Identification of Polymerase and Processivity Inhibitors of Vaccinia DNA Synthesis Using a Stepwise Screening Approach

Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that i) screen for compounds that block vaccinia DNA synthesis, ii) eliminate trivial inhibitors, e.g. DNA intercalators, and iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2,222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola

The Role of D4 in vaccinia virus processive DNA synthesis

Genome replication is inefficient without processivity factors, which tether DNA polymerases to their templates. The vaccinia virus DNA polymerase E9 cannot efficiently incorporate nucleotides in the absence of its processivity factors. When I began my studies, the protein A20 was implicated in conferring processivity onto E9, but the two proteins together were unable to synthesize extended DNA strands. In this dissertation, I present my efforts to determine the mechanism of vaccinia processive DNA synthesis. In the first chapter, I identify the requirement of a third vaccinia protein, D4, for processivity, and establish that A20, D4, and E9 are necessary and sufficient for processive DNA synthesis. In the second chapter, I focus on the role of D4, which also functions as a uracil DNA glycosylase (UDG) repair enzyme. I generated a series of D4 mutants to discover which sites are important for processivity, and identified three point mutants (K126V, K160V, and R187V) which did not function in processive DNA synthesis. These mutants retained UDG catalytic activity and could compete with wild-type D4 in processivity assays. They also retained the ability to bind both A20 and DNA, the two interactions thought to be required for processivity. The crystal structure of mutant R187V was resolved and revealed that while the local charge distribution around the substituted residue is altered, the protein has no major structural distortions. This, along with my data, suggests that the positive charges of residues 126, 160, and 187 are required for D4 to function in processive DNA synthesis. I have identified mutants K126V, K160V, and R187V, which function as wild- type D4 in all aspects but processive DNA synthesis. They are the first vaccinia mutants to be functional in UDG activity but deficient in processivity. My research has provided unique insights into the basis of the function of D4 in processivity

The Role of D4 in vaccinia virus processive DNA synthesis

Genome replication is inefficient without processivity factors, which tether DNA polymerases to their templates. The vaccinia virus DNA polymerase E9 cannot efficiently incorporate nucleotides in the absence of its processivity factors. When I began my studies, the protein A20 was implicated in conferring processivity onto E9, but the two proteins together were unable to synthesize extended DNA strands. In this dissertation, I present my efforts to determine the mechanism of vaccinia processive DNA synthesis. In the first chapter, I identify the requirement of a third vaccinia protein, D4, for processivity, and establish that A20, D4, and E9 are necessary and sufficient for processive DNA synthesis. In the second chapter, I focus on the role of D4, which also functions as a uracil DNA glycosylase (UDG) repair enzyme. I generated a series of D4 mutants to discover which sites are important for processivity, and identified three point mutants (K126V, K160V, and R187V) which did not function in processive DNA synthesis. These mutants retained UDG catalytic activity and could compete with wild-type D4 in processivity assays. They also retained the ability to bind both A20 and DNA, the two interactions thought to be required for processivity. The crystal structure of mutant R187V was resolved and revealed that while the local charge distribution around the substituted residue is altered, the protein has no major structural distortions. This, along with my data, suggests that the positive charges of residues 126, 160, and 187 are required for D4 to function in processive DNA synthesis. I have identified mutants K126V, K160V, and R187V, which function as wild- type D4 in all aspects but processive DNA synthesis. They are the first vaccinia mutants to be functional in UDG activity but deficient in processivity. My research has provided unique insights into the basis of the function of D4 in processivity

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

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