Selection of Polymerases with Altered Activity and Fidelity

Selection strategies exploit conditions that favor the exclusive survival of desired variants. Most often this is achieved by genetic complementation of a host that is deficient in a certain pathway or activity (Figure 4.3.1a). Inventing a selection scheme for ''low-fidelity DNA synthesis'' is, however, a difficult task. Loeb and coworker Joann Sweasy made a major breakthrough in this field when they employed an E. coli host strain that encoded a temperature-sensitive mutant of DNA polymerase I (genotype: recA718polA12). At elevated temperatures this mutant strain fails to form colonies unless complemented by a DNA polymerase that can effectively substitute for DNA polymerase I [20]. Using strain E. coli recA718polA12, the researchers first identified active mutants of rat DNA polymerase b [21, 22], of Ther-mus aquaticus DNA polymerase I [23], and of HIV reverse transcriptase [24-26]. Shortly thereafter, Sweasy and coworkers extended this genetic selection system to identification of different DNA polymerase b mutator mutants, that is, error-prone variants of the enzyme (Figure 4.3.1b) [27, 28].

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