Mechanism of DNA maintenance in human mitochondria

Most of the body's energy is generated in the compartment of the cell called mitochondria. Since human and other animals are highly dependent on energy supply from mitochondria, mitochondria are indispensable for all aspects of our life activity. Within mitochondria are small circles of DNA that contain the blueprint for proteins which are essential for energy production. The DNA in mitochondria is physically separated from nuclear DNA in nucleus. While genes in nuclear DNA are present at two copies per cell, there are typically a thousand or more copies of mitochondrial DNA per cell. Nuclear DNA duplicates only once when a cell divides into two daughter cells, so that one copy of nuclear DNA is distributed to each daughter cell. This process is strictly coordinated with the cell division timing. Interestingly, mitochondria let their DNA replicate in a quite different way, although the copy number is kept constant between a parental cell and a daughter cell. Observations to date suggest that some of the mitochondrial DNA molecules replicate more than once while others do not replicate at all during a round of cell division and the replication of mitochondrial DNA occurs at any time, unlike nuclear DNA replication. The factors controlling mitochondrial DNA replication are unknown. Another interesting feature is that the number of mitochondrial DNA molecules varies considerably between different tissues of the human body whereas nuclear DNA number is always the same, gametes aside. Also, continuous exercise results in the increase of mitochondrial DNA copy number in muscle tissue, indicating that mitochondria can change the copy number in response to the energy requirement of cells. Since mitochondria are a energy production factory of the cell and the blueprint of essential proteins for the factory are written in mitochondrial DNA, understanding of the replication mechanism and copy number maintenance of mitochondrial DNA is a significant subject of bioscience. This programme of research focuses on the molecular mechanisms of the above unique features of mitochondrial DNA. Investigation of these subjects is particularly important, since defects in mitochondrial DNA are now recognised as a common cause of human genetic disease, which frequently manifests dysfunction in neuronal system, skeletal muscle and heart muscle. These disorders are invariably progressive, and deterioration of patients often correlates with the extent of abnormality of mitochondrial DNA. Furthermore, this programme of research would be beneficial to general human health since dysfunction of mitochondria caused by mitochondrial DNA abnormality has recently been linked to the ageing process and male infertility. Through basic research on the mechanisms of mitochondrial DNA replication and the copy number regulation, I aim to contribute to the development of rational therapies for mitochondrial DNA disorders. These therapies need not be genetic, as it has recently become clear that altering substrate concentrations for mitochondrial DNA synthesis can profoundly impact on the amount and integrity of mitochondrial DNA. Therefore, it is realistic to expect that pharmacological intervention can be devised for mitochondrial DNA disorders, once the selection process is elucidated.

Copy number (CN) of mitochondrial DNA (mtDNA) varies in different tissues. It also changes in response to cellular status and energy demand in a cell. The regulation of mtDNA CN is poorly understood. Here I propose to study the mechanism of mtDNA regulation and the coordination of the regulation with mitochondrial ATP production through (i) characterization of mitochondrial lagging-strand, (ii) identification of factors for mtDNA replication and CN control and (iii) understanding of mitochondrial proteins with multiple functions. My recent studies of mammalian mtDNA replication suggested that a significant fraction of the replication intermediates has properties of conventional coupled leading- and lagging-strand DNA synthesis. To understand mtDNA replication, lagging-strand (Okazaki) fragments will be examined by labelling of mtDNA nascent strands under conditions where mitochondrial ligase is suppressed. Determination of the length of the fragments will clarify whether this mode of mtDNA replication is of the prokaryotic type, the eukaryotic (nuclear DNA) type, or unique to mitochondria. In mitochondria, replication factors essential for other replication systems, such as primase, initiation factors, and clamp and clamp loader proteins remain to be identified and regulatory factors for mtDNA CN are largely unknown. To understand mtDNA replication and CN control, relevant factors will be identified by comparing gene expression of cells devoid of mtDNA or cells with raised mtDNA CN with that of control cells. Several mitochondrial proteins are bifunctional, some such proteins may regulate mtDNA CN in response to mitochondrial status by sensing it through their other function. To verify this mechanism, recently implicated new functions of three mitochondrial proteins will be elucidated since their known functions or implied functions are either related to mtDNA regulation or mitochondrial and cellular biogenesis.