The Layered Structure of Human Mitochondrial DNA Nucleoids

Abstract

Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to crosslink to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.Eukaryotic cells typically contain thousands of mtDNA 2 genomes assembled into hundreds of aggregates known as nucleoids (1-3). These nucleoids show considerable dynamic stability, raising the possibility that this higher order organization is very important to the inheritance and segregation of mtDNAs. Nucleoid organization is thought to contribute to the rapid segregation of mtDNA mutations during oocyte maturation, the so-called "bottleneck" (4, 5), since early primordial germ cells appear to have only a small number of segregation units (6). The degree of mtDNA heteroplasmy varies considerably during embryonic development of somatic tissues as well, reflecting an uneven distribution of mtDNA genotypes that may be influenced by the clustered organization of mtDNA. A high rate of mitochondrial fusion appears to be essential for the maintenance of mtDNA in nucleoids (7,8). One of the consequences of active cycling of mitochondrial fusion and fission may be to permit an individual nucleoid to have access to a larger pool of diffusible proteins required for replication or transcription than it would if it were trapped within a single mitochondrion.Quantitative analysis of the size and mtDNA content of nucleoids in cultured mammalian cells suggests that an average nucleoid may contain 5-7 mtDNA genomes packed in a space with a diameter of only 70 nm (9). A human mtDNA nucleoid with seven 16.6-kb mtDNA genomes is a large structure containing 70 million daltons of DNA and a comparable mass of protein. These nucleoids resemble their counterparts in eubacteria, which employ abundant basic DNA binding proteins to package their genomes in a restricted volume. The packing density of bacterial nucleoids is impressive, since 4700 kb of E. coli DNA is folded into a volume of 0.08 -0.24 m 3 (10). If 5-7 mtDNA molecules are packed in a sphere with a diameter of 70 nm, the ca...

Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA

“…The finding that TFAM is very abundant is consistent with several other reports, demonstrating one TFAM molecule per 10 to 20 bp of mtDNA (13)(14)(15). In addition, biochemical characterization of purified nucleoids has shown that TFAM is the main protein component (20). TFAM is thus likely the main factor packaging and organizing mtDNA into nucleoids, but this finding does not exclude that other proteins may also be of importance.…”

“…Confocal microscopy has shown that mtDNA and TFAM colocalize in mammalian cells and are present in punctuate aggregates corresponding to nucleoids (18,19). A large number of putative nucleoid proteins have been identified by using biochemical approaches to identify proteins that can be cross-linked to or copurified with mtDNA (20) or that colocalize with mtDNA on confocal microscopy (18,19). Association of a protein that is essential for mtDNA maintenance with mtDNA does not necessarily mean that it has a role in structural organization of the nucleoid.…”

Quality matters: how does mitochondrial network dynamics and quality control impact on mtDNA integrity?

“…It has been suggested on the basis of immunofluorescent microscopy [32] and nucleoid massspectrometry analysis (see [33,34] for a recent overview) that nucleoids might organize so as to locally facilitate nucleoid maintenance and organize transcription, translation and biogenesis in its immediate surroundings. This also forms the basis for the suggestion that each nucleoid has a limited sphere of influence that could result in a focal mitochondrial OXPHOS deficiency if it contains mutant mtDNA [35].…”

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

“…Mammalian nucleoid composition has been analyzed by several groups (30,31) and a common component identified is h-mtTFA, which is thought to be a major packaging component in addition to a transcription factor (32). However, in yeast and mammals, nucleoids are also associated with other proteins (33)(34)(35)(36) and exist as heterogeneous populations (35,36). At present differential nucleoid structure and function are poorly understood, but it is reported that only specific subsets of nucleoids (or mtDNA molecules) are undergoing transcription and replication at a given point in time (28,37,38).…”