During the previous grant period it was demonstrated that mutations at a lacZ-plasmid reporter locus accumulated during aging in an organ-specific manner. In liver and heart many of the accumulated mutations were large genome rearrangements with one breakpoint in the lacZ gene and the other elsewhere in the mouse genome. While mice with defects in transcription-related DNA repair (i.e., CSB and XPD mutants) did not show accelerated mutation accumulation during aging, they do display symptoms of premature aging, as demonstrated in Project 1 by the Hoeijmakers group. Mice defective in nucleotide excision repair (XPA mutant) or a combination of defective nucleotide excision repair and inter-strand crosslink repair (Erccl mutants) did show an accelerated increase in mutation frequency during aging. However, while the Erccl mutant displayed symptoms of accelerated aging, the Xpa mutant only showed increased cancer susceptibility, as demonstrated by Hoeijmakers and van Steeg. In the Erccl mutant, premature aging was accompanied by the accelerated accumulation of genome rearrangements mutations in liver, while in the Xpa mutant mainly point mutations were found to accumulate. In this renewal application we propose to first test the hypothesis that defects in the repair of double-strand lesions, such as double-strand breaks and cross-links, are associated with both accelerated aging and the accelerated accumulation of large genome rearrangements. Second, based on results obtained in collaboration with Campisi, indicating that accelerated cellular senescence in mouse embryonic fibroblasts is caused by oxidative stress, possibly through genomic instability, we will test if overexpression of genes encoding antioxidant enzymes can retard the formation of rearrangements and partly correct accelerated aging and/or cellular senescence in mice and cells derived from them. Then, to directly analyze individual cells from young and old mouse organs for the accumulation of genome rearrangements, single-cell methods will be applied, including cytogenetic methods and molecular assays based on whole genome amplification. Finally, to assess the hypothesized consequences of genomic instability, cell-to-cell variation in gene expression levels in young and old organs will be determined using real-time PCR and microarrays with amplified total mRNA as target.
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