Project Abstract Phenotypic manifestation of cancer arises and further develops from genetic and epigenetic mutations, and a potential source of these mutations is difficult to replicate (DTR) DNA sequences. These regions form DNA secondary structures which must be resolved for DNA replication to progress fully. When cells are unable to replicate DNA through these regions, genome instability may occur, which can lead to cancer initiation. Cells contain proteins that allow DNA synthesis to occur faithfully through these regions, among which are the nuclease/helicase DNA2 and the MutSα complex. Previous research has shown that these proteins both bind to DNA secondary structures and that these proteins interact to drive resolution of these structures. Using the Single Molecule Analysis of Replicated DNA (SMARD) technique, we will visualize the extent of DNA replication in centromeres lacking DNA2, MSH2 or MSH6, components of the MutSα complex. Additionally, environmentally contaminating compounds (ECCs) are known to bind to and stabilize these DNA secondary structures. We will further use this technique to probe the impact of these compounds on DNA replication, which may reveal a further source of genome instability. Altogether, our research will establish the role of DNA2 and the MutSα complex on maintaining genome stability, explore the impact of putative DNA secondary structure stabilizing compounds on DNA replication, and reveal a molecular basis for cancer manifestation and advancement.