The goal of this work is to define the mechanism by which DNA damage resulting from an inflammatory response or other environmental sources of damage contribute to dynamic DNA mutations. Dynamic mutations are characterized by the expansion of a triplet repeat sequence such as (CAG)n/(CTG)n and are known to be the primary pathogenic signature of several neurodegenerative disorders. As one example, 5-35 repeats are present in the huntingtin gene of normal individuals but there are more than 40 repeats in Huntington's disease (HD) patients. Using a mouse model of HD, recent work from other laboratories has implicated the oxidative DNA damage product 7,8-dihydro-8-oxoguanine (8-oxoG) in dynamic mutations. Furthermore, the DNA repair enzyme OGG1, which is known to remove 8-oxoG from DNA duplexes, has also been linked to dynamic mutations. Our central hypothesis is based on our preliminary data that (CAG)n and (CTG)n sequences adopt non-B conformations that are hyper-susceptible to DNA damage relative to duplex, including the formation of 8-oxoG. In addition to containing hot spots for DNA damage we have also found that these non-B conformations are not efficiently repaired by OGG1. We postulate that formation of an OGG1- DNA misrepair complex stabilizes the non-B conformation and the DNA is then misreplicated and polymerase incorporates excess triplet repeats. This proposal outlines four specific aims to test our hypothesis. First, studies are proposed to identify the conformation of a family of repetitive CAG/CTG sequences that are prone to dynamic mutations. These experiments will be performed both with oligonucleotide substrates and also DNA in nucleosome core particles (NCP) which model the packaging of DNA in chromatin. Second, the patterns of damage in the triplet repeat DNA will be established using peroxynitrite as the damaging agent. Peroxynitrite is the reactive species produced during inflammation and is known to convert G to 8-oxoG. Third, the OGG1-mediated repair of 8-oxoG-containing DNA substrates, both oligonucleotide and NCP, will be characterized.
The final aim of this proposal is to define the processing of damage-containing CAG/CTG sequences by the replication machinery and elucidate the role of OGG1 in modulating replication fidelity.
The goal of this research is to define how the DNA damage derived from an inflammatory response or other environmental sources contribute to dynamic DNA mutations. In particular, this work will describe how the DNA damage product 7,8-dihydro-8-oxoguanine contributes to disease-initiating dynamic mutations. This knowledge is expected to provide the information needed to rationally design interventions to prevent or delay the onset of multiple diseases that are caused by dynamic mutations.
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