DNA Damage-binding Protein Research Articles

Detection of DNA damage-recognition proteins using the band-shift assay and southwestern hybridization.

We describe electrophoresis and biochemical conditions that allow detection of damaged DNA-binding proteins in cell extracts. In addition, we present an overview of the damage-recognition DNA-binding proteins from eukaryotic cells and discuss their hypothetical role in DNA repair.

Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2.

The biochemical processes responsible for the recognition and repair of cisplatin-damaged DNA in human cells are not well understood. We have developed a damaged DNA affinity precipitation technique that allows the direct visualization and characterization of cellular proteins that bind to cisplatin-damaged DNA. The method separates damaged DNA-binding proteins from complex radiolabeled cell mixtures and further resolves them into individual polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique is complementary to gel retardation and Southwestern blotting analyses that have been previously used to identify cellular components that specifically bind to cisplatin-damaged DNA. Using this technique, we have characterized a set of HeLaS3 nuclear proteins of 26.5, 28, 90, and 97 kDa that specifically bind to cisplatin-DNA adducts. Competition studies with soluble cisplatin-damaged DNA confirmed these findings. The major cisplatin-damaged DNA-binding proteins of 26.5 and 28 kDa recognized adducts of DNA modified with cisplatin but not with its trans-isomer or with UV radiation. These proteins were purified 450-fold to near homogeneity by ion-exchange and cisplatin-damaged DNA affinity chromatography. Amino-terminal sequence analysis showed that the 26.5- and 28-kDa proteins were identical to high mobility group (HMG) proteins HMG-2 and HMG-1, respectively.

Biochemical heterogeneity in xeroderma pigmentosum complementation group E

Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p < 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.