News and Publications

Role of DNA-Dependent Protein Kinase in DNA Repair and Transcription

P. Labhart

DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is activated by DNA ends and other discontinuous DNA structures. Recently, it was established that DNA-PK is required for the repair of double-stranded breaks in DNA and for V(D)J recombination during lymphoid development. However, the precise role of the kinase in these processes remains unknown, and additional roles in other cellular processes, such as transcription, cannot be ruled out.

We are using the Xenopus system, which is amenable to experimental manipulation of oogenesis and embryogenesis, to study the role of DNA-PK in DNA repair and in transcription. We found that DNA-PK specifically inhibited transcription by RNA polymerase I in vitro. Partial fractionation of the in vitro transcription system suggested that transcription factor Rib1, the Xenopus equivalent of human SL1, mediates the repression of transcription by DNA-PK. Furthermore, TATA-box binding protein, the core component of Rib1, is hyperphosphorylated at its N-terminal domain by DNA-PK in vitro. Whether this phosphorylation is responsible for the transcriptional repression is unknown.

In parallel experiments, we are using an extract from activated Xenopus eggs that efficiently joins nonhomologous DNA ends of added substrate DNA. We found that this reaction is inhibited by antibodies to DNA-PK and after immunodepletion of DNA-PK from the extract. Furthermore, joining of nonhomologous DNA ends and DNA-PK have a similar sensitivity to wortmannin, a known inhibitor of DNA-PK. The inhibitory effects on joining of DNA ends occur with DNA-repair substrates bearing pairs of cohesive, blunt, and mismatched ends. The Xenopus egg extract can thus be used to investigate in more detail the molecular mechanisms of joining of nonhomologous DNA ends and the specific role that DNA-PK plays in this process. It will be particularly important to determine the DNA-PK substrates that are involved in the DNA repair.

The physiologic significance of the repression of ribosomal gene transcription by DNA-PK is unknown. However, the combined results led to the formulation of a model in which the downregulation of RNA polymerase I transcription is part of the physiologic response to DNA damage. Possibly, the shutdown of transcription near the DNA breaks is necessary for efficient repair. This hypothesis can be tested in the Xenopus system.

PUBLICATIONS

Alexander, J., Oseroff, C., Sidney, J., Wentworth, P., Keogh, E., Hermanson, G., Chisari, F.V., Kubo, R.T., Grey, H.M., Sette, A. Derivation of HLA-A11/Kb transgenic mice: Functional CTL repertoire and recognition of human A11-restricted CTL epitopes. J. Immunol. 159:4753, 1997.

Bertoni, R., Sette, A., Sidney, J., Guidotti, L.G., Shapiro, M., Purcell, R., Chisari, F.V. Human class I supertypes and cytotoxic T lymphocyte repertoires extend to chimpanzees J. Immunol., in press.

Bertoni, R., Sidney, J., Fowler, P., Chisari, F.V., Sette, A. Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis. J. Clin. Invest. 100:503, 1997.

Cavanaugh, V.J., Guidotti, L.G., Chisari, F.V. Inhibition of hepatitis B virus replication during adenovirus and cytomegalovirus infections in HBV transgenic mice. J. Virol. 72:2630, 1998.

Chang, K.-M., Rehermann, B., Chisari, F.V. Immunopathology of hepatitis C. Springer Semin. Immunopathol. 19:57, 1997.

Chang, K.-M., Rehermann, B., McHutchison, J.G., Pasquinelli, C., Southwood, S., Sette, A., Chisari, F.V. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J. Clin. Invest. 100:2376, 1997.

Chisari, F.V. Hepatitis B virus pathogenesis: What have we learned from the transgenic mouse? In: 2nd International Meeting on Therapy in Liver Diseases. Arroyo, V. (Ed.). Masson­Little, Brown, Barcelona, Spain, in press.

Chisari, F.V. The immunobiology of viral hepatitis. In: T Cells in the Liver: Biology, Immunopathology and Host Defense. Crispe, N. (Ed.). Wiley, New York, in press.

Chisari, F.V., Lai, M.M.C. Hepatitis B and delta virus pathogenesis. In: Persistent Viral Infections. Ahmed, R., Chen, I. (Eds.). Wiley, New York, in press.

Franco, A., Guidotti, L.G., Hobbs, M.V., Pasquetto, V., Chisari, F.V. Pathogenetic effector function of CD4-positive T-helper 1 cells in hepatitis B virus transgenic mice. J. Immunol. 159:2001, 1997.

Guidotti, L.G. Mouse genetics at work: A new model of chronic hepadnavirus infection. Hepatology 28:268, 1998.

Hu, J.-F., Cheng, Z., Chisari, F.V., Vu, T.H., Hoffman, A.R., Campbell, T.C. Repression of hepatitis B virus transgene and HBV-induced liver injury by low protein diet. Oncogene 15:2795, 1997.

Jakubczak, J.L., Chisari, F.V., Merlino, G. Synergy between transforming growth factor and hepatitis B virus surface antigen in hepatocellular proliferation and carcinogenesis. Cancer Res. 57:3606, 1997.

Kuhröber, A., Wild, J., Pudollek, H.-P., Chisari, F.V., Reimann, J. DNA vaccination with plasmids encoding the intracellular (HBcAg) or secreted (HBeAg) form of the core protein of hepatitis B virus primes T cell responses to two overlapping Kb- and Kd-restricted epitopes. Int. Immunol. 9:1203, 1997.

Labhart, P. mRNA encoding the catalytic subunit of DNA-dependent protein kinase is widely expressed in Xenopus cells. Gene 203:235, 1997.

Livingston, B.D., Crimi, C., Grey, H., Ishioka, G., Chisari, F.V., Fikes, J., Grey, H., Chesnut, R.W., Sette, A. The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection. J. Immunol. 159:1383, 1997.

Nakamoto, Y., Guidotti, L., Kuhlen, C., Fowler, P., Chisari, F.V. Immune pathogenesis of hepatocellular carcinoma. J. Exp. Med. 188:341, 1998.

Nakamoto, Y., Guidotti, L.G., Pasquetto, V., Schreiber, R.D., Chisari, F.V. Differential target cell sensitivity to cytotoxic T lymphocyte-activated death pathways in hepatitis B virus in transgenic mice. J. Immunol. 158:5692, 1997.

Rehermann, B., Chisari, F.V. Cell-mediated immune response to the hepatitis C virus. Curr. Top. Microbiol. Immunol., in press.

Rehermann, B., Chisari, F.V. The immunology of chronic hepatitis B virus. In: Hepatitis B Virus: Molecular Mechanisms in Disease and Novel Strategies for Therapy. Koshy, R., Caselmann, W. (Eds.). World Scientific, River Edge, NJ, in press.