Alternative DNA secondary structures are mutagenic and prone to breakage, which can lead to theetiology of human genetic diseases and cancers. Formation of these structures can occur when the DNAduplex is unwound during metabolic DNA processes such as DNA replication, and cause abnormalities inthese processes. Both nucleotide sequences and cellular activities can determine the formation of a highlystable secondary structure at a given DNA region, and the influence of environmental exposures is alsoreported. With increasing recognition of the importance of DNA secondary structures in promoting generearrangements, it is timely and critical to carry out a bias-free assessment of the ability of the entire humangenome sequence to form secondary structures. This structure database can serve as a basis for futurestudies, such as exploration of structure-function relationships of chromosome components, investigation ofthe influence of DNA structure on DNA metabolic processes, and the impact of environmental exposures onDNA fragility. In this proposal, we will first analyze the propensity to form DNA secondary structures in a genome-wide analysis, and use it to identify structural characteristics of fragile sites. The entire available humangenome sequence will be evaluated using the MFOLD program for the potential to form DNA secondarystructures, to create a structure database. This information will be used to directly examine whether thesecondary structure-forming ability correlates with DNA fragility. Our analysis of chromosome 10 revealedexciting findings in which all fragile sites induced by aphidicolin display a higher propensity to fold into stablesecondary structures compared to the rest of the chromosome. Also, the current cytogenetically-defined largefragile sites can be refined, while additional fragile sites in non-fragile regions are identified. The goal is tocompile a list of gene regions possessing high potential to fold into stable secondary structures. Theseregions will be validated for the secondary structure formation in vitro and for DNA breakage in cells, todirectly test whether the propensity to form highly stable secondary structure is an underlying factor for DNAfragility. Then, to examine the effect of these regions on DNA replication and its instability, we will usethe SV40 replication system to evaluate cis-elements such as replication direction, and trans-factors such ascomponents of the cell cycle checkpoints in causing DNA breakage and replication delay. Finally, we willexamine whether environmental and therapeutic agents generate DNA breaks at these secondarystructure-rich and cancer-specific translocation-participating gene regions. This experiment will pavethe way for the clinical application of using fragile site breakage in diagnostics. This proposal will generate useful tools for structural studies, address the nature of DNA fragility, andfurther advance our knowledge about the impact of environmental exposures in human disease development.

Public Health Relevance

DNA fragility generated by alternative secondary structures is a known cause of many human diseases; andcan be affected by nucleotide sequences; cellular activities; and environmental exposures. We propose tocreate a genome-wide DNA secondary structure database for compiling a panel of gene regions with a highpotential to form stable secondary structures; and to examine these regions for the effect of both cellularactivities and environmental exposures on fragility. This aspires toward future applications of measuring DNAfragility in a personalized approach to diagnostics and treatment.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Genomics, Computational Biology and Technology Study Section (GCAT)
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Krasnewich, Donna M
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University of Virginia
Schools of Medicine
United States
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Reddy, Kaalak; Schmidt, Monika H M; Geist, Jaimie M et al. (2014) Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability. Nucleic Acids Res 42:10473-87