Engineering Polymerases for New Functions

Abstract

DNA polymerases are critical tools in biotechnology, enabling efficient and accurate amplification of DNA templates, yet many desired functions are not readily available in natural DNA polymerases. New or improved functions can be engineered in DNA polymerases by mutagenesis or through the creation of protein chimeras. Engineering often necessitates the development of new techniques, such as selections in water-in-oil emulsions that connect genotype to phenotype and allow more flexibility in engineering than phage display. Engineering efforts have led to DNA polymerases that can withstand extreme conditions or the presence of inhibitors, as well as polymerases with the ability to copy modified DNA templates. In this review we discuss polymerases for biotechnology that have been reported along with tools to enable further development.

Keywords: DNA damage; DNA modification; DNA polymerase; fidelity; processivity.

FIGURE 1.

Structures of representative DNA polymerases with DNA in dark gray and the incoming nucleotide in yellow. Conserved polymerase domains are colored similarly in each structure: the thumb domain is in green, the palm domain is in cyan, and the fingers domain is in blue. A) A-family Thermus aquaticus polymerase I (PDB ID: 1TAQ), the 5′−3′ exonuclease domain is in light purple and the 3′−5′ exonuclease domain is in dark purple [79]. B) B-family Enterobacteria phage RB69 (PDB ID: 4FK4), the N-terminal domain is in maroon and the 3′−5′ exonuclease domain is in dark purple [80]. C) Y-family Escherichia coli polymerase IV (PDB ID: 4IRK), the little finger domain is in magenta [81].

FIGURE 2:

Polymerization reaction and substrates. A) General polymerization reaction. B) Structures of nucleic acid sugars and modifications. C) Modified nucleobases.

FIGURE 3:

Polymerase engineering strategies. In order to generate polymerases with new or altered activity, the addition of new residues or mutations at residues already present are usually made, candidates for which can be identified from a variety of sources. The replacement of full domains is one engineering route that relies on the swapping or addition of domains from other enzymes to incorporate new activity or function. Like domain swapping, many protein engineering methods involve rational design based on information-transfer methods from comparison to other proteins or computational analysis, random mutagenesis followed by selection, or a combination of the two approaches. Targeted mutations are often knowledge-based and rely on previous studies for an understanding on how to achieve a desired effect. Random mutagenesis relies on making mutations in a less targeted fashion, often through error-prone PCR or use of random primers. Due to the large sequence space of enzymes, often large numbers of variants must be screened or selected to identify the desired functionality. Methods such as directed evolution and compartmentalized-self replication have been developed and used due to their increased sampling abilities.