Impact of Short Inverted Repeats on Genetic Instability at Mutation Hotspots

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DESCRIPTION (provided by applicant): Genetic instability is a hallmark of many human diseases, including cancer. There are "hotspot" regions in the genome that are particularly susceptible to DNA double-strand breaks (DSBs) that can result in instabilities such as chromosomal deletions and translocations. However, the mechanisms underlying these events are not clearly understood. Thus, the long-term objective of this study is to fill this gap in knowledge by elucidating the mechanisms involved in genetic instability at endogenous mutation hotspots in mammals. Interestingly, sequences with the capacity to adopt alternative DNA structures often co-localize with these mutation "hotspots". These alternative DNA secondary structures (i.e. non-B DNA) can form on tracts of repeat sequences, have a wide range of biological functions, and are possible causative factors in a number of human diseases. For example, cruciform DNA structures can form at palindromic or inverted repeat (IR) sequences, and are often found at genomic "hotspots" for deletion and rearrangement events in human cells. To assist in achieving our long-term objective, the immediate goal is to develop a novel transgenic mutation-reporter mouse model to determine the impact of alternative DNA structures (that co-localize with chromosome breakage hotspots) on genetic instability in mammals. Previously, we found that other naturally occurring non- B DNA structures (H-DNA and Z-DNA) from translocation breakpoint hotspots in the human c-MYC and BCL-2 genes, are highly mutagenic and can induce DSBs in mammalian cells and in mice, implicating these structures in translocation-related disease etiology. Recently, we determined that short cruciform-forming sequences (28 bp), which are abundant in the human genome, are mutagenic and stimulate DSBs in mammalian cells. However, we have not yet tested the mutagenic potential of short cruciform-forming sequences in a living organism. Thus, in this study, we will test the hypothesis that short DNA cruciforms are mutagenic in a tissue-specific fashion in mice. Specifically, we will: 1) develop a novel cruciform DNA-mutation reporter transgenic mouse model; and 2) determine the cruciform DNA-induced mutation frequencies and spectra in various tissues from transgenic mice. Results from these studies will assist in elucidating the molecular mechanisms involved in genetic instability at endogenous mutation hotspots in mammals, and will allow the development of novel strategies to reduce DNA structure-induced genetic instability to prevent and/or treat disease. Additionally, this work may result in the identification of novel targets for the prevention and/or treatment of translocation-related cancers.