Normal and Defective Repair of Damaged DNA in Human Cells: A Sensitive Assay Utilizing the Photolysis of Bromodeoxyuridine

A new technique has been developed for studying the extent of repair of UV-radiation damage to DNA in human cells. It is easy to use, has excellent sensitivity, and provides rapid quantitative estimates of repair. UV-irradiated cells whose DNA has been previously labeled with a radioisotope are grow...

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Bibliographic Details
Published inProceedings of the National Academy of Sciences - PNAS Vol. 68; no. 4; pp. 708 - 712
Main Authors Regan, J D, Setlow, R B, Ley, R D
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences of the United States of America 01.04.1971
National Acad Sciences
Subjects
Animals
Biological Sciences: Biochemistry
Bromodeoxyuridine
Cell Line
Cell lines
Centrifugation, Density Gradient
Culture Techniques
Dimers
DNA
DNA - radiation effects
DNA damage
DNA Replication
Enzymes
Fibroblasts - radiation effects
Humans
Irradiation
Lesions
Methods
Molecular Weight
Photochemistry
Photolysis
Radiation damage
Radiation dosage
Radiation Injuries, Experimental
Tritium
Ultraviolet Rays
Xeroderma Pigmentosum - radiotherapy
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Summary:A new technique has been developed for studying the extent of repair of UV-radiation damage to DNA in human cells. It is easy to use, has excellent sensitivity, and provides rapid quantitative estimates of repair. UV-irradiated cells whose DNA has been previously labeled with a radioisotope are grown after irradiation in nonradioactive bromodeoxyuridine, which is incorporated at the breaks induced by repair enzymes. After a period of growth in the thymidine analog the cells are exposed to a large flux of 313 nm radiation and then lysed on top of an alkaline sucrose gradient. Bromodeoxyuridine-containing sections of the DNA are thus selectively photolysed. Sedimentation in the alkaline gradient reveals the average molecular weight of disrupted segments and gives a measure of the number of breaks induced by repair enzymes over the whole period allowed for repair. The large change in average molecular weight observed upon exposure of normal repairing cells to 313 nm radiation is not observed in the repair-deficient cells from patients with xeroderma pigmentosum. The quantitative aspects of this assay for repair and its sensitivity should make it applicable to the study of repair induced by agents other than UV radiation.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.68.4.708