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

Abstract

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...

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

“…Site-directed mutagenesis in the D4 subunit identified residues important for the processivity of the E9⅐A20⅐D4 holoenzyme (40). These mutations changing positively charged resi- FIGURE 4.…”

“…Valine residues at these positions are likely to reduce affinity of D4 for the DNA. Finally, Lys-126, which seemed critical for processive activity (40), is far away from the DNA (12.5 Å; Fig. 7), and its effect can only be explained by a long range structural change induced by the introduction of the hydrophobic valine residue or by the participation of this specific residue in a transient contact with the DNA.…”

“…The current model of D4 tethering the polymerase holoenzyme to the DNA may be too simplistic because of its weak interaction with DNA at physiological ionic strength, although mutants affecting polymerase processivity point to a role of the DNA-binding site of D4 (40). The simultaneous presence of two DNA interacting domains, the polymerase active site in one hand and the D4 DNA binding site on the other hand, may still lead to an increased avidity, but this effect is hard to predict because of the long distance (ϳ150 Å) between the two DNA interacting domains (47).…”

“…D4 point mutants defective in processivity. The mutated residues (40) are mapped onto the D4 surface (yellow). A20 1-50 is shown in violet.…”

A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

“…All DNA Pols, from phage to human, function with a single cognate PF. However, the prototypic poxvirus vaccinia virus (VV) is somewhat unusual in that a heterodimer comprising the A20 and D4 viral proteins constitutes the functional PF (14)(15)(16)(17)(18). D4, which can also function as a uracil DNA glycosylase repair enzyme, binds to its PF partner A20 but not to E9 Pol (14,15,18).…”