Bst polymerase - a humble relative of Taq polymerase

Abstract

DNA polymerases are a superfamily of enzymes synthesizing DNA using DNA as a template. They are essential for nucleic acid metabolism and for DNA replication and repair. Modern biotechnology and molecular diagnostics rely heavily on DNA polymerases in analyzing nucleic acids. Among a variety of discovered DNA polymerases, Bst polymerase, a large fragment of DNA polymerase I from Geobacillus stearothermophilus, is one of the most commonly used but is not as well studied as Taq polymerase. The ability of Bst polymerase to displace an upstream DNA strand during synthesis, coupled with its moderate thermal stability, has provided the basis for several isothermal DNA amplification methods, including LAMP, WGA, RCA, and many others. Bst polymerase is one of the key components defining the robustness and analytical characteristics of diagnostic test systems based on isothermal amplification. Here, we present an overview of the biochemical and structural features of Bst polymerase and provide information on its mutated analogs.

Keywords: Bst polymerase; DNA polymerase; Directed evolution; Fidelity; Fusion proteins; Processivity; Site-directed mutagenesis; Strand displacement.

Graphical abstract

Fig. 1

Schematic representation of LAMP amplification (A), NEAR (B), and RCA.

Fig. 2

Schematic representation of Bst polymerase structure (A) and functionally important amino acid residues (B).

Fig. 3

3D-structure of Bst polymerase without N-terminal 5′–3′ exonuclease domain. The separate domains are marked in color: 3′–5′‐exonuclease is red, palm is green, thumb is blue, and fingers are yellow. The image was taken from the RCSB PDB (RCSB.org) of PDB ID 1XWL .

Fig. 4

3D-structure of the 3′–5′ exonuclease domain. The residues corresponding to those catalytically active in the Klenow fragment are labeled and marked in blue, with their side chains also shown. The secondary structures are also marked by the corresponding numbers and letters. The image was taken from the RCSB PDB (RCSB.org) of PDB ID 1XWL .

Fig. 5

3D structure of the polymerase domain. A represents the polymerase subdomains. The separate subdomains are marked in color: the palm is beige, the thumb is red, and the fingers are blue. The catalytically active residues are labeled and marked by magenta, with their side chains also shown. B represents the important α-helices. The helices are marked in color: H in red, I in yellow, O in violet, and O1 in orange. C and D show several amino acid residues participating in the polymerase functioning. The residues are labeled and marked by yellow, with their side chains also shown. The image was taken from the RCSB PDB (RCSB.org) of PDB ID 1XWL .