Saturday, May 10, 2025
5/10/2025
Genome-wide DNA Secondary Structure Analysis to Investigate DNA Fragility - Project Summary/Abstract: Both physiological DNA breaks occurring during DNA metabolic processes and pathological DNA breaks responding to a wide range of stresses, contribute to the outcome of human genome instability. DNA fragility generated by alternative DNA secondary structures is a known cause of many human diseases, and also occurs in normal DNA processes. Formation of these structures can arise from single-stranded DNA when the DNA duplex is unwound during DNA processes such as replication and transcription, and thus can be affected by cellular activities, nucleotide sequences, and chemical exposures. Here we will examine if DNA regions having potential to form stable secondary structures when unwound during cell processes, can serve as signals for topoisomerase II (TOP2) to recognize and cleave, and lead to the removal of the excessive supercoiling. We have carried out a computational evaluation of the entire available human genome sequence for optimal ability to fold single-stranded sequences into multiple-hairpin structures, and identified sites of highly stable DNA secondary structures throughout the genome. We will measure TOP2-mediated DNA breaks at these sites upon changes in DNA supercoiling from cell activities, then analyze TOP2-cleaved sites to identify structural features, and examine if DNA secondary structures influence the removal of TOP2 cleavage complexes. TOP2-mediated breaks are also often associated with pathological damage due to the use of TOP2 inhibitors as anticancer drugs. Many DNA secondary structure-rich and fragile regions are located within cancer- specific translocation-participating gene regions, including acute myeloid leukemia (AML)-rearranged regions. We will determine whether DNA fragility at these regions can serve as a biomarker for assessing the potential development of cancer-causing rearrangements. We will first test if DNA fragility at gene regions of AML rearrangements is sensitive to various chemotherapeutic agents, and if this sensitivity leads to the formation of AML rearrangements in human cells. Then, to test if this sensitivity can predict the rearrangement formation in patients, we will examine DNA breakage at these regions in normal cells of AML patients with the AML rearrangements, compared to that of normal individuals, as a means to evaluate individual susceptibility to AML. These experiments will facilitate the clinical application of using DNA fragility as a biomarker. With personalized medicine in mind, we will evaluate the effect of naturally occurring sequence variants on the fragility of the break-prone and AML translocation-participating gene regions, to further identify structure characteristics contributing to DNA fragility, and to reveal an unexploited consequence of non-coding variants. Our preliminary results suggest that sequence variants can influence DNA break frequency of the region by changing the extent or the type of secondary structure forming ability. This proposal will elucidate the mechanistic and functional features of DNA structure-driven fragility and provide a foundation for future clinical use of fragile site breakage in disease diagnostics.
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