Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1971 Apr;68(4):708–712. doi: 10.1073/pnas.68.4.708

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

James D Regan *, R B Setlow *, R D Ley *
PMCID: PMC389025  PMID: 5279512

Abstract

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 non-radioactive 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.

Full text

PDF
708

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BOYCE R., SETLOW R. The action spectra for ultravioletlight inactivation of systems containing 5-bromouracil-substituted deoxyribonucleic acid. I. Escherichia coli 15 T A U. Biochim Biophys Acta. 1963 Mar 26;68:446–454. doi: 10.1016/0006-3002(63)90166-5. [DOI] [PubMed] [Google Scholar]
  2. Cleaver J. E. Defective repair replication of DNA in xeroderma pigmentosum. Nature. 1968 May 18;218(5142):652–656. doi: 10.1038/218652a0. [DOI] [PubMed] [Google Scholar]
  3. Cleaver J. E. Xeroderma pigmentosum: a human disease in which an initial stage of DNA repair is defective. Proc Natl Acad Sci U S A. 1969 Jun;63(2):428–435. doi: 10.1073/pnas.63.2.428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. EAGLE H. Amino acid metabolism in mammalian cell cultures. Science. 1959 Aug 21;130(3373):432–437. doi: 10.1126/science.130.3373.432. [DOI] [PubMed] [Google Scholar]
  5. Hutchinson F., Hales H. B. Mechanism of the sensitization of bacterial transforming DNA to ultraviolet light by the incorporation of 5-bromouracil. J Mol Biol. 1970 May 28;50(1):59–69. doi: 10.1016/0022-2836(70)90103-8. [DOI] [PubMed] [Google Scholar]
  6. Lion M. B. Search for a mechanism for the increased sensitivity of 5-bromouracil-substituted DNA to ultraviolet radiation. II. Single-strand breaks in the DNA of irradiated 5-bromouracil-substituted T3 coliphage. Biochim Biophys Acta. 1970 May 21;209(1):24–33. doi: 10.1016/0005-2787(70)90657-x. [DOI] [PubMed] [Google Scholar]
  7. Painter R. B., Umber J. S., Young B. R. Repair replication in diploid and aneuploid human cells. Normal replication of repaired DNA after ultraviolet irradiation. Radiat Res. 1970 Oct;44(1):133–145. [PubMed] [Google Scholar]
  8. Pfeiffer S. E., Tolmach L. J. Inhibition of DNA synthesis in HeLa cells by hydroxyurea. Cancer Res. 1967 Jan;27(1):124–129. [PubMed] [Google Scholar]
  9. Regan J. D., Trosko J. E., Carrier W. L. Evidence for excision of ultraviolet-induced pyrimidine dimers from the DNA of human cells in vitro. Biophys J. 1968 Mar;8(3):319–325. doi: 10.1016/S0006-3495(68)86490-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. SETLOW R., BOYCE R. The action spectra for ultraviolet-light inactivation of systems-containing 5-bromouracil-substituted deoxyribonucleic acid. II. Bacteriophage T4. Biochim Biophys Acta. 1963 Mar 26;68:455–461. doi: 10.1016/0006-3002(63)90167-7. [DOI] [PubMed] [Google Scholar]
  11. STUDIER F. W. SEDIMENTATION STUDIES OF THE SIZE AND SHAPE OF DNA. J Mol Biol. 1965 Feb;11:373–390. doi: 10.1016/s0022-2836(65)80064-x. [DOI] [PubMed] [Google Scholar]
  12. Setlow R. B., Regan J. D., German J., Carrier W. L. Evidence that xeroderma pigmentosum cells do not perform the first step in the repair of ultraviolet damage to their DNA. Proc Natl Acad Sci U S A. 1969 Nov;64(3):1035–1041. doi: 10.1073/pnas.64.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Setlow R. B. The photochemistry, photobiology, and repair of polynucleotides. Prog Nucleic Acid Res Mol Biol. 1968;8:257–295. doi: 10.1016/s0079-6603(08)60548-6. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES