Protein Engineering Protocols - Mycobacteriology research center

238 Ghadessy and Holligeraffinity maturation to protection against DNA damage by ultraviolet radiation(2), and, possibly, RNA interference (3). Aberrant polymerase function hasbeen implicated in the pathogenesis of cancer (4) and many polymerases, inparticular, viral polymerases, are important drug targets. Finally, polymeraseshave been central to the development of modern biology, enabling DNAsequencing, polymerase chain reaction (PCR), site-directed mutagenesis, andcomplementary DNA cloning, and are also crucial for emerging technologies,such as molecular computing and nanobiotechnology. We reasoned that a betterunderstanding of polymerase function may, therefore, not only provideinsights into fundamental cellular processes, but may also enable novel applicationsin biotechnology and potentially accelerate the design of antiviral drugs.Although great progress has been made in the understanding of polymerasefunction through the pioneering structural studies of T. Steitz and others, andthrough careful biochemical and kinetic analysis of wild-type and mutant polymerases(5), the ability to alter polymerase properties in a predictable manneror to tailor polymerases for existing or novel applications has lagged behind.1.1. Polymerase Engineering by DesignAttempts have been made to alter polymerase function by the use of proteinengineering. For example, variants of Taq polymerase (Stoffel fragment andKlentaq) have been generated by full or partial deletion of its 5′ to 3′ exonucleasedomain; these show improved thermostability and fidelity, although at thecost of reduced processivity (6,7). In addition, the availability of high-resolutionstructures has allowed the rational design of mutants with improved properties(for example, Taq mutants with improved properties of dideoxynucleotide incorporationfor cycle sequencing; ref. 8). Site-directed mutagenesis has also yieldedpolymerase variants with an increased capability to incorporate ribonucleotides(9,10), reduced pausing (11), as well as numerous polymerases with alteredfidelity (12). Grafting of the thioredoxin-binding loop of T7 DNA polymeraseonto the Escherichia coli Pol I Klenow fragment lead to an impressive increasein processivity (13).1.2. Polymerase Engineering by Repertoire SelectionGenetic approaches have also been used for polymerase design. For example,Loeb and co-workers have selected active mutant polymerases by complementationof a polA12, recA718 bacterial strain with Taq polymerase (14), but alsowith human immunodeficiency virus reverse transcriptase (15) and human pol β(16). Complementation selection was used to probe the mutability of the polymeraseactive site, and screening of the selected mutants has yielded polymerasevariants with a range of properties, including reduced fidelity or an increasedcapability to incorporate ribonucleotides. Recently, the same approach was used