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    A method to assay inhibitors of DNA polymerase IIIC activity

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    Authors
    Butler, Michelle Marie
    Wright, George E.
    UMass Chan Affiliations
    Department of Biochemistry and Molecular Pharmacology
    Graduate School of Biomedical Sciences
    Document Type
    Journal Article
    Publication Date
    2008-04-26
    Keywords
    Animals; Bacillus subtilis; Bacterial Proteins; purification; Cattle; Cloning, Molecular; DNA; DNA Polymerase III; purification; DNA Primers; Enzyme Inhibitors
    Life Sciences
    Medicine and Health Sciences
    
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    Link to Full Text
    http://dx.doi.org/10.1007/978-1-59745-246-5_3
    Abstract
    The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE.DNA polymerases are unique enzymes with respect to the five substrates (four dNTPs, one of which is radiolabeled, and primer:template DNA) that they typically utilize. Variations of the assay, e.g., by leaving out one dNTP but allowing measurable incorporation of the remaining substrates, or use of homopolymer primer:templates, may be used to simplify the assay or to obtain mechanistic information about inhibitors. Use of gel analysis of primer extension assays can also be applied to study alternate substrates of DNA polymerases. Methods to isolate pol IIIC from Gram-positive bacterial cells and to clone and express the polC gene are described in this chapter. In addition, the assay conditions commonly used to identify and study the mechanism of inhibitors of pol IIIC are emphasized.
    Source
    Methods Mol Med. 2008;142:25-36. Link to article on publisher's site
    DOI
    10.1007/978-1-59745-246-5_3
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/32939
    PubMed ID
    18437303
    Related Resources
    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1007/978-1-59745-246-5_3
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    Morningside Graduate School of Biomedical Sciences Scholarly Publications

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