Mitochondrial dysfunction has been implicated in many human diseases. A subset of these are caused by mutations in mitochondrial DNA (mtDNA) which result in multi-system degenerative diseases affecting heart, muscle, and the nervous system. In addition, mtDNA mutations have been found in association with cardiomyopathy, bone marrow-pancreas disease (Pearson's syndrome), diabetes, several common neurodegenerative diseases, and even normal aging tissues. The overall goal of this project is to understand the interplay between the nuclear and mitochondrial genomes necessary for proper expression and maintenance of mtDNA and its relationship to the complex phenotypes of mtDNA mutations.
The specific aims are toward the characterization of regulatory events that control transcription and replication of human mtDNA. This requires the dissection of a variety of protein/protein and protein/nucleic acid interaction occurring in the displacement-loop (D-loop) regulatory region of the mtDNA molecule and characterization of the nuclear genes encoding products that are targeted to the mitochondria to function at this mtDNA control site. The D-loop region of mtDNA contains several regulatory loci including two transcriptional promoters, the leading-strand origin of mtDNA replication (OH), and a replication termination-associated sequence (TAS). Transcription and DNA replication are linked in human mitochondria because RNA transcripts initiated at a promoter upstream of OH are processed to generate primers for DNA synthesis by mitochondrial DNA polymerase. DNA replication is regulated further by a termination event that occurs downstream of OH. Transcription initiation, RNA processing, DNA replication,, and termination all require nuclear gene products. A major aim will be to isolate genes encoding protein components involved in these processes. The recent isolation of genes encoding human mitochondrial RNA and DNA polymerase catalytic subunits will facilitate these efforts and allow questions concerning the mechanism of transcription-primed DNA replication and replication termination to be addressed effectively in vitro. Having the genes encoding mtDNA regulatory molecules in hand allows new questions to be addressed concerning how nuclear gene expression controls mtDNA copy-number in vivo. The applicant's ability to pinpoint, and perhaps counteract, the pathogenic effects of defective mtDNA will come from elucidating pathways that connect the nuclear and mitochondrial genetic compartments in this manner.
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