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The genomes of eukaryotes are punctuated by long stretches of sequence which do not code for proteins or RNAs. Although some of these regions do contain crucial regulatory sequences, the vast majority of this DNA serves no known function. Typically, these regions of the genome are the ones in which the fastest change, in evolutionary terms, is observed, because there is typically little to no selection pressure acting on these regions to preserve their sequences.
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Molecular Evolution of the Tre Recombinase
12:02

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Published on: May 29, 2008

Polymerase evolution: efforts toward expansion of the genetic code.

Aaron M Leconte1, Liangjing Chen, Floyd E Romesberg

  • 1Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Journal of the American Chemical Society
|September 8, 2005
PubMed
Summary

Researchers evolved a DNA polymerase to efficiently replicate DNA with unnatural base pairs, specifically the PICS self-pair. This advancement expands the genetic code by improving the synthesis and extension of DNA containing artificial nucleobases.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Biochemistry

Background:

  • The genetic code, typically a four-letter alphabet, can be expanded using unnatural nucleobase analogues.
  • The PICS self-pair is a stable, hydrophobic nucleobase analogue candidate for expanding the genetic code.
  • Optimizing DNA polymerases is crucial for efficiently replicating unnatural base pairs.

Purpose of the Study:

  • To evolve a DNA polymerase capable of efficiently replicating DNA containing the PICS self-pair.
  • To enhance the incorporation and extension of unnatural nucleobases within DNA.

Main Methods:

  • An activity-based phage display selection system was employed to evolve DNA polymerases.
  • The system utilized co-displayed polymerase libraries and a DNA substrate with the PICS self-pair.
  • Isolation was achieved by selecting polymerases that incorporated biotin-dUTP to the primer, enabling streptavidin capture.

Main Results:

  • A mutant Sf polymerase, named P2, was evolved with significantly enhanced PICS self-pair replication capabilities.
  • P2 successfully inserted dPICSTP opposite dPICS and extended the unnatural primer terminus with natural nucleotides.
  • P2 is a triple mutant (F598I, I614F, Q489H) exhibiting increased affinity for the PICS-containing DNA primer-template.

Conclusions:

  • The evolved P2 polymerase efficiently replicates DNA containing the PICS self-pair, a key step in expanding the genetic code.
  • The identified mutations suggest a mechanism for increased affinity towards unnatural base pairs.
  • This work demonstrates the power of directed evolution for creating polymerases tailored for synthetic biology applications.