Damage-specific DNA-binding proteins from human cells
The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1% NP-40.
- Research Organization:
- Pennsylvania State Univ., University Park, PA (United States)
- OSTI ID:
- 5580346
- Resource Relation:
- Other Information: Thesis (Ph.D.)
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
DNA
BIOLOGICAL RADIATION EFFECTS
DAMAGE
PROTEINS
CPB
DNA REPAIR
GAMMA RADIATION
GENE RECOMBINATION PROTEINS
IRRADIATION
SENSITIVITY
ULTRAVIOLET RADIATION
XERODERMA PIGMENTOSUM
BIOLOGICAL EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CONGENITAL DISEASES
DISEASES
ELECTROMAGNETIC RADIATION
HEREDITARY DISEASES
IONIZING RADIATIONS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
RADIATION EFFECTS
RADIATIONS
REPAIR
SKIN DISEASES
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture
550200 - Biochemistry