Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Burmeister, W.P.; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N.; Iseni, Frédéric

doi:10.1074/jbc.m115.648352

Cited by 27 publications

(50 citation statements)

References 50 publications

(62 reference statements)

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“…Protein-protein interactions, in addition to protein-DNA interactions, may contribute to the association of these proteins with nascent DNA. For example, D4 can bind DNA (87,88) and, together with A20 (which has not been shown to bind DNA) and E9, forms a holoenzyme complex (23). H5 has affinity for DNA and interacts with both A20 (77) and the B1 protein kinase (77,89).…”

Section: Discussionmentioning

confidence: 99%

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Senkevich

Katsafanas

Weisberg

et al. 2017

J Virol

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Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2=-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor-or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate-and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases II␣ and II␤ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected. IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNAdependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases II␣ and II␤ and PCNA, which were found associated with nascent DNA.

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Section: Discussionmentioning

confidence: 99%

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Senkevich

Katsafanas

Weisberg

et al. 2017

J Virol

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“…In fact, D4 shares only ~ 20% sequence identity with human and E. coli uracil DNA glycosylases (Schormann et al, 2013; Schormann et al, 2007) and is resistant to the UGI protein, a well-studied inhibitor of many prokaryotic and eukaryotic UDGs (Ellison et al, 1996). D4 has been the subject of numerous crystallography studies (see below), the results of which have revealed that despite the limited primary amino acid sequence homology to other Family I UDGs, the structure of the VACV protein adopts the characteristic alpha / beta fold of DNA glycosylases (Burmeister et al, 2015; Contesto-Richefeu et al, 2014; Schormann et al, 2013; Schormann et al, 2007). …”

Section: Assembly Of a Processive Holoenzyme: The A20 And D4 (Udg)mentioning

confidence: 99%

“…Several groups have undertaken a detailed investigation of the protein structure of D4 alone, in complex with the N′-terminus of A20 and/or in complex with DNA oligonucleotides (Burmeister et al, 2015; Contesto-Richefeu et al, 2014; Contesto-Richefeu et al, 2016; Sartmatova et al, 2013; Schormann et al, 2013; Schormann et al, 2015; Schormann et al, 2007). In 2007, the first crystal structure of the D4 uracil DNA glycosylase was published (Schormann et al, 2007).…”

Section: : Structural Analysis Of the D4 D4/dna D4/a20 D4/a20/dnamentioning

confidence: 99%

The vaccinia virus DNA polymerase and its processivity factor

Czarnecki

Traktman

2017

Virus Research

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Vaccinia virus is the prototypic poxvirus. The 192 kilobase double-stranded DNA viral genome encodes most if not all of the viral replication machinery. The vaccinia virus DNA polymerase is encoded by the E9L gene. Sequence analysis indicates that E9 is a member of the B family of replicative polymerases. The enzyme has both polymerase and 3′–5′ exonuclease activities, both of which are essential to support viral replication. Genetic analysis of E9 has identified residues and motifs whose alteration can confer temperature-sensitivity, drug resistance (phosphonoacetic acid, aphidicolin, cytosine arabinsode, cidofovir) or altered fidelity. The polymerase is involved both in DNA replication and in recombination. Although inherently distributive, E9 gains processivity by interacting in a 1:1 stoichiometry with a heterodimer of the A20 and D4 proteins. A20 binds to both E9 and D4 and serves as a bridge within the holoenzyme. The A20/D4 heterodimer has been purified and can confer processivity on purified E9. The interaction of A20 with D4 is mediated by the N′-terminus of A20. The D4 protein is an enzymatically active uracil DNA glycosylase. The DNA-scanning activity of D4 is proposed to keep the holoenzyme tethered to the DNA template but allow polymerase translocation. The crystal structure of D4, alone and in complex with A201–50 and/or DNA has been solved. Screens for low molecular weight compounds that interrupt the A201–50/D4 interface have yielded hits that disrupt processive DNA synthesis in vitro and/or inhibit plaque formation. The observation that an active DNA repair enzyme is an integral part of the holoenzyme suggests that DNA replication and repair may be coupled.

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“…Surface plasmon resonance (SPR) experiments were carried out on a Biacore 3000 instrument (GE Healthcare) using CM5 sensor chips with immobilized streptavidin to which the 5=-end-biotinylated primer 5=-CCG AAT CAG GAA GAT AAC AGC GGT TTA GCC-3= was bound directly or after annealing to the complementary sequence as described previously (33). Experiments were performed in the appropriate gel filtration buffers described above at a flow rate of 15 l/min.…”

Section: Sprmentioning

confidence: 99%

Domain Organization of Vaccinia Virus Helicase-Primase D5

Hutin

Ling

Round

et al. 2016

J Virol

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Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae. We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single-and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins. IMPORTANCESince the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design. V iruses of the family Poxviridae have a large linear doublestranded DNA (dsDNA) genome coding for up to 250 open reading frames. The double-stranded DNA genome is covalently circularized at both ends and is fully replicated in the cytoplasm. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) (1) and in the Phycodnaviridae (reviewed in reference 2). Like poxviruses, these viruses are also members of the nucleocytoplasmic large DNA virus (NCLDV) clade (3).We are working on vaccinia virus, which was used for the vaccination campaign that led to the eradication of smallpox. It is a convenient and rather safe system for the study of general poxviru...

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Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Cited by 27 publications

References 50 publications

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

The vaccinia virus DNA polymerase and its processivity factor

Domain Organization of Vaccinia Virus Helicase-Primase D5

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