Directed DNA Polymerase Evolution: Effects of Mutations in Motif C on the Mismatch‐Extension Selectivity of Thermus aquaticus DNA Polymerase

Abstract

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele-specific PCR, as demonstrated by real-time PCR experiments and add to our understanding of DNA polymerase selectivity.

Engineered DNA Polymerases in Biotechnology

“…Reprinted from ref. [37]. Copyright: Wiley-VCH, 2007. 2080 www.chembiochem.org allelic discrimination resulting in a more reliable detection system.…”

“…[41] Marx and co-workers increased the selectivity of a Taq DNA polymerase by nonpolar substitution mutations of three amino acids QVH (Gln, Val, His; residues 782-784) of motif C, which is directly neighboured to catalytically essential amino acid residues. [36,37] Screening of ã 10,000 member mutant library yielded several mutants with respective amino acid mutations like ILL (Ile, Leu, Leu) instead of QVH showing increased discrimination properties of cognate versus non-cognate primer/template pairs (see Figure 4). Moreover, the hypothesis concerning the involvement of motif C in selectivity mechanisms has been corroborated by a successful design transfer of the finding for Taq DNA polymerase to Pfu DNA polymerase, a member of a different DNA polymerase sequence family.…”

“…Subsequent screening reactions of mutants can be processed by nucleotide-incorporation assays, [35] primer-extension reactions, [36] or PCR. [37] PCR screening results in a very high sensitivity due to the exponential enrichment of the product. Screening by primer extension is feasible by exploiting the fluorescence increase of minor-groove-binding dyes such as Sybr Green I [36] or by incorporation of radioactive-labelled triphosphates.…”

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DN

“…2, А) [38–40]. The domains of the A–family DNA–polymerases consist of six evolutionarily conserved motifs ( А , В , С , 1, 2 and 6), which are thought to play the main role in the formation of the active site and the network of specific bonds with the DNA–substrate [39–42]. The most conserved motifs are А , В and С , two of which ( А and С ) are present in all the known DNA– and RNA–polymerases.…”

“…The fragments responsible for the capture of the 3’–terminus of the primer, the inserted nucleotide, and the two magnesium ions needed for the catalysis are all localized on the inner surface of the “fingers” (motifs В , 6) and on the surface of the “palm” at the base of the “fingers” (motifs А , С ). The polymerase active site, which accomplishes the addition of nucleotides to the growing strand, is situated in the “palm” domain [40, 42]. Some DNA–polymerases also have additional domains, which can, for instance, add 3’ → 5’ exonuclease activity.…”