Biochemistry - Chemical Reactions in Living Cells, Volume 3 - D. Metzler 1980

Biochemical Genetics and the Synthesis of Nucleic Acids and Proteins
DNA Replication
Enzymes of DNA Synthesis

Once DNA polymerase I had been identified (Section A, 3, a), it was widely believed that the primary enzyme responsible for chain elongation during DNA Synthesis had been discovered. However, the discovery of an E. coli amber mutant lacking the Gene that encodes polymerase I (the polA gene; Fig. 15-1), yet still capable of normal DNA synthesis, stimulated an intensive search for new DNA polymerases. Two Other Enzymes were subsequently discovered—DNA polymerase II (encoded by the polB gene) and DNA polymerase III—whose intracellular levels did not exceed 25% of that of DNA polymerase I [195, 196]. While both enzymes resembled DNA polymerase I in many properties, they differed significantly in several respects.

One property of DNA polymerase I not mentioned previously is that the enzyme not only catalyzes the growth of DNA chains from the 3' end of the primer but also brings about a 10-fold slower hydrolytic Cleavage of NUCLEOTIDES from the 3' end. Furthermore, this same enzyme can catalyze the hydrolytic cleavage of nucleotides from the 5' end of DNA chains. It has been shown that these distinct catalytic activities reside in different domains of the same protein molecule, as if DNA polymerase I were the product of two fused genes [197]. Unlike DNA polymerase I, DNA polymerases II and III do not catalyze the hydrolytic removal of nucleotides from the 5' end.

It is hypothesized that the 3'–5' exonuclease activity enables DNA polymerase I to function as a "proofreader." This polymerase acts at the 3' end of the growing DNA chain. According to the proofreading hypothesis, before moving on to the next position¹⁾, the enzyme verifies whether the preceding base pair has been formed correctly. If the previous pair is mismatched, the enzyme acts as an exonuclease, excising the incorrectly incorporated nucleotide and allowing the polymerase to then insert the correct one. Thus, each base pair is checked twice: first prior to polymerization, and a second time after polymerization. This process is illustrated schematically in Fig. 15-30, which demonstrates how the 3'–5' exonuclease activity can operate once the enzyme reaches the end of a gap.

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FIG. 15-30. Schematic diagram illustrating three Types of Enzymatic activity of DNA polymerase I. The top panel depicts the 3'–5' exonuclease, or "proofreading," activity of the enzyme. Although the diagram shows the enzyme advancing by one position before the next step occurs, the precise timing of this translocation has not been definitively established. The bottom panel illustrates the polymerization reaction and the 5'–3' exonuclease action.

¹⁾ It remains uncertain whether the polymerase verifies base-pairing fidelity before or after translocation to the next polymerization site. The only definitively established fact is that the enzyme excises any mismatched nucleotide from the 3' end.

Important insights into the Replication process have been gained through genetic approaches [196, 198]. A series of Temperature-sensitive E. coli mutants defective in DNA synthesis were isolated, allowing the identification of the dnaA, B, C, D, E, F, and G genes at various loci on the chromosome map. The products of gene A, and likely gene C, are required for the initiation of replication but are dispensable for elongation. Genes B, D, E, and G are involved in elongation. Genes C and D are mapped very close to each other (at 89 min) and are now believed to constitute a single gene. This gene may encode a bifunctional protein capable of catalyzing both initiation and elongation. The product of gene F was identified as Ribonucleotide reductase [Eq. (14-50)]. This leaves the products of genes B, D, E, and G, which evidently play crucial roles in elongation. None of these genes determines the synthesis of DNA polymerase I. The dnaE gene has been identified as encoding DNA polymerase III, thereby providing genetic confirmation of its key role. However, this polymerase by itself is incapable of replicating double-stranded DNA; other Proteins are required for its function.

Eukaryotic DNA polymerases are classified into a, ß, and y forms, which are found in the nuclei (with the a form also detected in the Cytoplasm), as well as a mitochondrial (mt) enzyme [199].

While it is well established that METABOLISM/36.html">DNA replication in E. coli begins with a specific initiation event followed by bidirectional elongation along the chromosome, the mechanisms governing the termination of replication are much less understood. A series of experiments has demonstrated that termination somehow triggers the Synthesis of specific mRNA and proteins required for Cell Division [200]. Thus, the Cell Cycle essentially comprises a sequence of successive events, each of which "triggers" the next.