In situ detection of stalled cleavage complexes for studies in aging Abstract This project will introduce and validate the first quantitative in situ approach detecting stalled covalent cleavage complexes (SCCs), which spontaneously originate in mammalian cells during their normal aging. These covalent topoisomerase-DNA strand break intermediates are continually produced and slowly accumulate in aging cells. Their formation is accelerated by age-related degenerative diseases, such as Alzheimer’s. SCCs are generated in abortive catalysis and were recently revealed to be a critical part of the aging process in eukaryotes. In spite of the essential role of SCCs in the normal aging process and in the pathologies associated with aging, at present time, there are no specific approaches detecting and quantitating these DNA alterations in situ with the individual complex sensitivity. The existing bulk biochemical approaches are not applicable to the in situ formats, such as fixed cells and tissue sections. In this project, we will overcome this limitation by developing a new and enabling technology for molecular analysis of aging. The innovative in situ method will be based on novel labeling principles, and will simultaneously co-detect several characteristic features of SCCs. This study will close a significant technological gap and will introduce the first assay for quantitative assessment of SCCs in situ. The project will create a new tool for molecular research in aging and age-related disorders, with broad and general utility in biomedicine at large. The project will reach these Specific Aims: 1. To introduce the first quantitative in situ approach detecting covalent stalled cleavage complexes (SCCs), which spontaneously originate in mammalian cells during their normal aging. To test and validate the labeling principles of the new technology by using in situ models with regulated production of SCCs. To ensure the quantitative ability of the new approach and its high specificity for SCCs. 2. To test and validate the new method in the cell and tissue section models with biochemical and in vivo generation of SCCs. To assess and confirm the quantitative ability of the new approach and its high specificity for SCCs. To ensure robust and reliable assay performance in various samples.