Screening Polymerase Libraries for Altered Activity

Obviously, an adaptation of Loeb's scheme to the selection of RNA polymerases (DNA-dependent transcriptases, or RNA-dependent replicases), reverse tran-scriptases, or DNA polymerases with altered substrate tolerance is difficult because these enzymes hardly enable the conferring of a growth advantage to host cells. In an exceptional approach to this problem, Andrew Ellington and colleagues focused

Fig. 4.3.1. Schemes for genetic selection of DNA polymerase function based on complementation. (a) Host cells of E. coli recA718polA12 that encodes a temperature-sensitive variant of DNA polymerase I (PolIts) are transformed with a polymerase mutant library. Active polymerase mutants substitute for DNA polymerase I at the non-permissive temperature (37 °C). (b) The host strain E. coli recA718polA12trpE65 has an additional chromosomal ochre mutation (that is, a stop codon). As a consequence, this strain is unable to synthesize anthranilate synthase, an enzyme needed to produce tryptophan (Trp). Transformants surviving on tryptophan-deficient media might occur by direct reversion of the stop codon because of an active and error-prone polymerase variant [27].

Fig. 4.3.1. Schemes for genetic selection of DNA polymerase function based on complementation. (a) Host cells of E. coli recA718polA12 that encodes a temperature-sensitive variant of DNA polymerase I (PolIts) are transformed with a polymerase mutant library. Active polymerase mutants substitute for DNA polymerase I at the non-permissive temperature (37 °C). (b) The host strain E. coli recA718polA12trpE65 has an additional chromosomal ochre mutation (that is, a stop codon). As a consequence, this strain is unable to synthesize anthranilate synthase, an enzyme needed to produce tryptophan (Trp). Transformants surviving on tryptophan-deficient media might occur by direct reversion of the stop codon because of an active and error-prone polymerase variant [27].

on a method opposite to that of Loeb - and simply picked clone duplicates that did not grow if the expressed polymerase variant fulfilled their requirements [29]. In searching for T7 RNA polymerase variants with altered promoter specificities they used the so-called autogene [30], that is, a T7 RNA polymerase (T7 RNAP) gene linked to a T7 promoter. The activity of the autogene can be initiated by basal level expression of T7 RNAP in the cell, and can lead to cell death if induced by IPTG.

Usually, however, identifying active polymerases requires a strategy that involves active searching of the library. Again, Loeb and coworkers pioneered this field by starting with a combination of selection and screening [31, 32]. A Taq DNA poly-merase library was preselected for activity by genetic complementation of the above-mentioned selection scheme. Mutants that supported bacterial growth at the non-permissive temperature were isolated and screened for other interesting activity, for example RNA polymerase activity. They identified mutants with low effi ciency of RNA synthesis only, however, because their two-step procedure aimed at substituting for a DNA polymerase in the first place. Floyd Romesberg, Peter Schultz, and coworkers therefore invented a phage display-based approach for converting the Stoffel fragment of Taq DNA polymerase I (SF) into an RNA polymerase [33]. They displayed polymerase variants and primer-template duplexes on phage surfaces, assayed these constructs for incorporation of ribonucleoside triphosphates, including biotinylated UTP, and captured successful candidates on streptavidine-coated magnetic beads. Thus, the researchers were able to identify three SF variants that polymerized ribonucleoside triphosphates as efficiently as the wild-type incorporates dNTP substrates.

Another challenging approach for screening pools of polymerase variants was presented by Philipp Holliger and coworkers [34]. They constructed a simple feedback loop consisting of a polymerase that replicates only its own encoding gene within compartmentalized individual self-replication reactions. To co-localize gene and encoded protein the researchers emulsified E. coli cells, the "carriers" of polymerase variants, within droplets of a water-in-oil emulsion. Using this technique, Holliger was able to identify variants of Taq DNA polymerase with 11-fold greater thermostability or with 130-fold increased resistance to the potent inhibitor heparin.

In principle, the screening concepts introduced here could be applied to other polymerases, and other activity. As yet, however, the contexts of low polymerase fidelity or of tolerance versus non-natural substrates have not been sufficiently targeted. In the course of our studies, we tackled these problems and presented solutions for both, selecting, or screening, polymerase libraries. Thereby, we detected an error-prone polymerase variant and polymerase activity in the sole presence of sterically demanding substrates.

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