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

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 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.Xeroderma pigmentosum (XP) is an ultraviolet-sensitive, hereditary disease of the skin caused by an autosomal recessive mutation (1). The mutation results in a defect in the enzyme mechanism responsible for the repair of UV-induced lesions in DNA. Cleaver (2) first called attention to XP as possibly due to a repair-deficient mutation by his demonstration of decreased or absent unscheduled synthesis and repair replication in UV-irradiated "XP cells" (fibroblasts cultured from skin biopsy specimens of patients with XP). We demonstrated that XP cells do not excise UV-induced pyrimidine dimers (3), in contrast to normal cells, which can excise dimers (4). The excision process reflects early steps in repair; Cleaver (5) presented indirect evidence and we (3) provided direct evidence suggesting that a UV-specific endonuclease, the enzyme presumed to initiate the repair process, is nonfunctional in XP cells.The steps in repair are believed to be the following (6-8): (a) an ultraviolet-specific nuclease makes a single-strand break near a lesion (usually a pyrimidine dimer); (b) an exonuclease, perhaps associated with the DNA polymerase molecule, excises the dimer and other nucleotides as well; (c) DNA polymerase inserts new nucleotides into the gap produced by excision; and (d) DNA ligase closes the gap. Repair may be measured in many ways: for example, as loss of dimers from the DNA. This is a difficult measurement because of the small amounts of radioactive thymine associated with dimerized pyrimidines and the fact that the excision of dimers is only about 50% efficient in human cells (3,4). Repair may also be measured by centrifugation in alkali of DNA from normal and XP cells. After irradiation of normal cells a shift to lower molecular weight is observed, followed by the return to higher molecu...

Unscheduled DNA synthesis in cell lysate system reflecting DNA repair in vivo.

“…The reaction mixture was exposed to black light through a thymidine solution. This photolysis method is known to introduce single strand breaks in the BrdUrd-DNA at the BrdUrd residues with no direct effect on the thymidine-DNA (14). The photolysed Fig.…”

DNA repair, DNA replication and human disorders: A personal journey

“…Dick Setlow had developed an elegant procedure for measuring gap sizes during nucleotide excision repair, by allowing the cells to incorporate bromodeoxyuridine (BrdU) into the gaps. BrdU-containing DNA is susceptible to breakage by UV light of 313 nm, so the filled-in gaps could be reconverted into breaks, and the size of the filled-in gap was inversely proportional to the dose of 313 nm irradiation needed to cleave the patch [12,13]. I realised that I could adapt this method to measure the size of daughter strand gaps during postreplication repair (PRR) and to determine whether the gaps were filled in by recombination with parental DNA, in which case they would not contain BrdU and would not be cleavable by 313 nm light.…”

DNA Repair in Thymus Lymphocytes after Irradiation

“…Such stimulated cells are more radioresistant (Schrek and Stefani 1964) . These enzymes have been implicated in repair mechanisms (Town, Smith andKaplan 1971, Regan, Setlow andLey 1971) . Furthermore, repair replication of DNA after u.v.…”