title: Analysis of Mitochondrial DNA Copy Number and Its Regulation Through DNA Methylation of POLGA.
abstract: Replication of mitochondrial DNA (mtDNA) is important for ensuring that cells have sufficient mtDNA copy number to meet their specific requirements for the generation of cellular energy through oxidative phosphorylation. A number of transcription and replication factors are required for this process, with a key factor being the nuclear-encoded mtDNA-specific DNA polymerase γ. DNA polymerase γ has a catalytic subunit (POLGA), whose gene has been shown to be DNA methylated at exon 2. This methylation is considered to be one of the key mechanisms that regulate mtDNA copy number. These findings have made it of great importance to establish optimal methods for investigating the effects of DNA methylation on mtDNA replication. Here, we provide methods to determine the extent of DNA methylation at exon 2 of POLGA as well as other gene targets of interest. We also show how mtDNA copy number is assessed and, from these two outputs, define the efficiency of mtDNA replication by calculating the mtDNA-replicative efficiency index.
authors.affiliation: Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.affiliation: Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors.forename: William
authors.initials: W
authors.lastname: Lee
authors.affiliation: Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia.
authors.forename: Vijesh
authors.initials: V
authors.lastname: Vaghjiani
authors.affiliation: Mitochondrial Genetics Group, Centre for Genetic Diseases, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia. justin.stjohn@hudson.org.au.|The Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, VIC, 3168, Australia. justin.stjohn@hudson.org.au.
authors.forename: Justin C
authors.initials: JC
authors.lastname: St John
mesh_terms: D002478:Cells, Cultured; D019175:DNA Methylation / Q000235:genetics*; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D018628:Gene Dosage / Q000235:genetics*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008928:Mitochondria / Q000235:genetics; D055544:beta-Globins / Q000235:genetics
chemical_list: D004272:DNA, Mitochondrial; D055544:beta-Globins; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human
keywords: DNA methylation; MeDIP; POLGA; mtDNA copy number