Research Summary

The focus of this laboratory is directed at understanding the biochemical basis of DNA repair in mammalian cells and its relationship to mutagenic and carcinogenic phenomena. Our approach has been to isolate mammalian cell mutants that are defective in DNA repair and to use these mutants as molecular and biochemical tools for studying these processes. One of these mutants, XR-1, is abnormally sensitive to killing by x-rays and is deficient in DNA double strand break repair (DSB). This deficiency in DSB repair is due to a lack of XRCC4 protein which interacts with DNA ligase IV enzyme and stabilizes it against proteolytic degradation. (6)

In other work, we have found that a large (>2 x 106 dalton) "DNA PK-Ku" DNA-end binding protein complex is present in human nuclear extracts and preferentially binds to nuclear matrix attachment region sequences in genomic DNA. These observations raise the possibility that thiscomplex functions in a DNA repair and/or damage signaling pathway (1).

In another project, we have investigated the effect of a cell’s oxidation state on x-ray sensitivity and DNA double-strand-break repair. These studies suggest that oxidation of cysteine thiols in cellular protein increases cellular sensitivity to killing by x-rays and reduces DNA double-strand-break repair capacity(4, 5). Further the primary target of thiol oxidation are the cystein thiols in the Ku repair protein. (3)

Selected Publications

1. Mauldin, S.K., Getts, R.C., Liu, W. and Stamato, T.D. (2002). DNA-PK-dependent binding of DNA-ends to plasmids containing nuclear matrix attachment region DNA sequences: evidence for Assembly of a repair complex. Nucleic Acids Res 30:4075-4087.
2. Morrish, T.A., Gilbert, N., Myers, J.S., Vincent, B.J., Stamato, T.D., Taccioli, G.E., Batzer, M.A., Moran, J.V. (2002). DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat. Genet. 31: 159-165.
3. Ayene, I.S., Stamato, T.D., Mauldin, S.K., Biaglow, J.E., Tuttle, S.W., Jenkins, S.F., Koch, C.J. (2002). Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress. J. Biol. Chem. 277:9929-9935.
4. Ayene, I.S., Koch, C.J., Tuttle, S.W., Stamato, T.D., Perez, M.L., Biaglow, J.E. (2000). Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells. Int. J. Radiat Biol. 76: 1523-1531.
5. Tuttle, S.W., Stamato, T.D., Perez, ML, Biaglow, J. (2000). Glucose-6-phosphate dehydrogenase and the oxidative pentose phosphate cycle protect cells agains apoptosis induced by low doses of ionizing radiation. Radiat. Res. 153:781-787.
6. Bryans, M., Valenzano, M.C., Stamato, T.D. (1999). Absence of DNA ligase IV protein in XR-1 cells: evidence for stabilization by XRCC4. Mutat. Res. 433:53-58.

Spring Colloquium 2004 Virtual Poster

DNA-PK Dependent Targeting of DNA-ends to a Protein Complex Assembled on Matrix Attachment Region DNA Sequences