Gilbert Chu

Publications

Chu G, Chang E (1988) Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA. Science 242: 564-567.

Abstract. The disease xeroderma pigmentosum is characterized by deficient repair of damaged DNA. Fusions of cells from different patients have defined nine genetic complementation groups (A through I), implying that DNA repair in humans involves multiple gene products. In this report, an extension of the gel electrophoresis binding assay was used to identify at least one nuclear factor that (i) bound to DNA damaged by ultraviolet radiation or the antitumor drug cisplatin, but (ii) was notably absent in cells from complementation group E. Therefore, the factor appears to participate in a versatile DNA repair pathway at the stage of binding and recognition.

Hwang B, Chu G (1993) Purification and characterization of a human protein that binds to damaged DNA. Biochemistry 32: 1657-1666.

Abstract. Xeroderma pigmentosum group E binding factor (XPE-BF) binds to damaged DNA and is defective in complementation group E of xeroderma pigmentosum, an inherited disease characterized by defective DNA repair. XPE-BF was purified to near homogeneity by affinity chromatography on UV-irradiated DNA cellulose. Two methods were used to identify XPE-BF as a 125 kDa polypeptide. First, the denatured protein migrated as a 125 kDa polypeptide on SDS-PAGE. Second, the native protein behaved primarily as a 134 ± 20 kDa protein on gel filtration and glycerol gradient sedimentation. Damaged DNA binding was dependent on unmodified cysteine residues, stimulated by magnesium, and inhibited by zinc. Sedimentation revealed a major peak in binding activity at 6.8 S, corresponding to the major monomeric form, and a minor peak at 14.5 S, suggesting a homodimeric form. These peaks did not change in the presence of magnesium, but in the presence of zinc, were replaced by a broad peak centered at 18 S, consistent with higher order aggregates. The possible significance of these forms for the recognition and repair of damaged DNA is discussed.

Chu G (1994) The cellular response to cisplatin. J. Biol .Chem. 269: 787-790.

Abstract. The anticancer drug cisplatin provokes a complex response in the cell. A lethal dose of the drug kills cells primarily by forming DNA adducts, causing G2 arrest in the cell cycle and then triggering apoptosis. A sublethal dose induces drug resistance by several mechanisms, including changes in drug uptake and efflux, glutathione and metallothionine levels, and DNA repair. Cisplatin-DNA adducts bind several cellular proteins, including some that enhance survival of the cell by mediating DNA repair and others that hasten its death by conferring sensitivity to the drug.

Rathmell WK, Chu G (1994) A DNA-end-binding factor involved in double-strand break repair and V(D)J recombination. Mol. Cell .Biol. 14: 4741-4748.

Abstract. We have identified a nuclear factor that binds to double-stranded DNA ends, independently of the structure of the ends. It had equivalent affinities for DNA ends created by sonication or by restriction enzymes leaving 5', 3', or blunt ends, but had no detectable affinity for single-stranded DNA ends. Since X-rays induce DNA double-strand breaks, extracts from several complementation groups of X-ray sensitive mammalian cells were tested for this DNA end-binding (DEB) activity. DEB activity was deficient in three independently derived cell lines from complementation group 5. Furthermore, when the cell lines reverted to X-ray resistance, expression of the DNA end-binding factor was restored to normal levels. Previous studies had shown that group 5 cells are defective for both double-strand break repair and V(D)J recombination (33, 43) . The residual V(D)J recombination activity in these cells produces abnormally large deletions at the sites of DNA joining (33, 43) , consistent with deficiency of a factor that protects DNA ends from degradation. Therefore, DEB factor may be involved in a biochemical pathway common to both double-strand break repair and V(D)J recombination.

Rathmell WK, Chu G (1994) Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks. Proc. Natl. Acad. Sci. USA 91: 7623-7627.

Abstract. The Ku autoantigen is a well characterized heterodimer of 70 and 86 kDa that binds to DNA ends, but its cellular function has been obscure. An electrophoretic mobility shift assay and Ku antisera were used to show that Ku or a closely related protein was deficient in three mutant hamster cell lines from X-ray sensitive complementation group 5, which is characterized by defects in DNA double-strand break repair and V(D)J recombination. Furthermore, Ku protein expression was restored when the cells reverted to X-ray resistance. The Ku p86 gene maps to human chromosome 2q33-35, and group 5 cells are rescued by almost precisely the same region, 2q34-36. Thus, biochemical and genetic evidence suggest that Ku is involved in pathways for DNA recombination and repair. By its association with a DNA-dependent protein kinase activated by DNA ends, Ku may also initiate a signaling pathway induced by DNA damage, perhaps for cell cycle arrest.

Smider V, Rathmell WK, Lieber M, Chu G (1994) Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science 266: 288-291.

Abstract. Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen. Transfection of cDNA that encoded the 86 kD subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage.

Hwang BJ, Liao J, Chu G (1996) Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells. Mutation Res 362: 105-117.

Abstract. XPE binding factor (XPE-BF) is deficient in a subset of patients from xeroderma pigmentosum complementation group E. Binding activity copurifies with a 125 kDa polypeptide (p125) that binds to DNA damaged by ultraviolet (UV) radiation and many other agents. We isolated cDNA encoding a polypeptide with a predicted amino acid sequence that matched the sequences of eleven tryptic peptides derived from digestion of XPE-BF purified from human placenta. In vitro transcription and translation of the cDNA yielded a polypeptide of 125 kDa that bound specifically to UV-damaged DNA. Therefore the cDNA encodes either all or the major component of XPE-BF.