PRINCIPAL SCIENTIST
 Chen, D

 SCIENTISTS
 Burma, S
Chen, B
Chen, F
Gilley, D
Lio, Y
Wang, D
Yannone, S

 POSTDOCTORAL FELLOWS
Asaithamby, A
Ding, L
Kobayashi, J
Miroshnychenko, O
Ortega, L
Tanaka, H
Wang, J
Yano, K

 RESEARCH ASSOCIATES
 Patterson, M
Yano, K

 STUDENTS
Ho, E
Hsieh, C
Koai, E
Ng, E
Park, J
Panowski, S
Yau, C

 STAFF
 Staff Names Coming Soon

 


Deficiencies in DNA-damage signaling and repair pathways are fundamental to the etiology of most human cancers. Of the many types of DNA damage that occur within the cell, DNA double-strand breaks (DSBs) are particularly dangerous.

 


DSBs are caused by both endogenous (byproducts of cellular metabolism) and exogenous (ionizing radiation and chemotherapeutic drugs) threats. An inability to respond properly to DSBs or to repair them correctly can lead to cell death or promote tumorigenesis. Our research is focused on the mechanisms by which cells recognize, respond to, and repair DSBs. We are motivated by the fact that a more complete understanding of these pathways will provide insights into how cancer is triggered, and may lead to the development of more effective cancer therapies.

Eukaryotic cells have evolved two distinct mechanisms for repairing DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). These mechanisms differ primarily in that HR requires a homologous DNA template, while NHEJ acts independently of homologous DNA. We have begun to study how the Rad51 family of proteins interact and assemble to facilitate homologous recombinational repair (1). The majority of our research focuses on the NHEJ repair pathway. The DNA dependent protein kinase (DNA-PK) complex, consisting of the DNA-binding subunit Ku and the catalytic subunit DNA-PKcs, are central to NHEJ repair. Utilizing transgenic mouse models (2, 3), we demonstrated that while the kinase activity of DNA-PKcs is absolutely essential for the repair of DNA DSBs (4), DNA-PKcs is not required for the signaling of DNA damage to the cell cycle machinery (5). Although it has been established for many years that the kinase activity of DNA-PK is stimulated by DNA damage, the biologically relevant substrates of DNA-PK have remained elusive. We have recently identified WRN as a biologically relevant substrate for DNA-PK. We have further shown that DNA-PK assembles into a complex on DNA with WRN protein and regulates WRN activities (6). WRN deficiency is causative in a human disease characterized by "premature aging" and increased incidence of cancer. These studies suggest a link between the DNA repair and aging processes. Very recently, we demonstrated that DNA-PKcs is autophosphorylated in response to DNA damage and that this very early event is essential for the repair of DSBs. In addition, we demonstrated, for the first time, the localization of DNA-PKcs at the sites of DNA damage in vivo (7). We are also investigating how some of the proteins involved in NHEJ control other processes such as telomere maintenance. Telomere maintenance is a critical component of cellular senescence, telomerase is activated in most cancers, and telomere dysfunction may be an early event causing genomic instability during the progression of certain cancers. Recently, we discovered that Ku is associated with the mammalian telomere and that Ku functions in a unique way at the telomere to prevent end joining (8, 9). Using a transgenic mouse model we have recently determined that the DNA-PKcs kinase domain is critical for telomere capping (10) and we are in the process of identifying specific telomere proteins that are phosphorylated by DNA-PKcs in vivo. Ongoing research is directed toward dissecting the molecular functions of the kinase activity of DNA-PKcs and the biological significance of DNA-PKcs-mediated auto and trans-phosphorylation in mammalian cells.

In addition to the study of DNA-PK-mediated phosphorylation events, we are also interested in understanding how a related kinase, ATM, functions in the response of mammalian cells to DNA damage. ATM, which is defective in the cancer-predisposition syndrome ataxia telangiectasia, functions in preventing cells with damaged DNA from dividing thereby allowing sufficient time for DNA repair. We have identified ATM as the kinase responsible for histone H2AX phosphorylation at the sites of DNA damage, a very early and important response to DSBs (11). Our current research focuses on how ATM is activated by ionizing radiation and we have identified the Ku component of DNA-PK as a modulator of the ATM-activation process.

In an attempt to understand the global cellular response to DSBs and to identify novel genes and proteins involved in these responses, we are employing the latest advances in genomic and proteomic technologies. This research, sponsored by the US Dept. of Energy, takes advantage of newly established high-throughput mass spectrometry (Pacific Northwest National Laboratory) and cDNA microarray technologies (Lawrence Berkeley National Laboratory) to precisely characterize the cellular responses to low dose rates of ionizing radiation (LLIR) with respect to transcription, protein levels, and posttranslational modifications. As part of this effort, we are also managing the Lawrence Berkeley National Laboratory Life Sciences Division microarray facility which providies microarray facilities and expertise for the Breast Cancer Center for Excellence Program funded by the Department of Defense.

David Jen-Chi Chen
Senior Staff Scientist/
Life Sciences Division

One Cyclotron Rd.
Mailstop: 74-157
Berkeley, CA 94720
tel: (510)495-2861
email: DJChen@lbl.gov
 

 

1. Lio, Y. C., Mazin, A. V., Kowalczykowski, S. C., and Chen, D. J. Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro. J Biol Chem, 2002.

2. Kurimasa, A., Ouyang, H., Dong, L. J., Wang, S., Li, X., Cordon-Cardo, C., Chen, D. J., and Li, G. C. Catalytic subunit of DNA-dependent protein kinase: impact on lymphocyte development and tumorigenesis. Proc Natl Acad Sci U S A, 96: 1403-1408, 1999.

3. Ouyang, H., Nussenzweig, A., Kurimasa, A., Soares, V. C., Li, X., Cordon-Cardo, C., Li, W., Cheong, N., Nussenzweig, M., Iliakis, G., Chen, D. J., and Li, G. C. Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo. J Exp Med, 186: 921-929, 1997.

4. Kurimasa, A., Kumano, S., Boubnov, N. V., Story, M. D., Tung, C. S., Peterson, S. R., and Chen, D. J. Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining. Mol Cell Biol, 19: 3877-3884, 1999.

5. Burma, S., Kurimasa, A., Xie, G., Taya, Y., Araki, R., Abe, M., Crissman, H. A., Ouyang, H., Li, G. C., and Chen, D. J. DNA-dependent protein kinase-independent activation of p53 in response to DNA damage. J Biol Chem, 274: 17139-17143, 1999.

6. Yannone, S. M., Roy, S., Chan, D. W., Murphy, M. B., Huang, S., Campisi, J., and Chen, D. J. Werner syndrome protein is regulated and phosphorylated by DNA-dependent protein kinase. J Biol Chem, 276: 38242-38248, 2001.

7. Chan, D. W., Chen, B. P., Prithivirajsingh, S., Kurimasa, A., Story, M. D., Qin, J., and Chen, D. J. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev, 16: 2333-2338, 2002.

8. Hsu, H. L., Gilley, D., Blackburn, E. H., and Chen, D. J. Ku is associated with the telomere in mammals. Proc Natl Acad Sci U S A, 96: 12454-12458, 1999.

9. Hsu, H. L., Gilley, D., Galande, S. A., Hande, M. P., Allen, B., Kim, S. H., Li, G. C., Campisi, J., Kohwi-Shigematsu, T., and Chen, D. J. Ku acts in a unique way at the mammalian telomere to prevent end joining. Genes Dev, 14: 2807-2812, 2000.

10. Gilley, D., Tanaka, H., Hande, M. P., Kurimasa, A., Li, G. C., Oshimura, M., and Chen, D. J. DNA-PKcs is critical for telomere capping. Proc Natl Acad Sci U S A, 98: 15084-15088, 2001.

11. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., and Chen, D. J. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem, 276: 42462-42467, 2001.