Vaccinia E9 Polymerase
Vaccinia E9 Polymerase - Related Videos
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A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors
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Cited by 13 •
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Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.
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Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression
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We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.
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Translesion DNA Polymerases
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2020
Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication. TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
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Eukaryotic RNA Polymerases
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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action. All three eukaryotic RNAPs require specific transcription factors, of which the...
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Bacterial RNA Polymerase
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2020
Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity. In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...
FAQs
The enzyme uses a primer-template structure to guide selection of complementary deoxynucleoside triphosphates. Once the appropriate nucleotide is positioned, E9 forms a phosphodiester bond that extends the existing primer. This coordination between template recognition, nucleotide selection, and bond formation provides a biochemical framework for analyzing how viral polymerases achieve accurate genome copying.
The 3′-to-5′ exonuclease activity allows E9 to proofread newly synthesized DNA. Incorrectly incorporated nucleotides can therefore be removed from the growing strand before synthesis continues. Studying this activity helps distinguish the polymerase’s chain-extension function from its error-correction function and clarifies how both activities contribute to reliable vaccinia genome replication.
D4 associates with E9 and supports efficient genome replication by promoting processive synthesis, meaning the polymerase can continue extending DNA effectively. This interaction shows that catalytic activity alone does not fully determine replication performance. In biochemical studies, examining E9 together with D4 can reveal how accessory protein contacts regulate the efficiency of viral DNA synthesis.
Biochemical experiments can follow primer-template extension to assess nucleotide incorporation and phosphodiester-bond formation, while separate analysis of the 3′-to-5′ exonuclease function examines proofreading. Comparing E9 alone with E9 associated with D4 can further reveal effects on synthesis efficiency. These measurements connect individual enzyme activities with the broader process of viral genome replication.
E9 provides a model for investigating viral DNA synthesis and enzyme function within the poxvirus replication system. Researchers can use it to study how a family B polymerase copies DNA, how proofreading contributes to synthesis, and how accessory proteins influence catalysis. This focused biochemical system helps relate molecular enzyme behavior to vaccinia genome replication mechanisms.
Because E9 performs the catalytic steps required for DNA strand extension and D4 supports efficient synthesis, experiments can test whether compounds interfere with either polymerase activity or the polymerase–accessory protein relationship. Such studies provide a biochemical basis for evaluating compounds that disrupt viral DNA replication and for identifying which part of the replication process is affected.