Both parameters are enhanced when the pH is decreased from 9.8 to 6.2 (9). Both the fidelity and processivity of the Klenow fragment are influenced by reaction conditions. Mutant Klenow fragments that have nonfunctional proofreading activity have an approximate fidelity of 10 (8). The fidelity of Klenow polymerase during DNA-templated DNA polymerization is approximately 1/50,000 (i.e., one misincorporation per 50,000 nucleotides), and this fidelity is derived in part from its 3-5′ editing activity. The Klenow fragment also acts as a reverse transcriptase by copying RNA templates (7), albeit distributively (i.e., incorporating one to two nucleotides per binding event), but the biological relevance of this activity is not known. The Klenow fragment incorporates individual deoxyribonucleotides at a relatively modest rate of 50 nucleotides/s (6), and is moderately processive (on average, it synthesizes 50 nucleotides after binding and before dissociation). Much of the biochemical understanding of the polymerase and 3′-5 exonuclease functions stems from studies of the Klenow fragment. The N-terminal 5′-3′ exonuclease domain contains 323 amino acid residues, and the C-terminal fragment contains 605 residues (4, 5), which is frequently called the Klenow fragment. Pol I is cleaved into two subunits by mild proteolysis with trypsin. coli K12 chromosome (3), is a single-subunit enzyme that functions autonomously of other replicative factors. Subunits of Pol I and Their PropertiesĭNA polymerase I, a product of the PolA gene which maps at minute 86 of the E. The early cloning and overexpression of the Klenow fragment (1) and the determination of its three-dimensional structure (2) have allowed better understanding of polymerization mechanisms, molecular mechanisms of 3′-5′ exonuclease activity, and structure-function relationships of polymerases. coli (400 molecules per cell), and it functions primarily to fill DNA gaps during repair and replication. Pol I is the most abundant polymerase in E. coli DNA polymerase I (103 kDa) results in forming of two products: (1) the large Klenow fragment (68 kDa) that contains both the DNA polymerase and 3′-5′ exonuclease (proofreading) activities and (2) a smaller fragment (35 kDa) that contains the 5′-3′ exonuclease activity. This enzyme contains three activities: 5′-3′ polymerase, 3′-5′ exonuclease (for proofreading), and 5′-3′ exonuclease (for nick translation, excision repair, and hydrolysis of the RNA primers during DNA replication). Pol I, the first polymerase discovered in bacteria, is required for rapid, efficient growth in rich media. coli, has important roles during DNA replication, genetic recombination, and DNA repair. Pol I, one of three such polymerases found in E. Escherichia coli DNA polymerase I (pol I) has served for several decades as the prototype DNA-dependent DNA polymerase.
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