Engineering DNA polymerases with fusion protein technology

Fusion DNA polymerases are genetically engineered enzymes comprising two constituent elements: a DNA polymerase and double-stranded DNA binding protein. These chimeric proteins are powerful enzymes that have significantly improved modern PCR by enhancing its efficiency and enabling a variety of new applications.

The idea of creating fusion DNA polymerases was inspired by the natural cellular replication machinery. Replication systems in cells rely on highly processive DNA polymerases that are capable of incorporating thousands of nucleotides without dissociating from the DNA template. Their high processivity is achieved through interaction with so called “processivity-enhancing proteins” that either bind directly to dsDNA to increase polymerase-DNA binding efficiency or, that mechanically prevent the polymerase from dissociating from its DNA template.

Artificial in vitro systems for DNA amplification reconstitute the replication machinery with core components but often lack the critical DNA polymerase processivity factors. DNA polymerases used in PCR commonly have very low processivity and are able to incorporate only a few nucleotides per binding event. As a consequence, these polymerases are slow, typically requiring more than 1 minute to amplify 1 kb of DNA template; they are unable to amplify amplicons longer than 4–5 kb and are easily affected by inhibitors such as ethanol, EDTA, or other factors in DNA samples. Consequently, low processivity of PCR enzymes is known to be a key limiting factor for PCR efficiency, and therefore, increasing DNA polymerase processivity has been necessary to improve PCR performance and enable new applications.

Protein engineering based on fusion technology was employed in an attempt to improve performance of DNA polymerases used in PCR(1). Mimicking natural replication machinery, a dsDNA-binding protein was fused to a non-processive DNA polymerase in order to serve as its processivity-enhancing domain. The dsDNA-binding protein of choice was Sso7, derived from the thermophilic archea, Sulfolobus sulfactaricus. Sso7 is a very small (7 kDa) sequence non-specific DNA–binding protein that naturally functions in chromatin remodeling. When Sso7 was fused to non-processive DNA polymerases, the polymerases not only became highly processive, but they also demonstrated numerous other favorable features including higher efficiency, faster overall performance, and the ability to amplify amplicons as long as 15 kb. In addition, polymerases with Sso7 displayed surprising resistance to PCR inhibitors from tissues and DNA samples. This feature has further enabled new applications such as Direct PCR which allows DNA amplification directly from tissue samples without the need for any DNA purification or extraction steps.

The best known and widely used fusion PCR enzyme is the Thermo Scientific™ Phusion™ High-Fidelity DNA Polymerase. Phusion DNA polymerase was created by fusing the Pyrococcus-like proofreading DNA polymerase to Sso7. This powerful combination resulted in an enzyme that has superior features including very high processivity capable of amplifying templates as long as 20 kb and allowing very short reaction times (15 seconds/kb), high fidelity (52x Taq DNA polymerase), robustness, and inhibitor tolerance. With its introduction in 2003, Phusion DNA polymerase established a new standard for high-performing PCR. Phusion products are referenced in thousands of publications and have become the first-choice DNA polymerases for a multitude of applications ranging from reconstruction(2), design(3) and massively-parallel, high-throughput sequencing of whole genomes(4).


References

  1. Y. Wang et al., (2004) A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nuc. Acids Res. 32:1197-1207.
  2. D.G. Gibson et al., (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215-1220.
  3. D.G. Gibson et al., (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52-56.
  4. Kinde et al., (2011) Detection and quantification of rare mutations with massively parallel sequencing. PNAS 108(23):9530–953.

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