PROJECT SUMMARY Cancer incidence increases with age in a tissue-specific fashion. Genetic instability and epigenetic alterations (e.g., cytosine methylation) are hallmarks of both cancer etiology and aging, thus linking aging to cancer. Importantly, alternative DNA structure-forming sequences (i.e., non-B DNA) have been identified as endogenous mutation hotspots associated with cancer etiology in an age-related and tissue-specific fashion. Further, methylation can impact non-B DNA formation, its mutagenic potential, and DNA repair mechanisms; and the mutagenic processing of non-B DNA requires DNA repair proteins. However, the mechanisms involved in the age-related and tissue-specific generation of these mutation “hotspots” remain largely unknown. With the aging population increasing, there is a critical need to fill this fundamental gap in knowledge. Our long-term goal is to elucidate the mechanisms of age-associated, tissue-specific DNA structure-induced genetic instability to guide future studies to develop new strategies to prevent and/or treat cancer. Thus, the overall objective of this application is to determine the mechanisms involved in differential DNA structure-induced genetic instability with age and tissue type to inform on cancer etiology. We will test the novel hypothesis that the formation of non-B DNA and their mutagenic processing differ with age in a tissue-specific fashion due to alterations in DNA repair processing and cytosine methylation. The rationale is that determining the mechanisms associated with age-related, tissue-specific DNA structure-induced genetic instability will offer a novel scientific framework whereby new strategies to prevent and/or treat age-associated diseases, such as cancer, can be developed. The hypothesis will be tested in the following aims: 1) Measure the amount of non-B DNA formed and its mutagenic potential with age in mouse tissues; 2) determine age- and tissue-associated alterations in cytosine methylation that alter non-B DNA structure formation and mutagenesis; and 3) identify the DNA repair-associated mechanisms of mutagenic processing of endogenous mutation hotspots with age in mice. Novel mutation-reporter mice containing human non-B DNA sequences from cancer-associated mutation hotspots will be used to determine the effects of age and tissue type on the mutagenic processing of non-B DNA. This is innovative because it will test the novel hypothesis that DNA structure-induced genetic instability is altered with age and tissue type in mammals, dependent on age-related modulations in epigenetics and DNA repair. The expected contribution is the elucidation of the impact of age and tissue type on DNA structure- induced genetic instability, which is significant because the results will inform on the etiology of various cancer types with age. This is expected to have a significant positive impact because the results will help achieve our long-term goal to understand the mechanisms of age-associat...