Processes of DNA Replication and Protein Synthesis

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).

Compare and contrast DNA replication and protein synthesis?

The key to iPOND is the use of “click” chemistry, a copper-catalyzed reaction between an azide and an alkyne, that has been employed extensively to label and retrieve biomolecules from the complex cellular environment (Figure 2). The Cortez lab took advantage of the fact that cells readily incorporate the alkyne-containing nucleotide 5′-ethynyl-2′-deoxyuridine (EdU) in place of thymidine as they replicate DNA. Reaction of DNA containing EdU with a biotin azide reagent (designed and synthesized in the lab of VICB member Ned Porter) under click chemistry conditions attaches a biotin label to the alkyne moiety of the incorporated EdU in the DNA. Following fragmentation of the DNA, biotin-labeled DNA fragments are captured on streptavidin-coated beads, providing a rapid and efficient method for purification (Figure 3A). The use of formaldehyde to covalently cross-link DNA to any associated proteins provides a means to isolate and identify proteins bound to the DNA at the site of the EdU-biotin label.


DNA, RNA, DNA Replication, Protein Synthesis ..

02/01/2018 · DNA, RNA, DNA Replication, Protein Synthesis and Mutations Review Game

DNA replication is a complex process requiring the intricately timed interaction of a large number of enzymes and accessory binding proteins (Figure 1). Damage encountered during replication, termed replication stress, can stall replication and activate the DNA damage response (DDR). The DDR protects, repairs, and promotes the faithful completion of chromosome replication. Alternatively, if the damage burden is too large and cannot be repaired, the DDR targets the damaged cells for cell death. These DDR activities ensure that each daughter cell will be an exact and genomically intact replica of the parental cell. Mutations in DDR proteins lead to the accumulation of genomic instability that ultimately causes human diseases such as cancer. Therefore understanding the DDR is important to understanding mechanisms of disease etiology. The regulation of the DDR in response to insults such as irradiation and chemical toxins has been thoroughly studied; however attempts to investigate how the DDR is regulated under conditions of replication stress have been hampered by a lack of methods with sufficient sensitivity and resolution to explore the changes that occur over time at the replication fork. Now, Vanderbilt Institute of Chemical Biology (VICB) member David Cortez and his laboratory propose a new method, iPOND (isolation of Proteins On Nascent DNA), that provides a high resolution picture of DNA replication at both healthy and stalled forks [B. M. Sirbu et al. (2011) Genes and Development, 25, 1320].