Modes of mitochondrial DNA replication and its regulation in Drosophila melanogaster (573.3)

Abstract

Although replication of the mitochondrial DNA genome has been studied extensively in mammalian and yeast models, the mechanisms of mitochondrial DNA (mtDNA) replication in the widely‐used model organism Drosophila melanogaster remain largely unknown. We employ a comparative approach to analyze mtDNA replication intermediates using two‐dimensional neutral agarose gel electrophoresis (2DNAGE), and to visualize their architecture by transmission electron microscopy (TEM). Our studies target the nuclear‐encoded replisome proteins (DNA polymerase γ, mtDNA helicase and single‐stranded DNA‐binding protein), as well as transcription factors (mTTF and mTerf5), and their roles in the regulation of mtDNA replication through overexpression of both wild type proteins and biochemical variants in cultured insect cells and fly strains. Whereas 2DNAGE allows the analysis of minimally manipulated DNA preparations, TEM provides molecular details regarding the length of Okazaki fragments and on the presence of double‐stranded DNA and/ or RNA loops at the replication fork. In addition to the evaluation of the modes of replication in normal and perturbed cells, biotin labeling of the replisome proteins enables the determination of their positions on the replicating mtDNA molecule. Together, these methods can be used to identify novel structures and proteins involved in the progression of the replication process.Grant Funding Source: Supported by Academy of Finland and NIH GM45295