The genetic material making up genes is composed of deoxyribose nucleic acid (DNA). DNA has several properties that enable it to function as genetic material; it is able to (1) direct the synthesis of copies of itself (replicate itself), (2) store information that directs protein synthesis, and (3) direct the synthesis of structural and regulatory proteins. DNA is composed of nucleic acids. Therefore, appreciation of the structure of nucleic acids and DNA, and the process of protein synthesis are essential prerequisites to the understanding of control of cellular activity, drug action, and various disease processes.
DNA transcription is the process of making a single strand complementary RNA copy of DNA. Data is copied from the DNA to the RNA with the aid of the enzyme RNA polymerase. Using this process, the genetic information stored in the DNA is carried in the form of RNA to other parts of the cell. In eukaryotic cells a gene begins with a promoter region and an initiation code and ends with a termination code. However, the intervening gene sequence contains patches of nucleotides that have no meaning. If they were used in protein synthesis, the resulting proteins would be worthless. Eukaryotic cells prune these segments from the mRNA after transcription. RNA polmerase synthesizes a strand of pre-mRNA that initially includes copies of the meaningful mRNA coding sequences (exons) and the meaningless mRNA coding sequences (introns). Soon after its manufacture, this pre-mRNA molecule has the meaningless introns clipped out and the exons spliced together in the final version of mature mRNA
The purpose of this exercise is to become familiar with the structure of nucleic acids, DNA, RNA and to reinforce the role of DNA and RNA in the process of protein synthesis.
In 1969, Jovin et al. elucidated the amino acid composition (Jovin et al. 1969a, b). That same year, DeLucia and Cairns isolated an E. coli strain with a mutation that affected the DNA polymerase and surprisingly found that the mutant synthesized DNA normally. This discovery cast doubts on the role of DNA polymerase in replication and led groups to search for other replication enzymes. At the same time, Klenow and colleagues showed that the treatment of DNA polymerase with the proteolytic enzyme subtilisin (type Carlsberg) resulted in an increase of polymerase activity and decrease of exonuclease activity. The resulting DNA polymerase was isolated and was named the “Klenow fragment” (Klenow and Henningsen 1970a, and Klenow and Overgaard-Hansen 1970).
The Klenow fragment is a proteolytic product of E. coli DNA polymerase I that retains polymerization and 3’ to 5’ exonuclease activity, but has lost 5’ to 3’ exonuclease activity.
DNA polymerase I is the predominant polymerizing enzyme found in E. coli. It contains a single disulfide bond and one sulfhydryl group (Jovin et al. 1969b). Five distinct DNA polymerases have been isolated from E. coli and have been designated I, II, III, IV, and V. DNA polymerase I functions to fill DNA gaps that arise during DNA replication, repair, and recombination. DNA polymerase II also functions in editing and proofreading mainly in the lagging strand (Kim et al. 1997, Wagner and Nohmi 2000). DNA polymerase III is the main replicative enzyme. DNA polymerase IV and V have large active sites that allow for more base misincorporation, and are therefore more error-prone. They also lack proofreading-exonuclease subunits to correct misincorporations (Nohmi 2006, and Hastings et al. 2010). DNA polymerase V is present at significant levels only in SOS-induced cells and over-expression restricts DNA synthesis (Marsh and Walker 1985).