Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149

Friefeld, Beth R.; Lichy, Jack H.; Hurwitz, Jerard; Horwitz, Marshall S.

doi:10.1073/pnas.80.6.1589

Cited by 38 publications

(18 citation statements)

References 20 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%

“…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).…”

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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%

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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“…Three of the required proteins are encoded by the adenovirus genome. Of these, the 80-kilodalton (kDa) preterminal protein (pTP) (6,43) and the 140-kDa adenovirus polymerase (15,16,35,44) have been shown to be involved in the initiation reaction (15,35,43). The third virus-encoded replication protein, the 72-kDa single-stranded DNA-binding protein (48), is dispensable for initiation, but is required, together with the adenovirus polymerase, for elongation of nascent DNA chains (9,15,35,41,44,45).…”

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confidence: 99%

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

Wides¹,

Challberg²,

Rawlins³

et al. 1987

Mol. Cell. Biol.

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The initiation of adenovirus DNA takes place at the termini of the viral genome and requires the presence of specific nucleotide sequence elements. To define the sequence organization of the viral origin, we tested a large number of deletion, insertion, and base substitution mutants for their ability to support initiation and replication in vitro. The data demonstrate that the origin consists of at least three functionally distinct domains, A, B, and C. Domain A (nucleotides 1 to 18) contains the minimal sequence sufficient for origin function. Domains B (nucleotides 19 to 40) and C (nucleotides 41 to 51) contain accessory sequences that significantly increase the activity of the minimal origin. The presence of domain B increases the efficiency of initiation by more than 10-fold in vitro, and the presence of domains B and C increases the efficiency of initiation by more than 30-fold. Mutations that alter the distance between the minimal origin and the accessory domains by one or two base pairs dramatically decrease initiation efficiency. This critical spacing requirement suggests that there are specific interactions between the factors that recognize the two regions.

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“…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).…”

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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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Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149

Cited by 38 publications

References 20 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.

Adenovirus origin of DNA replication: sequence requirements for replication in vitro.

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

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