SpringerProtocols: Abstract: 2. Use of Pol I-Deficient E. coli for Functional Complementation of DNA Polymerase

2. Use of Pol I-Deficient E. coli for Functional Complementation of DNA Polymerase

By: Manel Camps³, Lawrence A. Loeb⁴

Abstract

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The E. coli JS200 strain carries a temperature-sensitive allele of DNA polymerase I that renders this strain conditional lethal. Growth under restrictive conditions is restored by small amounts of DNA polymerase activity. Even mutants with greatly reduced (1–10% of wild-type) catalytic activity or distantly-related polymerases of bacterial, eukaryotic, or viral origin effectively complement JS200 cells. The versatility of this complementation system makes it advantageous for selection of active polymerase mutants, for screening of polymerase inhibitors, or for screening of mutants with altered properties. Here we describe complementation of JS200 cells with the wild-type E. coli DNA polymerase I to illustrate such functional polymerase complementation.

Affiliation(s): (3) Department of Pathology, University of Washington, Seattle, WA
(4) Department of Biochemistry, University of Washington, Seattle, WA

Book Title: Directed Enzyme Evolution: Screening and Selection Methods

Series: Methods in Molecular Biology | Volume: 230 | Pub. Date: May-16-2003 | Page Range: 11-18 | DOI: 10.1385/1-59259-396-8:11

Subject: Cell Biology

References

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