While most DNA exists in the canonical B-DNA form, repetitive DNA sequences can form alternatively structured DNA (or non-B-DNA) such as H-DNA and Z-DNA. These non-B DNA-forming sequences are abundant in mammalian genomes and are often associated with diseases such as Fragile X Syndrome, Huntington's disease and myotonic dystrophy. We have found that H-DNA and Z-DNA-forming sequences are highly mutagenic in mammalian cells and in mice, and often co-localize with chromosome breakage hotspots, such as in the promoter region of the c-MYC gene that maps to a translocation breakpoint hotspot in Burkitt's lymphoma. Furthermore, the mutagenic H-DNA and Z-DNA-forming sequences stimulate DNA double-strand breaks (DSBs), leading to deletions and translocations, implicating them in translocation-related disease etiology. In addition to their intrinsic instability, it has been demonstrated that DNA damage from exogenous sources may be enriched in non-B-forming sequences and this damage may be refractory to repair. UV irradiation from the sun is a ubiquitous environmental carcinogen that we are exposed to on a daily basis and has been associated with cancers such as melanoma. In B-DNA, UV irradiation causes the formation of DNA lesions such as 6-4 photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs). However, how exogenous DNA damaging agents such as UV irradiation affect H-DNA and Z-DNA structure formation, stability, and mutagenic potential is not well understood. Our long-term objectives are to understand how UV irradiation affects non-B DNA structure-induced genetic instability in human disease. The immediate objectives of this proposal are to test the hypothesis that non-B DNA regions will be more prone to UV irradiation damage, and more refractory to repair than B-DNA. We will test this hypothesis using a mammalian cell system and a mutation reporter assay our lab has previously developed.
Specific Aim 1 addresses the mutagenic potential of non-B DNA sequences after UV exposure. The mutagenic potential of non-B DNA after UV irradiation will be tested in mammalian COS-7 cells using our mutation reporter assay.
Specific Aim 2 addresses the potential of UV lesion formation within and surrounding non-B DNA-forming sequences and structures. We will determine how non-B DNA structures affect lesion formation and how lesion formation affects the formation of non-B DNA structures.
Specific Aim 3 characterizes the repair pathways involved in processing UV lesions at the non-B DNA structures using cell lines deficient in different repair proteins.
Since non-B DNA-forming sequences are abundant in mammalian genomes and we are constantly exposed to UV irradiation, understanding how these two mutagenic factors influence each other by increasing or decreasing genetic instability is crucial for understanding the etiology of disorders related to genetic instability, such as cancer. The results of these studies will help to determine the susceptibility of certain DNA sequences to mutagenesis and ultimately carcinogenesis in order to develop improved cancer treatment and/or prevention strategies.
