Overexpression and Purification of the Vaccinia Virus DNA Polymerase

McDonald, William F.; Traktman, Paula

doi:10.1006/prep.1994.1059

Cited by 40 publications

(52 citation statements)

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“…Restriction endonucleases, T4 DNA ligase, Taq polymerase, and calf intestinal phosphatase were purchased from Roche Applied Science, whereas Geneticin (G418 sulfate) was purchased from Invitrogen. Protein molecular weight markers, 14 …”

Section: Materials Cells Viruses and Dna Plasmidsmentioning

confidence: 99%

“…For generation of vT7fUDG, the overall strategy was to place the T7-driven 3XFLAG-UDG cassette within the non-essential TK locus of wt virus. Generation of this virus was achieved by selection of TK Ϫ viruses after transfecting wt-infected cells with the plasmid described above; the strategy followed has been previously described (13,14,18). vvT7fUDG⅜ ‫گ‬ : Recombinant Virus Enabling T7 Polymerase-mediated Overexpression of 3ϫ-FLAG-tagged Catalytically Null UDG-For generation of pTM-3XfUDG⅜ ‫گ‬ , two rounds of overlap PCR were used to introduce two mutations into the D4R ORF that render the UDG catalytically inactive (Gly 202 3 Ala and Ala 542 3 Thr).…”

Section: Construction Of Recombinant Vaccinia Virusesmentioning

confidence: 99%

“…2) as appropriate (5,14). Infections were initiated at 37°C and shifted to 31.5°C at 4 hpi to increase the yield of soluble protein.…”

Section: Overexpression and Purification Of Fudg A20 And Dna Polymementioning

confidence: 99%

“…Processivity, which enables polymerases to replicate long templates rapidly and accurately, is not an intrinsic property of most polymerases but rather is conferred by accessory proteins. Indeed, the vaccinia virus E9 protein, which is the catalytic subunit of the polymerase, is inherently distributive under physiological conditions (4). In our efforts to purify and characterize the vaccinia virus processivity factor, we described the identification of A20 as a stoichiometric component of the processivity factor (5).…”

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confidence: 99%

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Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

Stanitsa

Arps

Traktman

2006

Journal of Biological Chemistry

126

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The vaccinia virus E9 protein, the catalytic subunit of the DNA polymerase holoenzyme, is inherently distributive under physiological conditions, although infected cells contain a highly processive form of the enzyme. The viral A20 protein was previously characterized as a stoichiometric component of the processivity factor, and an interaction between A20 and E9 was documented in vivo. A20 has been shown to interact with D4, the virally encoded uracil DNA glycosylase (UDG), by yeast-two hybrid and in vitro analysis. Here we confirm that UDG and A20 interact in vivo and show that temperature-sensitive viruses with lesions in the D4R gene show a profound defect in DNA synthesis at the non-permissive temperature. Moreover, cytoplasmic extracts prepared from these infections lack processive polymerase activity in vitro, implicating D4 in the assembly or activity of the processive polymerase. Upon overexpression of 3؋FLAG-UDG, A20, and E9 in various combinations, we purified dimeric and trimeric UDG-A20 and UDG-A20-polymerase complexes, respectively. These complexes are stable in 750 mM NaCl and can be further purified by Mono Q chromatography. Notably, the trimeric complex displays robust processive polymerase activity, and the dimeric complex can confer processivity on purified E9. Consistent with previous reports that the catalytic activity of UDG is dispensable for virus replication in tissue culture, we find that the role of UDG role in the polymerase complex is not diminished by mutations targeting residues involved in uracil recognition or excision. Our cumulative data support the conclusion that A20 and UDG form a heterodimeric processivity factor that associates with E9 to comprise the processive polymerase holoenzyme.Vaccinia virus, the prototypic orthopoxvirus, shows a significant degree of genetic autonomy from the host cell. Because all stages of the viral life cycle take place in the cytoplasm, vaccinia encodes most, if not all, of the factors involved in the replication of its 192-kb genome. The repertoire of essential replication functions appears to include: E9 (replicative DNA polymerase), A20 (stoichiometric component of the processivity factor), D5 (DNA-independent dNTPase), B1 (Ser/Thr protein kinase), I3 (single strand DNA-binding protein), A22 (Holliday junction resolvase), and D4 (uracil DNA glycosylase, UDG) 2 (Ref. 1, reviewed in Refs. 2 and 3). Other proteins implicated in the process of genome replication or maintenance include A50 (DNA ligase), H6 (topoisomerase), F2 (dUTPase), J2 (thymidine kinase), A48 (thymidylate kinase), and F4/I4 (ribonucleotide reductase) (reviewed in Refs. 2 and 3).In most cases, a processive DNA polymerase comprises the core of the replication machinery. Processivity, which enables polymerases to replicate long templates rapidly and accurately, is not an intrinsic property of most polymerases but rather is conferred by accessory proteins. Indeed, the vaccinia virus E9 protein, which is the catalytic subunit of the polymerase, is inherently distributive under phys...

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Section: Materials Cells Viruses and Dna Plasmidsmentioning

confidence: 99%

Section: Construction Of Recombinant Vaccinia Virusesmentioning

confidence: 99%

“…2) as appropriate (5,14). Infections were initiated at 37°C and shifted to 31.5°C at 4 hpi to increase the yield of soluble protein.…”

Section: Overexpression and Purification Of Fudg A20 And Dna Polymementioning

confidence: 99%

mentioning

confidence: 99%

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Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

Stanitsa

Arps

Traktman

2006

Journal of Biological Chemistry

126

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“…The polymerase catalyzes primerand template-dependent DNA synthesis and possesses 3Ј to 5Ј exonucleolytic activity (4,5). The essential role of D4 in DNA replication (6) is independent of its uracil DNA glycosylase activity (7), which presumably has a facultative repair function.…”

mentioning

confidence: 99%

Poxvirus DNA primase

Silva

Lewis²,

Berglund³

et al. 2007

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

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Poxviruses are large enveloped viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. At least six virus-encoded proteins are required for synthesis and processing of the doublestranded DNA genome of vaccinia virus, the prototype member of the family. One of these proteins, D5, is an NTPase that contains an N-terminal archaeoeukaryotic primase domain and a C-terminal superfamily III helicase domain. Here we report that individual conserved aspartic acid residues in the predicted primase active site were required for in vivo complementation of infectious virus formation as well as genome and plasmid replication. T he poxviruses comprise a large family of DNA viruses that include the causal agent of smallpox (1). Unlike most other DNA viruses, poxviruses replicate entirely in the cytoplasm. To accommodate this unique lifestyle, they encode enzymes and factors needed for genome replication and transcription, which are potential targets for antivirals (2). Poxvirus genomes are 130,000-300,000 bp long and consist of two complementary strands of DNA that are covalently linked to form hairpins at each end. A transcription system is packaged in infectious virus particles allowing early mRNAs to be synthesized soon after cell entry. Early proteins are used for host defense, genome replication, and transcription of intermediate stage genes. Intermediate proteins include late-stage transcription factors, whereas late proteins are mostly involved in virus assembly and dissemination.Most laboratory studies of poxviruses are carried out by using vaccinia virus (VACV). Studies with conditional lethal mutants indicate that five VACV early proteins are required for DNA replication, namely, E9 DNA polymerase, D4 uracil DNA glycosylase, A20 processivity factor, B1 protein kinase, and D5 NTPase (reviewed in ref.3). The polymerase catalyzes primerand template-dependent DNA synthesis and possesses 3Ј to 5Ј exonucleolytic activity (4, 5). The essential role of D4 in DNA replication (6) is independent of its uracil DNA glycosylase activity (7), which presumably has a facultative repair function. The A20 and D4 proteins physically interact (8, 9) and together provide processivity for the DNA polymerase (10). The B1 kinase phosphorylates a cellular DNA-binding protein called BAF and prevents the latter from blocking VACV DNA replication (11). The fast stop DNA replication phenotype of conditional lethal D5 mutants suggests a function at the replication fork (12). D5 also interacts with A20 (8, 9) and forms multimers (13). Extensive protein sequence analyses have indicated that the C-terminal region of the 90-kDa D5 protein belongs to the helicase superfamily III within the AAAϩ class of NTPases, which includes the replicative helicases of numerous other DNA and RNA viruses (14,15). Furthermore, the N-terminal domain of D5 has sequence and structural features that are common to the archaeoeukaryotic primase superfamily, the members of which have diverse roles in DNA replication and repair (16). Nevertheless, the onl...

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Characterization of a Processive Form of the Vaccinia Virus DNA Polymerase

1997

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We have previously shown that the purified, 116-kDa DNA polymerase encoded by vaccinia virus is inherently distributive, synthesizing only a few nucleotides per template binding event under moderate reaction conditions (W. F. McDonald and P. Traktman, J. Biol. Chem. 269, 31190-31197). These properties would be incompatible with efficient DNA replication in vivo and suggest that the polymerase most probably interacts with accessory proteins that stabilize the template/polymerase interaction. Here we show that a highly processive form of the enzyme is indeed present with cytoplasmic lysates prepared from infected cells, and demonstrate that this form of the enzyme is likely to comprise the DNA polymerase in association with an early viral protein with a native molecular weight of approximately 48K.

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Overexpression and Purification of the Vaccinia Virus DNA Polymerase

Cited by 40 publications

References 0 publications

Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

Vaccinia Virus Uracil DNA Glycosylase Interacts with the A20 Protein to Form a Heterodimeric Processivity Factor for the Viral DNA Polymerase

Poxvirus DNA primase

Characterization of a Processive Form of the Vaccinia Virus DNA Polymerase

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