MITOCHONDRIAL DNA REPLICATION FIDELITY & CARDIAC DISEASE

线粒体 DNA 复制保真度

• 批准号: 6056484
• 负责人: LAURIE SIMON KAGUNI
• 金额: $ 11.03万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6056484
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; Drosophilidae; affinity chromatography; arthropod genetics; gel electrophoresis; gene mutation; genetic regulation; genetically modified animals; heart disorder; helicase; mitochondrial DNA; nucleic acid chemical synthesis; nucleic acid sequence; polymerase chain reaction; site directed mutagenesis

The long-term objective of this proposal is to elucidate the mechanism of replication and its regulation in animal mitochondria. The proposed workfocuses on two major objectives. The first is to evaluate mechanistically the relationship between mitochondrial DNA replication fidelity and altered forms of mitochondrial DNA that accumulate in normal aging and diseased hearts. Two possible replicative mechanisms for generation of mitochondrial DNA molecules with deletions/duplications will be tested in vitro reactions employing two kepy replicative proteins, mitochondrial DNA polymerase and mitochondrial single-stranded DNA-binding protein. The models to be examined are transient strand misalignment and template DNA strand switching during DNA synthesis. Second, an error-prone bypass mechanism for generation of base substitution errors during DNA synthesis past sites of DNA damage will be tested. In addition to providing conclusive biochemical DNA is support of these models for mitochondrial DNA mutagenesis, an important goal will be to reintroduce into animals altered forms of these two key proteins, to establish a causal relationship between mitochondrial DNA mutations and cardiac disease. The second major objective is to identify new mitochondrial DNA replication proteins functioning at the replication fork. In both procaryotic and eucaryotic systems, DNA replication fidelity may be influenced greatly by auxiliary proteins. Protein affinity charomatography will be employed to identify proteins that interact physically with mitochondrial single-stranded DNA-binding protein. Biochemical assays will be developed in parallel to identify proteins that function in concert with mitochondrial DNA polymerase and single-stranded DNA-binding protein at the replication fork. Altered forms of mtDNA cause human disease. To date, very little mechanistic information is available to demonstrate how the mutant forms are generated. Many mtDNA diseases show significant cardiac manifestations and in several, cardiac disease is the major finding. Furhter, substantial clinical data now show that the heart tissue in otherwise healthy cardiac patients bears extensive damage of the same types as in mtDNA diseases with variable pathologies. This work will help establish the basic biochemical mechanisms involved in generating these altered forms, and also provide an animal model system to determine whether mtDNA mutations are a cause or a consequence of cardiac disease.

这项建议的长期目标是阐明