Question

1. Pol ε is used for ____________strand synthesis and Pol δ is used for __________ strand synthesis.

2. What is the role of PCNA in DNA replication?

3. What is an “epitope tag”?

4. What is the main component that recruits Pol δ for lagging strand synthesis?

Answer 1

1) DNA & Replicative and Repair Polymerases

2) PCNA (proliferating cell nuclear antigen) has been found in the nuclei of yeast, plant and animal cells that    undergo cell division, suggesting a function in cell cycle regulation and/or DNA replication. It subsequently became clear that PCNA also played a role in other processes involving the cell genome.

PCNA exerts a tripartite function by operating as (1) a sliding clamp during DNA synthesis, (2) a polymerase switch factor and (3) a recruitment factor. Most of its functions are mediated by its interactions with various proteins involved in DNA synthesis, repair and recombination as well as in regulation of the cell cycle and chromatid cohesion. Moreover, post-translational modifications of PCNA play a key role in regulation of its functions. Finally, a phylogenetic comparison of PCNA genes suggests that the multi-functionality observed in most species is a product of evolution.

Most plant PCNAs exhibit features similar to those found for PCNAs of other eukaryotes. Similarities include: (1) a trimeric ring structure of the PCNA sliding clamp, (2) the involvement of PCNA in DNA replication and repair, (3) the ability to stimulate the activity of DNA polymerase δ and (4) the ability to interact with p21, a regulator of the cell cycle. However, many plant genomes seem to contain the second, probably functional, copy of the PCNA gene, in contrast to PCNA pseudogenes that are found in mammalian genomes.

3)

An epitope is a portion of a molecule to which an antibody binds. Epitopes can be composed of sugars, lipids or amino acids. In most cases, epitope tags are constructed of amino acids. Epitope tags are added to a molecule (usually proteins) which an investigator wants to visualize. Visualization can take place in a gel, a western blot or labeling via immunofluorescence.

If you wanted to follow a particular protein but did not have an antibody that would bind your protein, you might consider adding an epitope tag onto your protein. Epitope tags range from 10 to 15 amino acids long and are designed to create a molecular handle for your protein. An epitope tag could be placed anywhere within your protein, but typically they are placed on either the amino or carboxyl terminus to minimize any potential disruption in teriary structure and thus function of your protein

4) Eukaryotes and Prokaryotes