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Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.
Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.
Cloning of a Human Damage-specific DNA Binding Protein
Anne F. Nichols, Tom Brody, Scott Keeney, and Stuart Linn
Department of Molecular and Cell Biology; University of California; Berkeley, California 94720. Department of Biochemistry and Molecular Biology; Harvard University; Cambridge, Massachusetts 02138.
DDB is a damage-specific DNA binding protein and potential DNA repair factor . This specific binding activity is absent from cells of some individuals with Xeroderma Pigmentosum Group E , a human hereditary disease characterized by defective nucleotide excision repair and a high incidence of skin cancer. When DDB was purified to near homogeneity from HeLa cells, the specific DNA damage binding activity copurified with polypeptides of 124 and 41 kDa . DNAse I footprints of the heterodimer of DDB on synthetic DNA substrates containing a single UV-photoproduct was different from that of the isolated p124 subunit. While the size of the footprint was unchanged, the appearance of an additional hypersensitive site in the presence of the heterodimer indicated that the p41 subunit is a functional subunit of the DDB protein .
We have isolated full-length cDNAs which encode both polypeptides of DDB from a human fibroblast lambdaZAP library. There is no significant homology of the 41 kDa subunit with any proteins in current databases. However, the translated region of human p124 DDB cDNA has 98% homology with the monkey p127 UV-DDB isolated by Takao et al. [NewGenBank, ACC L20216]. Their amino acid sequences have almost 100% identity, with a single conservative substitution. The p41 subunit has been expressed in an in vitro rabbit reticulocyte lysate system. Polyclonal antibodies will be produced in rabbits, using over-expressed DDB polypeptides as antigens.
This work was funded by DOE Grant FG03-92ER61458. A.N. was supported in part by an appointment to the Alexander Hollaender Distinguished Postdoctoral Fellowship Program sponsored by the DOE, Office of Health and Environmental Research, and administered by the Oak Ridge Institute for Science and Education.
 Keeney, S., Eker, A.P., Brody,T., Vermeulen, W., Bootsma, D., Hoeijmakers, J.H., and Linn,S. (1994) Correction of the DNA repair defect of xeroderma pigmentosum group E by injection of a DNA damage-binding protein. Proc. Natl. Acad. Sci., USA, 91, 4053-6.
 Keeney, S., Wein, H., and Linn, S. (1992) Biochemical heterogeneity in xeroderma pigmentosum complementation group E. Mutation Res. DNA Repair, 273, 49-56.
 Keeney, S., Chang, G.J., and Linn, S. (1993) Characterization of a human DNA damage binding protein implicated in xeroderma pigmentosum E. J.Biol. Chem., 268, 21293-21300.
 Reardon, J.T., Nichols, A.F., Keeney, S., Smith, C.A., Taylor, J-S. Linn S., and Sancar, A. (1993) J. Biol. Chem., 268, 21301-8.