The mitochondria contain their own genome, which is organized in a dynamic high-order nucleoid structure consisting of several copies of mitochondrial DNA (mtDNA) molecules associated with proteins. The mitochondrial nucleoids are the units of mtDNA inheritance, and are the sites of mtDNA transcription, replication and maintenance. Therefore, the integrity of mitochondrial nucleoids is a key determinant of mitochondrial metabolism and the bioenergetic state of the cell. Deciphering the interaction of mtDNA with proteins in nucleoprotein complexes is fundamental to understand the mechanisms of mtDNA segregation leading to mitochondrial dysfunction and to develop therapies to treat diseases associated with mtDNA mutations. The work presented in this dissertation provides essential insights into the dynamics of mtDNA interaction with nucleoid proteins. In order to unveil the organization of the mitochondrial genome, we have mapped major regulatory regions of the mtDNA in vivo using mitochondrial-targeted DNA methyltransferases. In chapter 2, we have demonstrated that DNA methyltranferases are powerful tools in probing mtDNA-protein interactions in living cells. The DNA methyltransferases' accessibility to their cognate sites in the mtDNA is negatively correlated with the frequency and binding strength that protein factors occupy a specific site. Our results show that the transcription termination region (TERM) within the tRNALeu(UUR) gene is consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mTERF1 (mitochondrial transcription termination factor 1). DNA methyltransferases have also been shown to be effective in detecting changes in mitochondrial nucleoid architecture due to nucleoid remodeling. We were able to determine changes in the packaging state of mitochondrial nucleoids by monitoring changes in mtDNA accessibility. The impact of altered levels of major nucleoid proteins was assessed by monitoring changes in mtDNA methylation pattern. We observed a more condensed nucleoid state causing a decrease in mtDNA methylation when the levels of the mitochondrial transcription factor A (TFAM) were altered. Changes in mtDNA methylation pattern were also evident when cells were treated with ethidium bromide (EtBr) and hydrogen peroxide. The mtDNA nucleoids adopted a less compact state during rapid mtDNA replication after EtBr treatment. In contrast, we observed a more compact mtDNA, less accessible to DNA methyltransferase after hydrogen peroxide treatment. Our results indicate that mitochondrial nucleoids are not static, but are constantly been modulated in response to factors that affect the nucleoid environment. In chapter 3, we identified the in vivo DNA binding sites of major transcription regulatory proteins, TFAM and mTERF3 using a targeted gene methylation (TAGM) strategy. In this approach, the mtDNA binding protein is fused to a DNA methyltransferase as an attempt to selectively methylate the sites adjacent to the protein target DNA region. Knowledge on how proteins interact with the mtDNA in high-order structures, which function as a mitochondrial genetic unit, will help elucidate the segregation and accumulation of mutated mtDNA in diseased tissues.