Loss of the Mitochondrial Nucleoid Protein, Abf2p, Destabilizes Repetitive DNA in the Yeast Mitochondrial Genome

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Loss of Abf2p, an abundant mitochondrial nucleoid-associated protein, results in increased mitochondrial frameshifts and direct-repeat mediated deletions but has no effect on the rate of mitochondrial point mutations. The instability of repeated sequences in this strain may be linked to the loss of mitochondrial DNA in abf2-Δ strains.

A functional mitochondrial genome is essential for the survival of eukaryotic cells. Several inherited neurodegenerative disease syndromes arise as a result of mitochondrial mutation. Deletions and rearrangements of the mitochondrial DNA (mtDNA) are easily detected and have been found in a variety of tissue types in aging mammals. Accumulation of deletions in mtDNA of mice lacking the proofreading domain of the mitochondrial polymerase, Polγ, has been linked to precocious aging of these animals (VERMULST et al. 2008), underscoring the importance of maintaining mitochondrial genome stability. Similarmutations are also observed in mitochondria of S. cerevisiae. When yeast cells are grown on a fermentable carbon source such as glucose, nonrespiring, "petite" variants arise that form smaller colonies than respiring cells (DUJON 1981). Analysis of deletion junctions in mtDNA from petite cells suggests that at least some mutations arise by recombination between directly repeated sequences (DUJON 1981).

mtDNA is organized into nucleoids, structures that aremore DNase resistant than naked DNA (WILLIAMSON 1976; MIYAKAWA et al. 1987, 1995; NEWMAN et al. 1996). Each nucleoid consists of multiple mitochondrial genomes and tightly associated proteins. In yeast, the most extensively studied nucleoid-associated protein is Abf2p, which shows homology to HMG (high mobility group) proteins that bend DNA and are involved in nuclear transcription and chromatin packaging. ABF2 is a nuclear gene whose deletion gives rise to rapid loss of mtDNA when cells are grown on a fermentable carbon source, although cells can maintain mtDNA when grown in media that selects for mitochondrial gene expression (DIFFLEY and STILLMAN 1991). Yeast abf2-Δ strains can be rescued by both the Escherichia coli HU protein and the human homolog of Abf2p, hmTFA, suggesting broad conservation of at least some functions (MEGRAW and CHAE 1993; PARISI et al. 1993). Chromatin immunoprecipitation experiments have demonstrated that Abf2p binds most regions of the mtDNA, with a higher affinity for GC-rich sequences. In addition, there is a decreased Abf2p to DNA ratio in glycerol grown cells relative to those grown in dextrose. This decrease correlates with the increased sensitivity of nucleoids to DNase I treatment, suggesting that the genome is more loosely condensed as the availability of Abf2p declines (KUCEJ et al. 2008).

Studies have implicated Abf2p in histone-like DNA packaging, replication, and in the localization of other mitochondrial proteins to nucleoids (NEWMAN et al. 1996; ZELENAYA-TROITSKAYA et al. 1998; FRIDDLE et al. 2004). In addition, a role for Abf2p in mtDNA recombination has been proposed. In crosses, mitochondrial recombinants were obtained approximately fivefold less frequently when both parents were deleted for ABF2 than in wild-type crosses. This suggests a role for Abf2p in promotion of recombination events; however, this analysis is complicated by the fact that altered mtDNA and protein mixing during mating was observed in these strains (ZELENAYA-TROITSKAYA et al. 1998). The significant changes to mitochondrial nucleoid structure and organization observed in abf2-Δ strains, even under conditions where mtDNA was retained, prompted us to test whether mutations accumulate in the mitochondrial genome under these conditions.

As previously reported, abf2-Δ strains rapidly lose mitochondrial function after cells are shifted to medium containing a fermentable carbon source. In our strain background, 81% of abf2-Δ cells are petite after ~18 generations of growth in dextrose, as compared to 0. …