Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly

Schormann, Norbert; Grigorian, Alexei; Samal, Alexandra B.; Raman, Krishnan; DeLucas, Lawrence J.; Chattopadhyay, Debasish

doi:10.1186/1472-6807-7-45

Cited by 40 publications

(77 citation statements)

References 35 publications

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“…The A20 binding site on D4 mapped to the N-terminal 25 residues of D4 (76). N-terminal residues 1 to 16 and C-terminal uracil DNA glycosylase residues 208 to 218 of D4 may also contribute to the interaction with A20 (141). Structural analysis supports a model with A20 functioning as a central scaffold protein with separate binding regions for D4, E9, D5, and H5 (141).…”

Section: Virus-virus Protein Interactionsmentioning

confidence: 53%

“…N-terminal residues 1 to 16 and C-terminal uracil DNA glycosylase residues 208 to 218 of D4 may also contribute to the interaction with A20 (141). Structural analysis supports a model with A20 functioning as a central scaffold protein with separate binding regions for D4, E9, D5, and H5 (141). VACV proteins G2, A18, and H5 also interact with each other in vitro and in vivo (16).…”

Section: Virus-virus Protein Interactionsmentioning

confidence: 64%

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Poxvirus Proteomics and Virus-Host Protein Interactions

Vliet

Mohamed

Zhang

et al. 2009

Microbiol Mol Biol Rev

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SUMMARY Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.

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Section: Virus-virus Protein Interactionsmentioning

confidence: 53%

Section: Virus-virus Protein Interactionsmentioning

confidence: 64%

Poxvirus Proteomics and Virus-Host Protein Interactions

Vliet

Mohamed

Zhang

et al. 2009

Microbiol Mol Biol Rev

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“…Recombinant R187V mutant D4 with an N-terminal His 6 tag was purified as described for wt D4 (39). Purified protein was crystallized by the hanging-drop vapor diffusion method using 1.5 M ammonium sulfate, 14% glycerol, and 0.1 M HEPES buffer (pH 7.5) in the reservoir.…”

Section: Methodsmentioning

confidence: 99%

Vaccinia Virus D4 Mutants Defective in Processive DNA Synthesis Retain Binding to A20 and DNA

Shudofsky

Silverman

Chattopadhyay

et al. 2010

J Virol

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Genome replication is inefficient without processivity factors, which tether DNA polymerases to their templates. The vaccinia virus DNA polymerase E9 requires two viral proteins, A20 and D4, for processive DNA synthesis, yet the mechanism of how this tricomplex functions is unknown. This study confirms that these three proteins are necessary and sufficient for processivity, and it focuses on the role of D4, which also functions as a uracil DNA glycosylase (UDG) repair enzyme. A series of D4 mutants was generated to discover which sites are important for processivity. Three point mutants (K126V, K160V, and R187V) which did not function in processive DNA synthesis, though they retained UDG catalytic activity, were identified. The mutants were able to compete with wild-type D4 in processivity assays and retained binding to both A20 and DNA. The crystal structure of R187V was resolved and revealed that the local charge distribution around the substituted residue is altered. However, the mutant protein was shown to have no major structural distortions. This suggests that the positive charges of residues 126, 160, and 187 are required for D4 to function in processive DNA synthesis. Consistent with this is the ability of the conserved mutant K126R to function in processivity. These mutants may help unlock the mechanism by which D4 contributes to processive DNA synthesis.Poxviruses are large, double-stranded DNA viruses that replicate exclusively in the cell cytoplasm in granular structures known as virosomes (31). Separated from the host nucleus, they rely on their own encoded gene products for DNA synthesis and replication (43). To efficiently synthesize its ϳ200,000-base genome, the poxvirus DNA polymerase must be tethered to the DNA template by its processivity factor. DNA processivity factors are proteins that stabilize polymerases onto their templates for effective genome replication (1, 22). Processivity factors are synthesized by nearly all replicating systems, ranging from bacteriophages to eukaryotes, yet each one is specific to its cognate polymerase. In the presence of these factors, polymerases are able to incorporate a great number of nucleotides per template binding event; in their absence, polymerases detach from their templates too frequently to successfully replicate the genome (14,20). E9, the DNA polymerase of the prototypical poxvirus, vaccinia virus, synthesizes approximately 10 nucleotides before dissociating from the viral DNA template (28). However, it can incorporate thousands of nucleotides when it is associated with its processivity factor (29). This extended strand synthesis, known as processivity, is necessary for vaccinia virus to effectively replicate its 192-kb genome.The protein A20 was first reported to be a component of the vaccinia virus processive DNA polymerase (19,37), yet we were unable to establish processivity in vitro using only A20 and E9. To identify which other proteins were required for processivity, we assessed six in vitro-synthesized proteins known to be involved in vac...

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“…D4 is the most evolutionarily distant member of family I sharing only ϳ20% sequence identity with the other UNGs (5). However, despite the relatively low degree of homology, the overall fold of D4 is well conserved when compared with other family members (19,20). Furthermore, sequence alignments of VACV D4 with related UNGs identified conserved active site residues that are predicted to form the uracil recognition pocket (i.e.…”

mentioning

confidence: 99%

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

Burmeister¹,

Tarbouriech²,

Fender³

et al. 2015

Journal of Biological Chemistry

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Background: D4 is a uracil-DNA glycosylase (UNG) and an essential component of the vaccinia virus DNA polymerase holoenzyme. Results: The crystal structure of D4 in complex with DNA is presented. Conclusion: The D4⅐DNA contacts exhibit major differences compared with the human UNG⅐DNA complex. Significance: This work allows a better understanding of the structural determinants required for UNG function.

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Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly

Cited by 40 publications

References 35 publications

Poxvirus Proteomics and Virus-Host Protein Interactions

Poxvirus Proteomics and Virus-Host Protein Interactions

Vaccinia Virus D4 Mutants Defective in Processive DNA Synthesis Retain Binding to A20 and DNA

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

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