Telomeres protect chromosome ends in eukaryotes. In the absence of telomerase, telomeres shorten, which eventually leads to senescence. A minority of cells, however, can escape senescence and stabilize telomeres by a recombination process called Alternative Lengthening of Telomeres (ALT). ALT is responsible for telomere maintenance in ~15% of cancers, but it also contributes to stabilization of telomeres in aging or stem cells. Thus, understanding the ALT mechanism is important to identify factors that can influence whether and how it occurs. It is also well known that exposure to various environmental stressors and air toxins influence all aspects of telomere biology and promote telomere-related diseases, including various types of cancer. However, the effects of environmental factors on ALT and telomere dynamics are difficult to study, owing to the absence of experi- mental systems to identify and follow the critical steps responsible for ALT establishment in human cells at the molecular level. A key gap in the study of ALT has been, until recently, the lack of quantitative assays. The goal of this research is to unravel the mechanisms of ALT by identifying genetic, structural, and environmental factors affecting ALT. This research takes advantage of a powerful system in yeast, Saccharomyces cerevisiae, where ALT was originally discovered and where ALT can be followed from the beginning of telomere erosion through formation of survivor cells. This research will employ a unique combination of methods that were recently devel- oped by the applicants that enabled a quantitative study of ALT. This research, so far, has yielded three firsts in the field: (i) a population genetics-based assay that determined the frequency of ALT, (ii) ultra-long sequencing described the detailed structure of individual chromosome ends in ALT survivors, and (iii) a combination of com- putational modeling, Southern blot analysis and PacBio sequencing uncovered “molecular milestones” repre- senting different steps of ALT in large populations of yeast cells. Using these new approaches, this research will determine the effects of genetic, chromosome-structural and environmental factors during the steps of ALT, including: (i) the formation of ALT precursor cell populations initiated by eroded chromosome ends; (ii) the de- velopment of ALT survivors taking place in such populations; (iii) the frequency of ALT outcomes, and (iv) the molecular structure of chromosome ends in ALT survivors. This research will test the effect of environmental stressors on ALT, including oxidative damage from paraquat and the effect of cadmium, a ubiquitous environ- mental pollutant and a type I carcinogen. This will provide insight into molecular effects of environmental factors as well as illuminate new molecular mechanisms of ALT formation, potentially uncovering opportunities for med- ical interventions. Furthermore, this research will establish a robust system to evaluate the influence of o...