Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis

Kwant, Marijke M.; Vliet, Peter C. van der

doi:10.1093/nar/8.17.3993

Cited by 38 publications

(21 citation statements)

References 28 publications

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“…Re-replication is especially extensive in cells infected with the E1B-55K-mutant virus (unpublished observations and Cherubini et al, 2006) and leads to double-stranded DNA breaks and regions of single stranded DNA that trigger the DNA-damage response pathway (Hook et al, 2007; Jones and Petermann, 2012). Because the adenovirus DNA polymerase is unaffected by a level of aphidicolin that blocks cellular DNA synthesis (Kwant and van der Vliet, 1980), double-mutant virus-infected cells were treated with a concentration of aphidicolin sufficient to suppress cellular but not viral DNA replication before measuring nuclear fragmentation (Fig. 8A).…”

Section: Resultsmentioning

confidence: 99%

E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

2014

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Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4orf3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4orf3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins.

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Section: Resultsmentioning

confidence: 99%

E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

2014

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“…Ad DNA replication in vitro with the aphidicolinresistant DNA polymerase and purified factors remains sensitive to aphidicolin, suggesting a second site of aphidicolin action, possibly one of the host-encoded stimulatory factors. Other investigators have also proposed a second site of aphidicolin action based on the observations that Ad DNA replication in vivo is less sensitive to aphidicolin than is host DNA replication and that the effect of aphidicolin on DNA polymerase a, but not on Ad DNA replication, is competitive with dCTP (16,17). The specific site and mechanism of aphidicolin action in inhibiting Ad DNA replication remain unknown.…”

Section: Methodsmentioning

confidence: 99%

Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication.

Lichy¹,

Field²,

Horwitz³

et al. 1982

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

114

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A complex containing the 80,000-dalton precursor to the adenovirus (Ad)-encoded terminal protein (pTP) and a 140,000-dalton protein is required for Ad DNA replication in vitro. This complex has been separated into subunits by glycerol gradient centrifugation in the presence ofurea. The isolated 140,000-dalton subunit contains a DNA polymerase activity which can be differentiated from all host DNA polymerases. No enzyme activity was detected with the isolated pTP. The requirements for reactions involved in the initiation of Ad DNA replication were determined by using the isolated subunits. The covalent addition ofdCMP, the first nucleotide in the DNA chain, to the pTP, which serves as the primer for replication, required the DNA polymerase subunit as well as the pTP. Synthesis of viral DNA in vitro also required both subunits. The properties of the DNA polymerase suggest that it may be a viral gene product.Studies with in vitro adenovirus (Ad) DNA replication systems have provided evidence in support of a model in which the virus-encoded 80,000-dalton pre-terminal protein (pTP) serves as the primer for the initiation of Ad DNA replication (1). According to the model, replication begins with the covalent addition of dCMP, the 5'-terminal nucleotide of each Ad DNA strand, to the pTP. The product of this reaction, pTP-dCMP, has a 3'-OH which can be used by a DNA polymerase as a primer terminus, allowing elongation ofthe nascent strand. The evidence favoring this model includes the observation that all nascent DNA chains are covalently linked to protein (2). In addition, the predicted intermediate, pTP-dCMP, is synthesized in vitro (3).Synthesis of pTP-dCMP provided an assay which permitted the purification ofpTP from cytoplasmic extracts ofAd-infected HeLa cells (4, 5). With the purified protein, the covalent addition ofdCMP to the pTP required MgCl2 and a DNA cofactor which could be either Ad DNA with terminal protein covalently linked to the 5' terminus ofeach strand (Ad DNA-pro) or a number of single-stranded DNAs, such as OX174 DNA (6). With Ad DNA-pro as a cofactor, the reaction was stimulated as much as 10-fold by ATP and a nuclear extract of uninfected cells; the virus-encoded DNA binding protein (Ad DBP) had no effect (4). In contrast, when single-stranded 4X174 DNA was used, the reaction was inhibited by ATP, nuclear extract, or Ad DBP. The pTP copurified with a DNA polymerase activity (Ad DNA polymerase) which resembled DNA polymerase a in its template preference and sensitivity to certain inhibitors. However, the Ad DNA polymerase was insensitive to aphidicolin, a potent inhibitor of DNA polymerase a. The most purified fraction of pTP contained approximately equimolar quantities of pTP and a 140,000-dalton protein. Because DNA polymerase a obtained from several sources has been reported to have a catalytic subunit of 140,000-160,000 daltons (7,8), it seemed likely that the 140,000-dalton protein would be the catalytic subunit of the Ad DNA polymerase. The sedimentation behavior of the purified pT...

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“…The initiation reaction is insensitive to ddNTPs, aphidicolin, and (S)-g-(3-hydroxy-2-phosphonylmethoxypropyl)adenine diphosphoryl, all strong inhibitors of elongation (21)(22)(23)(24)(25). Moreover, the K m for dCTP is 3.2-fold lower during initiation (1).…”

Section: Dissociation Is Accompanied With Changes In the Catalytic Prmentioning

confidence: 99%

Dissociation of the Protein Primer and DNA Polymerase after Initiation of Adenovirus DNA Replication

King

Teertstra

Vliet

1997

Journal of Biological Chemistry

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Initiation of adenovirus DNA replication occurs by a jumping back mechanism in which the precursor terminal priming protein (pTP) forms a pTP⅐trinucleotide complex (pTP⅐CAT) catalyzed by the viral DNA polymerase (pol). This covalent complex subsequently jumps back 3 bases to permit the start of chain elongation. Before initiation, pTP and pol form a tight heterodimer. We investigated the fate of this pTP⅐pol complex during the various steps in replication. Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonucleotides followed by glycerol gradient separation of the reaction products, we established that pTP and pol are separated during elongation. Whereas pTP⅐C and pTP⅐CA were still bound to the polymerase, after the formation of pTP⅐CAT 60% of the pTP⅐pol complex had dissociated. Dissociation coincides with a change in sensitivity to inhibitors and in K m for dNTPs, suggesting a conformational change in the polymerase, both in the active site and in the pTP interaction domain. In agreement with this, the polymerase becomes a more efficient enzyme after release of the pTP primer. We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employing synthetic oligonucleotide templates with a sequence repeat of two nucleotides (GAGAGAGA . . . instead of the natural GTAGTA . . . ) we show that G5 rather than G3 is used to start, leading to a pTP⅐tetranucleotide (CTCT) intermediate that subsequently jumps back. This indicates flexibility in the use of the start site with a preference for the synthesis of three or four nucleotides during initiation rather than two.The replication of the linear 36-kilobase pair adenovirus (Ad) 1 DNA initiates at two origins located at either molecular end employing a protein-priming mechanism. Initiation requires at least two viral proteins, the DNA polymerase (pol) and the precursor of the terminal protein (pTP), which acts as the primer and is linked covalently to the first nucleotide, a dCMP molecule. Initiation can be stimulated by the cellular transcription factors NFI and Oct-1 which guide the pTP⅐pol complex to the core origin employing their DNA binding domains. This leads to stabilization of the preinitiation complex and increases the number of initiation events by enhancing the V max of the reaction. In addition, the virus-coded singlestranded DNA-binding protein stimulates initiation by lowering the K m for dCTP (1).The virus uses a jumping back mechanism for initiation. At position 4 from the 3Ј-end of the template a pTP⅐CAT intermediate is synthesized which jumps back to be paired to the template residues 1-3 before elongation starts (2). This jumping or sliding-back mechanism was first described for 29 (3) and appears to be universal for protein-priming replication systems (4 -6), enabling errors made during initiation and small deletions to be restored. Subsequent elongation concomitant with the displacement of the non-template strand further requires the Ad DNA-binding protei...

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Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis

Cited by 38 publications

References 28 publications

E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication.

Dissociation of the Protein Primer and DNA Polymerase after Initiation of Adenovirus DNA Replication

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