Scientific Report 2005

Molecular Biology

DNA Damage Responses in Human Cells

C.H. McGowan, V. Blais, H. Gao, E. Langley, A. MacLaren, J. Scorah, E. Taylor

Complex multicellular organisms, such as humans, have large numbers of mitotically competent cells that are capable of renewal, repair, and, to some extent, regeneration. The advantages of being able to replace damaged or aged cells are off set by the inherent susceptibility of mitotic cells to acquiring mutations and becoming cancerous. DNA is inherently vulnerable to many sorts of chemical and physical modification; thus, as they duplicate and divide, cells can acquire mutations. Both spontaneous and induced DNA damage must be repaired with minimal changes if growth, renewal, and repair are to be successful. Our overall objective is to understand how mammalian cells protect themselves from DNA damage and thus from cancer.

Eukaryotic cells have evolved with a complex network of DNA repair processes and cell-cycle checkpoint responses that ensure that damaged DNA is repaired before it is replicated and becomes fixed in the genome. These pathways are highly conserved through evolution, and much information about human responses to DNA damage has been gained from studies of simple genetically tractable organisms such as yeast. We use a combination of molecular, cellular, and genetic techniques to determine how these pathways operate in human cells.

Checkpoints control the order and timing of events in the cell cycle; they ensure that biochemically independent processes are coupled so that a delay in a critical cell-cycle process will cause a delay in all other aspects of progression of the cycle. In addition, checkpoints coordinate repair with delays in progression of the cell cycle and promote the use of the most appropriate repair pathway. We used genetic models to identify 2 checkpoint kinases in humans that limit progression of the cell cycle when DNA is damaged. One of these kinases, Chk2, is activated in response to DNA damage. Chk2 physically interacts with Mus81-Eme1, a conserved DNA repair protein that has homology to the xeroderma pigmentosum F family of endonucleases. Xeroderma pigmentosum is a cancer-prone disorder that results from a failure to appropriately repair damaged DNA.

Biochemical analysis indicates that Mus81-Eme1 has associated endonuclease activity against structure-specific DNA substrates, including Holliday junctions. Enzymatic analysis, immunofluorescence studies, and the use of RNA interference have all contributed to the conclusion that Mus81-Eme1 is required for recombination repair in human cells. We are also using gene targeting to study the function of the Mus81-Eme1 endonuclease in mice. Inactivation of Mus81 in mice increases genomic instability and sensitivity to DNA damage but does not promote tumorogenesis. In addition, we showed that Mus81-Eme1 is specifically required for survival after exposure to cisplatin, mitomycin C, and other commonly used anticancer drugs. As a point of interaction between checkpoint control and DNA repair, the relationship between Mus81-Eme1 and Chk2 most likely will provide information critical to understanding the responses to DNA damage as a whole.

Anticancer therapy is largely based on the use of genotoxic agents that damage DNA and thus kill dividing cells. Coordination of cell-cycle checkpoints and DNA repair is especially important when unusually high amounts of DNA damage occur after radiation or genotoxic chemotherapy. Hence, a detailed understanding of cellular responses to DNA damage is essential to understanding both the development and the treatment of disease in humans.

Publications

Dendouga, N., Gao, H., Moechars, D., Janicot, M., Vialard, J., McGowan, C.H. Disruption of murine Mus81 increases genomic instability and DNA damage sensitivity but does not promote tumorigenesis. Mol. Cell. Biol. 25:7569, 2005.

McGowan, C.H., Russell, P. The DNA damage response: sensing and signaling. Curr Opin. Cell Biol. 16:629, 2004.

Zhang, R., Sengupta, S., Yang, Q,, Linke, S.P., Yanaihara, N., Bradsher, J., Blais, V,. McGowan, C.H., Harris, C.C. BLM helicase facilitates Mus81 endonuclease activity in human cells. Cancer Res. 65:2526, 2005.