Replication of DNA enzymology

The fundamental enzymology of DNA replication derives from both in vivo and in vitro studies with cells and extracts derived from E. coli. Many of the enzymes involved in DNA replication were identified by isolation of conditional lethal mutants of the bacterium, e.g., mutants that are unable to replicate DNA (and unable to grow) at high temperatures (42°C) but that replicate and grow normally at low temperatures (30°C). 

The synthesis of DNA is a complex process because of the need for faithful replication, enzyme specificity, and topological constraints. Approximately 20 different enzymes are utilized in bacteria to replicate DNA. In addition to polymerization reactions, DNA replication requires accurate initiation, termination, and proofreading to eliminate errors. 

All DNA polymerases select the nucleotide that is to be added to the 3 -OH end of the growing chain 

Biological activity RNA primer excision, DNA repair SOS DNA repair Replicase 

Even though the hydrolysis of the nucleoside triphosphates has a large negative AG, the polymerization reaction as written still has a positive AG at concentrations present in a cell and in laboratory reactions (+0.5 kcal/mol = 2.1 kJ/mol). Thus, in the absence of any other reaction DNA polymerases would catalyze depolymerization rather than polymerization. Indeed, if excess pyrophosphate and a polymerase are added to a solution containing a partially replicating DNA molecule, the polymerase acts like a nuclease. In order to drive the reaction to the right, pyrophosphate must be removed, and this is 

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To explain the enzymology of DNA replication, we first introduce the enzymes that degrade DNA rather than synthesize it. These enzymes are known as nucleases, or DNases if they are specific for DNA rather than RNA. Every cell contains several different nucleases, belonging to two broad classes exonucleases and endonucleases. Exonucleases degrade nucleic acids from one... 

Source Adapted from T. A. Baker and S. H. Wickner, Genetics and enzymology of DNA replication in Escherichia coli. Ann. Rev. Genetics, 26 447, 1992. 

The enzymology of DNA replication in prokaryotes 227 TABLE 18.1 The subunit structure of DNA polymerase III holoenzyme of E. coli... 

One area of basic biochemical research that has paid unexpected dividends is DNA replication. Enzymological work here has characterized the various DNA polymerases in bacterial and eukaryotic cells. With progress in the biochemical characterization of these enzymes, new applications have been found for them in research... 

From the complementary duplex structure of DNA described in chapter 25, it is a short intuitive hop to a model for replication that satisfies the requirement for one round of DNA duplication for every cell division. In chapter 26, DNA Replication, Repair, and Recombination, key experiments demonstrating the semiconservative mode of replication in vivo are presented. This is followed by a detailed examination of the enzymology of replication, first for how it occurs in bacteria and then for how it occurs in animal cells. Also included in this chapter are select aspects of the metabolism of DNA repair and recombination. The novel process of DNA synthesis using RNA-directed DNA polymerases is also considered. First discovered as part of the mechanisms for the replication of nucleic acids in certain RNA viruses, this mode of DNA synthesis is now recognized as occurring in the cell for certain movable genetic segments and as the means whereby the ends of linear chromosomes in eukaryotes are synthesized. 

The assay conditions provided below should be considered as starting points for further optimization. As the optimal conditions for each activity may differ, researchers are encouraged to optimize salt concentration, pH, template concentration, and temperature for each DNA polymerase in each assay. A thorough review of assay methods for mesophilic DNA polymerases can be found in a Methods of Enzymology volume devoted entirely to DNA replication proteins. ... 

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