DNA Repair

Edited by: Thomas W. Durso and Karen Young Kreeger C.U. Kirchgessner, C.K. Patil, J.W. Evans, C.A. Cuomo, L.M. Fried, T. Carter, M.A. Oettinger, J.M. Brown, "DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect," Science, 267:1178-83, 1995. (Cited in more than 140 publications as of February 1997) Comments by Cordula U. Kirchgessner and J. Martin Brown, department of radiation oncology, Stanford University School of Medicine A 1995 paper in Cell (T. Blunt et al., Cell, 80:813-23, 1995; discussed in Hot Papers, The Scientist, Jan. 6, 1997, page 15) described an enzyme called DNA-dependent protein kinase (DNA-PK), which catalyzes the repair both of DNA double-strand breaks and of specific DNA breaks occurring in the development of the immune system. QUICK FIX: Cordula Kirchgessner and Martin Brown presented genetic evidence of an enzyme that catalyzes DNA repair. Like the Cell paper, this work-by researchers from Stanford University's School of Medicine, Massachusetts General Hospital, and St. John's University-deals with severe combined immune deficient (SCID) mice, which cannot repair DNA double-strand breaks. Both papers concluded that an absence of the DNA-PK catalytic subunit (DNA-PKcs), known as p350, is responsible for the SCID defect. The difference, according to Cordula U. Kirchgessner, an assistant professor of radiation oncology at Stanford's School of Medicine, is that the Cell paper was primarily based on biochemistry, while this paper relied on genetic evidence. "The two papers complement each other nicely to present a complete story," she comments. Kirchgessner, who at the time of this paper was a postdoc in the laboratory of J. Martin Brown, a professor and director of radiation biology at Stanford, previously found that human chromosome 8 would complement the SCID defect to allow the SCID cells to resist the effects of harmful radiation (C.U. Kirchgessner et al., Cancer Research, 53:6011-6, 1993). For this study, the researchers created a panel of SCID cell lines retaining small pieces of the human chromosome 8. They identified the minimal region conferring radioresistance and showed that the gene for the catalytic subunit of DNA-PK was located in that region. Experiments on DNA-PKcs revealed 100 percent concordance between the expression of the DNA-PKcs and radioresistance. "Although not the ultimate proof, it made DNA-PKcs a very good candidate for the SCID gene," Kirchgessner states. "Finding that DNA-PKcs levels in wild-type radioresistant cell lines were 20 to 100 times greater than in SCID cell lines was a good indication that this truly is the protein deficient in SCID." Brown attributes the paper's citation popularity to the fact that DNA-PK is the first protein found to be involved in DNA double-strand break repair in mammalian cells, and to its identification of the long-sought gene underlying the SCID immune defect. He says Brown adds that identification of DNA-PKcs as the gene responsible for the radiosensitive phenotype of SCID mice was a gratifying conclusion to five years of research. The work began with the chance discovery by his and two other laboratories of the radiosensitive phenotype in SCID mice (K.A. Biedermann et al., Proceedings of the National Academy of Sciences, 88:1394-7, 1991). "This finding was like opening the door to a new world," Kirchgessner remarks. "We can finally study the biochemical pathway of double-strand break repair in mammalian cells. This is important, as an unrepaired DNA break can lead to a chromosome break. Chromosome aberrations are often a stepping stone to a cell being malignant. Knowing about the proteins involved in the repair will give us a possibility to study the repair pathways in cancer cells and their possible role in carcinogenesis." Since this paper was published, Kirchgessner and her colleagues have studied DNA-PK's role as a phosphorylating agent, catalyzing the addition of phosphogroups to specific proteins. Such a phosphorylation usually serves to regulate the activity of proteins. Specifically, the team looked at p53, the tumor suppressor gene, and replication protein A (RPA), two substrates that DNA-PK phosphorylates in vitro and that have been indicated in DNA repair because they are phosphorylated after DNA damage. The group examined the phosphorylation of p53 and RPA in vivo using the DNA-PKcs deficient SCID cells and a human cell line, MO59J-which is also deficient in DNA-PKcs-as well as the respective wild types. They showed that both p53 induction and RPA phosphorylation are normal in SCID cells, indicating either that these are not physiologically relevant substrates for DNA-PK in vivo or that a redundant pathway exists (L.M. Fried et al., PNAS, 93:13825-30, 1996). "The importance of this 'negative' result lies in the contradiction of premature conclusions drawn from in vitro work, which postulated the DNA double-strand break repair pathway to proceed from DNA-PK to p53," Kirchgessner observes.

DNA Repair

The Scientist Staff

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