Salmon sperm DNA, treated with the antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP), was enzymatically degraded to (oligo)nucleotides. Four Pt-containing products were identified by 1H NMR after preparative chromatography on a diethylaminoethyl-Sephacel column at pH 8.8. In all identified adducts, comprising approximately 90% of the total Pt in the DNA, Pt was linked to the N7 atoms of the nucleobases guanine and adenine. The two major adducts were cis-Pt(NH3)2d(pGpG) and cis-Pt-(NH3)2d(pApG), both derived from intrastrand cross-links of cis-DDP on neighboring nucleobases. Only the d(pApG) but not the d(pGpA) adduct could be detected. Two minor adducts were Pt(NH3)3dGMP, resulting from monofunctionally bound cis-DDP to guanine, and cis-Pt(NH3)2d(GMP)2, originating from interstrand cross-links on two guanines as well as from intrastrand cross-links on two guanines separated by one or more bases. For analytical purposes we developed an improved method to determine cis-DDP adducts. Routinely, 40-micrograms samples of enzymatically degraded cis-DDP-treated DNA are now analyzed by separation of the mononucleotides and Pt-containing (oligo)nucleotides on the anion-exchange column Mono Q (FPLC) at pH 8.8 (completed within 14 min) and subsequent determination of the Pt content in the collected fractions by atomic absorption spectroscopy. The method was used to optimize the digestion conditions for cis-DDP-treated DNA. In kinetic studies on the formation of the various adducts, a clear preference of the Pt compound to react with guanines occurring in the base sequence d(pGpG) was established.

Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however,-one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UVirradiation. The time taken to convert initially lowmolecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Recently, complementation studies have shown that there are several genetically different forms of XP, all deficient in excision-repair (3)(4)(5)(6)(7)(8). In addition there is a further class of XP patients (termed "XP variants" in this communication) which, while exhibiting the usual clinical symptoms, seem to be completely normal in excision-repair of pyrimidine dimers (9-14). All XP lines examined have normal rates of rejoining of DNA single-strand breaks induced by ionizing radiation (14).Despite very low levels of excision-repair in cells from most XP patients (and also in rodent cell lines), they can nevertheless tolerate the production in their DNA of over 105 pyrimi- In this communication we show that fibroblast cultures from three XP variants have normal levels of excision-repair, but are abnormal in postreplication repair. After UV-irradiation, the time taken for the newly synthesized DNA to attain a high molecular weight similar to that in unirradiated controls is much longer than in normal cells. Furthermore, this conversion of low-to high-molecular weight DNA is drastically inhibited by caffeine, which has very little effect in normal human cells. MATERIALS AND METHODSCell Lines used in these experiments were primary fibroblasts from healthy donors and XP patients listed in Table 1.Excision of Pyrimidine Dimers Measured by Loss of UVEndonuclease-Susceptible Sites. This procedure has been described in detail (24). Briefly; fibroblast cells cultured as described in ref. 24 were labeled with [3H]thymidine, UVirradiated, and incubated in the absence of radioactive label for various times. They were then mixed with an equal volume of unirradiated cells whose DNA had been labeled with [14C]-dT. DNA was extracted from the mixed cell population and incubated with or without the UV-specific endonuclease from Micrococcus luteus, which'specifically nicks DNA near pyrimidine dimers (24,25). The DNA products resulting from enzymic attack were centrifuged through 5-20% alkaline sucrose gradients at 40,000 rpm for 135 min at 210 in an SW56 rotor. After centrifugation, fractions were collected, radioactivity was determined, and the weight-average molecular weight of the DNA distributions ...

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52 ؊/؊ ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.Double-strand breaks (DSBs) in the DNA of living organisms occur during several physiological processes including meiotic recombination, mating-type switching in yeast, and V(D)J rearrangement in developing B and T lymphocytes. Agents such as ionizing radiation and certain chemicals also lead to the induction of DSBs in the genome. If left unrepaired, DSBs result in broken chromosomes and cell death, as has been shown convincingly in yeast (5). Alternatively, incorrect repair of DSBs may generate deletions, chromosome rearrangements, and cell transformation and eventually lead to the formation of tumors.Two main pathways are known to be involved in the repair of DSBs in eukaryotes: end-to-end rejoining, a homology-independent but error-prone process, and error-free repair via (homologous) recombination. Repair of DSBs in the yeast Saccharomyces cerevisiae occurs predominantly via recombination, whereas a contribution of end-to-end rejoining can be observed only in a recombination-deficient background (9, 27, 47). Recombinational repair in S. cerevisiae involves the genes of the RAD52 epistasis group, of which nine members have been identified thus far (ScRAD50, ScRAD51, ScRAD52, ScRAD54, ScRAD55, ScRAD57, ScRAD59, ScMRE11, and ScXRS2) (2,11,15,16,44). Interestingly, it has been shown that ScRAD50, ScMRE11, and ScXRS2 are also involved in end-to-end rejoining (10,28,55). Mutations in genes of the RAD52 group result in an increased sensitivity to ionizing radiation and defects in one or more types of recombination. Among these mutants, the Scrad51, Scrad52, and Scrad54 mutants display the most severe radiation sensitivity and defects in recombination.Biochemical experiments with S. cerevisiae have shown that the ScRad51 protein forms nucleoprotein filaments with single-stranded DNA and promotes pairing and limited strand exchange (51). The ScRad52 protein alone or a heterodimer of ScRad55 and ScRad57 functions as a cofactor in this reaction, ...

To study gene mutations in different organs and tissues of an experimental animal, we produced transgenic mice harboring bacteriophage lambda shuttle vectors integrated in the genome in a head-to-tail arrangement. As a target for mutagenesis, the selectable bacterial lacZ gene was cloned in the vector. The integrated vectors were rescued from total genomic DNA with high efficiency by in vitro packaging and propagation of the phages in a LacZ- strain of Escherichia coli C. The background mutation frequencies in brain and liver DNA appeared to be low, as was indicated by the absence of colorless plaques among 138,816 and 168,160 phage isolated from brain and liver DNA, respectively. Treatment of adult female transgenic mice with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of the frequency of mutated vectors isolated from brain DNA, up to 7.4 x 10(-5) at 250 mg of the alkylating agent per kilogram of body weight. At this dose, in liver DNA of the same mice, mutation frequencies were approximately 3 x 10(-5). DNA sequence analysis of four mutant vectors isolated from brain DNA indicated predominantly G.C----A.T transitions. These results demonstrate the value of this transgenic mouse model in studying gene mutations in vivo. In addition to its use in fundamental research, the system could be used as a sensitive, organ-specific, short-term mutagenicity assay.

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G⅐C to A⅐T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G⅐C to T⅐A, G⅐C to C⅐G, and A⅐T to C⅐G transversions and G⅐C to A⅐T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G⅐C to A⅐T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.