Telomerases.

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

DNA-Directed DNA Polymerase↗

PubMed2001

High-fidelity in vitro recombination using a proofreading polymerase.

We describe a convenient PCR-based protocol for in vitro recombination of homologous genes, thereby minimizing the rate of associated point mutations. High-fidelity recombination conditions were obtained using Vent DNA polymerase, which, in contrast to Taq DNA polymerase, shows significant proofreading activity and ranges among the slowest thermostable DNA polymerases, allowing tight control of the polymerase-catalyzed DNA extension. To determine the mutagenesis rate and to analyze the efficiency of recombination, 89 clones from a standard experiment were randomly selected for further analysis. Sequence comparison revealed that 21% (19/89) of the clones result from different recombination events in the marker-containing region (260 bp). The overall mutation rate is only 0.02%, which is the lowest rate thus far reported for in vitro recombination experiments.

DNA-Directed DNA Polymerase↗