Polymerase evolution: efforts toward expansion of the genetic code.

Polymerase evolution: efforts toward expansion of the genetic code. Research Abstract Details 

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Polymerase evolution: efforts toward expansion of the genetic code. Abstract Text:

Aaron M Leconte,Liangjing Chen,Floyd E Romesberg,

Genetic information is encoded by, but potentially not limited to, a four-letter alphabet. A variety of predominantly hydrophobic nucleobase analogues that form self-pairs in DNA have been examined as third base pair candidates. For example, the PICS self-pair is both stable in duplex DNA and synthesized by some wild-type polymerases with reasonable efficiency. These efforts to expand the genetic code are expected to be facilitated by optimizing both the unnatural nucleobase analogues and the polymerases that replicate them. Here, we report the use of an activity-based selection system to evolve a DNA polymerase that more efficiently replicates DNA containing the PICS self-pair. The selection system is based on the co-display on phage of DNA polymerase libraries and a DNA substrate containing the self-pair. Only polymerases that accept the unnatural substrate incorporate a biotin-dUTP to the attached primer and may then be isolated on a streptavidin solid support. A mutant of Sf polymerase, P2, was evolved which both inserts dPICSTP opposite dPICS in the template and extends the unnatural primer terminus by incorporation of the next correct natural dNTP, where the parental enzyme catalyzes neither step at detectable rates. P2 was found to be a triple mutant of Sf, with the mutations F598I, I614F, and Q489H. The evolved properties of P2, as well as the observed mutations, are consistent with an increased affinity for the DNA primer-template containing the self-pair.

Polymerase evolution: efforts toward expansion of the genetic code. Publishing Authors By Initials

AM Leconte,L Chen,FE Romesberg,

For similar organic chemicals research abstracts see: organic chemicals research

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Polymerase evolution: efforts toward expansion of the genetic code. Journal Published:

PUBLICATION TYPE: Research Support, U.S. Gov't,

Journal: Journal of the American Chemical Society

VOLUME: 127

Page Numbers: 12470-1

Journal Abbreviation: J. Am. Chem. Soc.

ISSN: 0002-7863

DAY: 14

MONTH: Sep

YEAR: 2005

Polymerase evolution: efforts toward expansion of the genetic code. Information

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LANGUAGE: eng

NlmUniqueID: 7503056

Polymerase evolution: efforts toward expansion of the genetic code. Keywords Mesh Terms:

KEYWORDS: Organic Chemicals

MESH TERMS: chemistry

Chemical & Substance for Abstract: Polymerase evolution: efforts toward expansion of the genetic code. Information

Substance Name: DNA-Directed DNA Polymerase

Registry Number: EC 2.7.7.7

Grant and Affiliation Information for Polymerase evolution: efforts toward expansion of the genetic code.

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

Country: United States

AGENCY: United States NIGMS

GRANT: GM 60005

ACRONYM: GM

MEDLINETA: J Am Chem Soc

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