Physical mapping of two temperature-sensitive adenovirus mutants affected in the DNA polymerase and DNA binding protein

Roovers, Dick J.; Young, C. S. H.; Vos, Hans L.; Sussenbach, J.S.

doi:10.1007/bf00308565

Cited by 12 publications

(8 citation statements)

References 49 publications

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“…Our results demonstrate that Exo II, and thus region IV, is essential for viral growth, as might be predicted from the degree of conservation of this region in a variety of DNA polymerases. This finding is consistent with results for T4 and adenovirus, in which certain region IV mutations confer temperature sensitivity or complete failure to grow (46,51,(54)(55)(56)73). It also demonstrates that region IV plays a critical role in DNA synthesis per se.…”

Section: Discussionsupporting

confidence: 80%

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.

Gibbs¹,

Weisshart²,

Digard³

et al. 1991

Mol. Cell. Biol.

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Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the a-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain a-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that a-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an a-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between a-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in a-like DNA polymerases.DNA polymerases are central to the replication of genetic material. Much of our information regarding DNA polymerases has come from detailed structural, enzymological, and genetic studies of Escherichia coli DNA polymerase I (Pol I) and its relative, T7 DNA polymerase (33, 61). However, most eukaryotic cellular and viral replicative DNA polymerases and certain bacterial and bacteriophage DNA polymerases are members of the (x-like polymerase family and share only very limited sequence similarity with Pol I (1,39,45,60,68). A fundamental question is: Do these enzymes carry out their functions similarly to or differently from Pol I?DNA polymerases usually contain both polymerase and exonucleolytic activities either on a single polypeptide or as separate subunits. The 3'-5' exonuclease activities associated with polymerases frequently perform proofreading functions, thereby reducing replication errors and mutation rates. A key feature of Pol I is that its polymerase and 3'-5' exonuclease activities reside on a single polypeptide (37) as separate domains with the active sites about 3 nm apart (47). This physical separation translates into functional independence, as revealed by analysis of mutations, including fairly gross deletions, of Pol I and T7 DNA polymerase that inactivate 3'-5' exonuclease activity without major deleterious effects on polymerase activity and processivity (11,12,19,61).Interestingly, a-like DNA polymerases share sequence similarities with active sites in the 3'-5' exonuclease domain of Pol I (2, 39, 45, 60). Bernad et al. (2) have termed these segments Exo I, Exo II, and Exo III (Fig...

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Section: Discussionsupporting

confidence: 80%

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.

Gibbs¹,

Weisshart²,

Digard³

et al. 1991

Mol. Cell. Biol.

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“…Testing of potentially lethal mutations by marker rescue The methods for marker rescue and the analysis of recombinant viruses have been described in detail (Volkert et al, 1989). Briefly, A549 cells in 35 mm culture dishes were co-transfected with 100 ng of DNA-protein complex from the temperature-sensitive mutant H5ts 149, which contains a C to T transition at bp 7563 in the DNA polymerase (Roovers et al, 1990), and with 300 ng of the pMR2 derivative containing the mutations of interest. After incubation of the transfected plates at the permissive temperature for 3 days, yields were harvested, and wild type viruses were selected by plaque assay on A549 cells at the restrictive temperature.…”

Section: Methodsmentioning

confidence: 99%

Transcription from the adenovirus major late promoter uses redundant activating elements.

Reach

Young

1991

The EMBO Journal

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The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.

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“…Four proteins [i.e., protein V, protein VII, l, and terminal protein (TP)] condense the Ad genome in the viral core, forming a chromatin-like organization [2][3][4]. The proteins IVa2, L4 33K, L4 22K, E2 72K, and L1 52/55K are involved in capsid assembly and/or genome packaging [5][6][7][8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Formation of adenovirus DNA replication compartments and viral DNA accumulation sites by host chromatin regulatory proteins including NPM1

et al. 2019

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The adenovirus (Ad) genome is believed to be packaged into the virion by forming a chromatin‐like structure. The replicated viral genome is likely to be condensed through binding with viral core proteins before encapsidation. Replicated viral genomes accumulate in the central region of the nucleus, which we termed virus‐induced postreplication (ViPR) body. However, the molecular mechanism by which the nuclear structure is reorganized and its functional significance in virus production are currently not understood. In this study, we found that viral packaging protein IVa2, but not capsid proteins, accumulated in the ViPR body. In addition, nucleolar chromatin regulatory proteins, nucleophosmin 1 (NPM1), upstream binding factor, and nucleolin accumulated in the ViPR body in late‐stage Ad infection. NPM1 depletion increased the nuclease‐resistant viral genome and delayed the ViPR body formation. These results suggested that structural changes in the infected cell nucleus depend on the formation of viral chromatin by host chromatin regulatory proteins. Because NPM1 depletion decreases production of the infectious virion, we propose that host factor‐mediated viral chromatin remodeling and concomitant ViPR body formation are prerequisites for efficient encapsidation of Ad chromatin.

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Physical mapping of two temperature-sensitive adenovirus mutants affected in the DNA polymerase and DNA binding protein

Cited by 12 publications

References 49 publications

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.

Transcription from the adenovirus major late promoter uses redundant activating elements.

Formation of adenovirus DNA replication compartments and viral DNA accumulation sites by host chromatin regulatory proteins including NPM1

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