Distinct Roles for Ku Protein in Transcriptional Reinitiation and DNA Repair

Abstract

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.