation between the first deoxynucleotide and pTP (pTP-dCMP) (3,16) for Ad replication but also be useful in understanding the mechanism of action of other DNA polymerases. Ad Pol has been successfully cloned, expressed in COS-1 cells from an expression vector (p91023), and shown to specify an enzymatically active DNA polymerase in a cell-free replication system (22). With the production of an active Ad Pol in vitro, it has become possible to systematically explore the functional domains of this polypeptide and investigate the significance of evolutionarily conserved amino acid sequences by rapid and selective in vitro mutagenesis of the cloned gene.We have constructed a series of 16 linker-insertion mutations within the coding region of the Ad Pol gene and determined the effect of each mutation in three independent in vitro reactions. The results indicate that essential regions fo...

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“…Apparently DBP protects the single-stranded DNA against a nuclease activity present in the reconstituted system, a protective function that has been described before for DBP [40]. The nuclease activity could originate from the DNA polymerase which possesses a 3'--* 5' exonuclease activity on single-stranded DNA [24,41].…”

Section: Comparison Of Replication Protein Requirements For Displacemmentioning

confidence: 99%

Adenovirus DNA replication in vitro: Duplication of single-stranded DNA containing a panhandle structure

Leegwater

Rombouts

Vliet

1988

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expres

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Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal Xma I fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.

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The In Vitro Replication of Adenovirus DNA

Cited by 41 publications

References 68 publications

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites.

Dissection of functional domains of adenovirus DNA polymerase by linker-insertion mutagenesis.

Adenovirus DNA replication in vitro: Duplication of single-stranded DNA containing a panhandle structure

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