Ataxia telangiectasia (AT) is an autosomal recessive disease, characterized by both neurological disorders and a high incidence of early-onset cancers. On a cellular level, cellular radiosensitivity and radioresistant DNA synthesis are the hallmarks of AT. While expression of cellular radiosensitivity varies somewhat among affected individuals, radioresistant DNA synthesis is seen consistently and, in fact, is the only end point used for assigning individuals to genetic complementation groups. For this reason, complementation-group-specific correction of radioresistant DNA synthesis in AT cells has long been thought to be an absolute requirement for confirmation of a bona fide clone of an AT gene. Since primary AT cells grow poorly in culture, SV40-immortalized AT fibroblasts are the usual recipients of transfected DNA in these studies. In experiments reported here, we demonstrate that SV40-immortalized AT fibroblasts have significantly reduced radioresistant DNA synthesis compared to primary AT fibroblasts, and their response to radiation is more like normal cells, in that both the radiosensitive and radioresistant components appear to be present. This suggests that there may be an interaction between SV40 proteins and the AT gene product or its downstream elements. This partial "complementation" of radioresistant DNA synthesis in SV40-immortalized AT cells complicates complementation cloning strategies, and should be considered when terminally screening putative AT gene clones by analysis of radioresistant DNA synthesis.

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is noncomplementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.

Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 ± 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.

Ewing's sarcoma cell lines were compared to other cell lines for induction of DNA strand breaks by ionizing radiation and their ability to repair those breaks. The alkali-unwinding assay and alkaline sucrose gradient analysis were used for these studies. The alkali-unwinding assay revealed that the amount of DNA unwound per strand break in Ewing's sarcoma cells was less than for other cells and was not influenced by high-salt denaturation conditions. Ewing's sarcoma cells had similar induction and repair rates for strand breaks compared with other cell lines. The kinetics of unwinding suggests there are constraints to DNA unwinding in the chromatin of Ewing's sarcoma cells, possibly related to high levels of poly(ADP-ribose) polymerase in these cells.

Gamma endonuclease is a ${\rm Mg}^{2+}\text{-independent}$ enzyme of Micrococcus luteus that recognizes and cleaves DNA at a variety of altered pyrimidines produced by ionizing radiation. The production of enzyme-recognizable sites (ERS) by ionizing radiation under different irradiation conditions was measured. Ionizing radiation produced the greatest number of ERS when irradiations were performed under anoxic conditions in the presence of the free radical scavenger KI. Since dihydrothymine is a major pyrimidine lesion produced in DNA during anoxic irradiation, the ability of γ endonuclease to excise this lesion was assessed. Dihydrothymine was released from DNA irradiated under anoxic conditions in a radiation dose-dependent manner, consistent with γ endonuclease's known DNA glycosylase activity. Gamma endonuclease was also shown to cleave heavily uv-irradiated DNA. When the sequence specificity of γ-endonuclease cleavage was studied using uv-irradiated DNA, cleavage was seen specifically at cytosines. The identity of this enzyme-recognizable cytosine photoproduct is not known.