Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa(-/-)) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa(-/-) and wild-type mice. Neither photolesion was removed in the Xpa(-/-) epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m(2) UVB increased the lacZ mutant frequency in the epidermis of Xpa(-/-) mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa(-/-) mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa(-/-) and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C --> T transitions at dipyrimidine sites; however, Xpa(-/-) mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC --> TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds.

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We have synthesized functional nanocomposites consisting of titanium dioxide (TiO 2 ) nanoparticles covalently bound to DNA oligonucleotides. The TiO 2 nanoparticle is approximately 4.5 nm in diameter and retains photocatalytic properties when bound to DNA via dopamine [1]. The DNA attached to TiO 2 also maintains its ability to bind complimentary DNA sequences. Excitation of these nanocomposites by electromagnetic radiation (above 3.2 eV) leads to accumulation of electropositive holes in the bound DNA causing cleavage [1]. We hypothesize that these novel nanocomposites will be capable of targeting sequence specific aberrant and foreign DNA which cause human disease (oncogenes, retroviruses, and genomes of endoparasites). In the present study we used optical microscopy and X-ray fluorescence microscopy to show that it is possible to achieve directed subcellular localization of the TiO 2 -DNA nanocomposites depending on the sequence of DNA bound to the nanoparticle.Oligonucleotides complimentary to genomic DNA encoding 18S rRNA (ttccttggatgtggt) were synthesized to be 3' conjugated to fluorescent molecule tetramethylrhodamine (TAMRA). Such fluorescent oligonucleotides were bound to TiO 2 nanocomposites as described [1]. Human cells with 2n chromosomes contain approximately 300 copies of 18S rRNA gene which are located in the nucleolus [2], while polyploidy cancer cell lines may contain even more copies of this gene in, frequently, several nucleoli. Rat pheochromocytoma PC12 cells at 60-80% confluence were transfected (Superfect, Qiagen) with the TiO 2 -R18S(TAMRA) nanocomposites according to the manufacturer's recommendations. The cells were then washed with PBS, scraped, and pelleted (2500rpm). The cell pellets were resuspened in 10-20µl of F12K media supplemented with 10% serum and seeded on formvar coated EM grids. Cells were allowed to attach to the EM grids for 2-3 hours and then fixed in cold 100% methanol at -20˚C. These cells were stained with Syto RNASelect green fluorescent cell stain to label the nucleolus (Molecular Probes S32703) and Hoechst (Molecular Probes, H3570). The cells were then visualized with the Zeiss LSM 510 Laser Scanning Confocal Microscope for the location of TAMRA labeled oligonucleotides. Upon completion of microscopy, the EM grids with the cells were washed from the mounting media by immersion in PBS for 10 minutes and then dehydrated in 100% ethanol for 10 minutes more. The same dried whole cells were then analyzed for the presence of titanium using the 2-ID-D X-ray beamline of the Advanced Photon Source at Argonne National Laboratory [1]. Figure 1 shows there is weak but detectable titanium X-ray fluorescence (Fig. 1B) that overlaps with the presence of fluorescent TAMRA signal in the cells transfected with TiO 2 -R18S(TAMRA) (Fig.