Ku Antigen-DNA Conformation Determines the Activation of DNA-Dependent Protein Kinase and DNA Sequence-Directed Repression of Mouse Mammary Tumor Virus Transcription

Abstract

Mouse mammary tumor virus (MMTV) transcription is repressed by DNA-dependent protein kinase (DNA-PK) through a DNA sequence element, NRE1, in the viral long terminal repeat that is a sequence-specific DNA binding site for the Ku antigen subunit of the kinase. While Ku is an essential component of the active kinase, how the catalytic subunit of DNA-PK (DNA-PK_cs) is regulated through its association with Ku is only beginning to be understood. We report that activation of DNA-PK_cs and the repression of MMTV transcription from NRE1 are dependent upon Ku conformation, the manipulation of DNA structure by Ku, and the contact of Ku80 with DNA. Truncation of one copy of the overlapping direct repeat that comprises NRE1 abrogated the repression of MMTV transcription by Ku–DNA-PK_cs. Remarkably, the truncated element was recognized by Ku–DNA-PK_cs with affinity similar to that of the full-length element but was unable to promote the activation of DNA-PK_cs. Analysis of Ku–DNA-PK_cs interactions with DNA ends, double- and single-stranded forms of NRE1, and the truncated NRE1 element revealed striking differences in Ku conformation that differentially affected the recruitment of DNA-PK_cs and the activation of kinase activity.

ACKNOWLEDGMENTS

We are grateful to B. Kemper and P. Hsieh for samples of T4 and T7 endonuclease, respectively. We thank Y. Lefebvre and our colleagues in the Haché laboratory for critical commentary on the manuscript.

This work was supported by an operating grant from the Medical Research Council of Canada to R.J.G.H. C.S.-P. has been supported by postdoctoral fellowships from the Medical Research Council and the Arthritis Society of Canada.