Study of the physical basis of DNA strand inheritance in human mitochondrial DNA – MITOMUTREP

A human cell, aside from nuclear DNA, contains thousands of copies of mitochondrial DNA (mtDNA), a double-stranded, circular molecule of 16,569 bp. It has been proposed that mtDNA is a critical target of reactive oxygen species: by-products of oxidative phosphorylation that are generated in the organelle during aerobic respiration. Indeed, oxidative damage to mtDNA are more extensive and persistent as compared to that to nuclear DNA. Although transversions are the hallmark of mutations induced by reactive oxygen species, paradoxically, the majority of mtDNA mutations that occur during ageing and cancer are transitions. Furthermore, these mutations show a striking strand orientation bias: T?C/G?A transitions preferentially occur on the light (L) strand, whereas C?T/A?G on the heavy (H) strand of mtDNA. Before, to explain the unusual pattern of somatic mutations in mtDNA, we proposed that the majority of mtDNA progenies, created after multiple rounds of replication, are derived from the H-strand only, owing to asymmetric replication of only one DNA strand. Earlier, the classical Meselson–Stahl experiment (1958) employed stable isotope labelling of bacterial chromosome to address fundamental question on the mode of DNA replication in cellular organisms. Here we propose to use this classical approach to investigate mtDNA replication. First, DNA will be labeled with stable isotope nitrogen-15 (15N) by growing human cells in 15N rich-media. Then cells will be transferred to normal 14N media and the 15N/14N ratio in H- and L-strand of mtDNA will be measured at different time points. Comparison of the heavy-to-light isotope ratio in L- and H-strand over time of growth will allow us to verify if the isotope ratio in H-strand would decrease at slower rate as compared to that in L-strand. The data obtained in this project will help to discriminate between symmetric and asymmetric DNA strand inheritance and bring insight into uncommon pattern of somatic mutations in human mtDNA