Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA Glycosylase

Abstract

BackgroundUracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized.ResultsThis manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This also represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. Comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms.ConclusionThe first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-015-0037-1) contains supplementary material, which is available to authorized users.

The vaccinia virus DNA polymerase and its processivity factor

“…Specifically, the protein consists of a core β-sheet, made up of two anti-parallel β-strands, surrounded by two α-helices, one on the N′- and one on the C′-terminus of the central β-sheet (Schormann et al, 2007). Comparison of this crystal structure, as well as those of UDG in complex with uracil and dsDNA, to those available for human and E coli uracil DNA glycosylases, reveals that the catalytic pocket of UDG is nearly identical to other Family I members, including the conservation of two key catalytic residues, Asp68 and His181 (Schormann et al, 2013; Schormann et al, 2015; Schormann et al, 2007; Schormann et al, 2011; Schormann et al, 2016). In 2015, Schormann et al defined the D4 residues responsible for mediating protein-DNA interactions, with the interface being made up of Ile67, Pro71, Gly128, Glu129, Thr130, Lys131, Gly159, Lys160, Thr161, Asp162, Tyr180, His181, and Ala183.…”

“…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).…”

“…Despite the evidence supporting the importance of this “tongue and groove” interaction, Schormann et al observed that when in complex with DNA, both Arg167 and Pro173 appear to move in an orientation that would not be favorable for the formation of this “groove and pocket” interaction (Schormann et al, 2015). This finding may imply that formation of a heterotrimeric complex requires the formation of the D4:A20 heterodimer prior to binding to DNA.…”

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

“…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).…”

Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod

“…The crystal structures have also been determined of UNG in complex with DNA from human [12][13][14], Vaccinia virus [15,16] and D. radiodurans [17]. These structures have revealed that the uracil is flipped out of the DNA and into the active site upon detection in a process where the enzyme binds the minor groove, compress the phosphate backbone and kink the DNA [13].…”