JBC Ideal method for primary cell transfection

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Originally published In Press as doi:10.1074/jbc.M010752200 on February 2, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15423-15433, May 4, 2001
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Distinct Roles for Ku Protein in Transcriptional Reinitiation and DNA Repair*

Robin L. WoodardDagger , Kyung-jong Lee, Juren Huang, and William S. Dynan§

From the Gene Regulation Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912

Transcriptional reinitiation is a distinct phase of the RNA polymerase II transcription cycle. Prior work has shown that reinitiation is deficient in nuclear extracts from Chinese hamster ovary cells lacking the 80-kDa subunit of Ku, a double-strand break repair protein, and that activity is rescued by expression of the corresponding cDNA. We now show that Ku increases the amount or availability of a soluble factor that is limiting for reinitiation, that the factor increases the number of elongation complexes associated with the template at all times during the reaction, and that the factor itself does not form a tight complex with DNA. The factor may consist of a preformed complex of transcription proteins that is stabilized by Ku. A Ku mutant, lacking residues 687-728 in the 80-kDa subunit, preferentially suppresses transcription in Ku-containing extracts, suggesting that Ku interacts directly with proteins required for reinitiation. The Ku mutant functions normally in a DNA end-joining system, indicating that the functions of Ku in transcription and repair are genetically separable. Based on our results, we present a model in which Ku is capable of undergoing a switch between a transcription factor-associated and a repair-active state.

* This work was supported by Public Health Service Grant GM 35866 and by National Science Foundation Grant MCB-9906440.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: University of Virginia's College at Wise, 1 College Avenue, Wise, VA 24293-4412.

§ Eminent Scholar of the Georgia Research Alliance. To whom correspondence should be addressed: Inst. of Molecular Medicine and Genetics, Rm. CB-2803, Medical College of Georgia, 1120 15th St., Augusta, GA 30912. Tel.: 706-721-8756; Fax: 706-721-8752; E-mail: wdynan@mail.mcg.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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