Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase

Roovers, Dick J.; Overman, Paul F.; Chen, Xiaoqing; Sussenbach, J.S.

doi:10.1016/0042-6822(91)90032-7

Cited by 23 publications

(41 citation statements)

References 57 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%

“…Nucleoside monophosphates, which inhibit the 3'-5' exonuclease activity of Pol I without affecting polymerization (49), reportedly inhibit the polymerization activity of HSV Pol (18). Numerous T4 and adenovirus mutations within the putative 3'-5' exonuclease domain appear to affect polymerizing activity (6,52,54,55), although it is not clear that these mutations do not exert global folding defects. None of the results presented above has conclusively shown that the polymerizing and exonuclease activities are inseparable; nevertheless, they support our suggestion that many a-like DNA polymerases do not contain polymerase and exonuclease domains as distinct and independent as those of Pol I and its relatives.…”

Section: Discussionmentioning

confidence: 99%

“…That these segments are important for 3'-5' exonuclease activity has been confirmed by mutational analyses of the a-like polymerases of bacteriophages +29 and T4. Several bacteriophage T4 mutations resulting in either altered mutation rates, reduced 3'-5' exonuclease activity, or both reside in these segments or elsewhere in a region extending from 90 amino acids upstream of Exo I to 20 amino acids downstream of Exo III (52)(53)(54)(55). Bernad et al (2) have shown that mutagenesis of specific residues within Exo I and Exo II of +29 DNA polymerase results in an enzyme that retains polymerase activity but is deficient in 3'-5' exonuclease activity.…”

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confidence: 99%

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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%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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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“…Moreover, an exonucleasedeficient mutant was constructed by mutating a conserved residue located at the proposed exonuclease domain of Ad pol. Combined with mutational studies (22,26,27), these results suggest a molecular architecture for Ad pol similar to that of RB69 DNA pol, a model enzyme for the family B polymerases (11). Furthermore, we demonstrate that pTP binds at the primer binding groove of Ad pol.…”

mentioning

confidence: 59%

“…Within this family, it is part of the subclass of protein-priming DNA pols (15). Mutational analysis of the polymerase domain has shown that, like other pol␣-like DNA pols, Ad pol is functionally conserved with the polymerase activity located at the C terminus (4,5,6,17,22,26,27). Biochemical analysis of Ad pol has shown that it replicates DNA in a processive manner but that it has a distributive 3Ј-5Ј exonuclease activity on single-stranded DNA, although removal of a mismatched nucleotide and subsequent switching to polymerization proceeds processively (18).…”

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confidence: 99%

Molecular Architecture of Adenovirus DNA Polymerase and Location of the Protein Primer

Brenkman

Breure

Vliet

2002

J Virol

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Adenovirus (Ad) DNA polymerase (pol) belongs to the distinct subclass of the pol␣ family of DNA pols that employs the precursor terminal protein (pTP) as primer. Ad pol forms a stable heterodimer with this primer, and together, they bind specifically to the core origin in order to start replication. After initiation of Ad replication, the resulting pTP-trinucleotide intermediate jumps back and pTP starts to dissociate. Compared to free Ad pol, the pTP-pol complex shows reduced polymerase and exonuclease activities, but the reason for this is not understood. Furthermore, the interaction domains between these proteins have not been defined and the contribution of each protein to origin binding is unclear. To address these questions, we used oligonucleotides with a translocation block and show here that pTP binds at the entrance of the primer binding groove of Ad pol, thereby explaining the decreased synthetic activities of the pTP-pol complex and providing insight into how pTP primes Ad replication. Employing an exonuclease-deficient mutant polymerase, we further show that the polymerase and exonuclease active sites of Ad pol are spatially distinct and that the exonuclease activity of Ad pol is located at the N-terminal part of the protein. In addition, by probing the distances between both active sites and the surface of Ad pol, we show that Ad pol binds a DNA region of 14 to 15 nucleotides. Based on these results, a model for binding of the pTP-pol complex at the origin of replication is proposed.

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Identification of a Cellular Repressor of Transcription of the Adenoviral Late IVa2 Gene That Is Unaltered in Activity in Infected Cells

Lin

Flint

2000

Virology

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The gene encoding the adenovirus type 2 IVa(2) protein, a sequence-specific activator of transcription from the viral major late promoter, is itself transcribed only during the late phase of infection. We previously identified a cellular protein (IVa(2)-RF) that binds specifically to an intragenic sequence of the IVa(2) transcription unit. We now report that precise substitutions within the IVa(2)-RF-binding site that decreased binding affinity increased the efficiency of IVa(2) transcription in in vitro reactions containing IVa(2)-RF. Consistent with the conclusion that this cellular protein represses IVa(2) transcription, mutations that led to more efficient transcription in the presence of IVa(2)-RF were without effect in reactions lacking this cellular protein. No change in the concentration or activity of IVa(2)-RF could be detected in adenovirus-infected cells during the period in which the IVa(2) gene is transcribed. We therefore propose that restriction of IVa(2) transcription to the late phase is the result of titration of this cellular repressor as the number of copies of the IVa(2) promoter increases upon replication of the viral genome.

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Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase

Cited by 23 publications

References 57 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.

Molecular Architecture of Adenovirus DNA Polymerase and Location of the Protein Primer

Identification of a Cellular Repressor of Transcription of the Adenoviral Late IVa2 Gene That Is Unaltered in Activity in Infected Cells

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