DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.Prolonged survival of individual species depends on the accurate transmission of genetic material from one generation to the next (1). However, in times of stress, the propensity to mutate and to rapidly create variants that can escape selection pressures facilitates survival of a small fraction of the original population (2). Thus, evolution may be characterized by periods of high fidelity DNA replication, as well as by the presence of transient mutators, which have a selective growth advantage during adverse conditions (3). Identifying mechanisms of generating potential mutators is crucial toward understanding the dynamic processes that govern evolution, as well as toward devising effective chemotherapeutic strategies against pathogenic bacteria (4, 5) and cells (6) that mutate at elevated rates.Cells have evolved multistep mechanisms to guarantee the exceptionally high fidelity of DNA replication that is required for the maintenance of species. The genetic sequence of organisms is maintained over prolonged evolution by the fidelity of DNA replication (7), the efficiency of DNA repair processes (8), and the recombination-mediated lateral transfer events (9). Quantitatively, nucleotide selection at the active site of DNA polymerases is the most significant contributor to the fidelity of DNA replication (10). Nucleot...
Objectives: To review the literature comparing operative techniques in tonsillectomy and postoperative pain. Methods: Randomized controlled trials comparing operative techniques and pain outcomes over a 10-year period were reviewed. Results: 44 trials met inclusion criteria for analysis. A total of nine different techniques for complete or partial tonsillectomy were compared. There were several studies with contradictory results. Many of the studies were performed under different parameters. Conclusions: Future studies comparing tonsillectomy technique and pain outcomes should be conducted using a standard protocol to allow for accurate comparisons between numerous techniques.
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