FEN1 activity is critical for the normal maintenance of nuclear DNA. This enzyme has demonstrated roles in genome replication, DNA repair, and recombination. A role for FEN1 in mtDNA maintenance has only recently been suggested [14
], however, the mitochondrial subcellular localization of FEN1 has been debated [13
]. Here we corroborate the findings of Liu et al.
] with our genetic analysis of RAD27
deletion strains, and validate the localization of FEN1 in yeast and mice. We have positively identified FEN1 in mitochondrial fractions of yeast and mice and found that it colocalizes with yeast mitochondrial structures by fluorescence microscopy.
Our comprehensive genetic analysis of RAD27
deletion strains supports a mitochondrial role for Rad27p, and shows that this protein may perform different functions in the nuclear and mitochondrial compartments. While previous studies of RAD27
mutant strains demonstrate increases in various types of nuclear mutations including recombination, point mutations, reversions, and frameshifts [17
]; our study reveals increases in mitochondrial point mutation accumulation but decreases in mitochondrial DRMD and mitochondrial microsatellite instability.
Unlike nuclear DNA, mtDNA in yeast is dispensable under laboratory growth conditions. As a result, mtDNA can be lost when cells are provided with a fermentable carbon source such as dextrose, resulting in non-respiring cells called petites
. If Rad27p plays a critical role in mtDNA replication, we would expect loss of the mitochondrial genome at a high frequency under non-selective conditions in a RAD27
mutant. Since the rad27
-Δ strain does not show an increase in the frequency of petites
, we conclude that Rad27p does not play a significant role in mtDNA replication, or that there is redundancy in its function in mtDNA replication in yeast. However, our genetic data do suggest a role for Rad27p in mtDNA repair, consistent with the in vitro
evidence implicating FEN1 in mitochondrial LP BER [14
We observe a significant increase in mitochondrial mutations resulting in erythromycin resistance. While our increase in mtDNA point mutations seems to be at odds with the observation that deletion of RAD27 does not increase respiration loss, it is important to note that respiration loss primarily results from large-scale deletions and rearrangements of the mitochondrial genome and is measured as percent of total cells. In contrast, erythromycin resistance results specifically from point mutations, and is measured on a scale of 10−7 cells. Therefore these events are mechanistically distinct, and even dramatic increases in mtDNA point mutations may not induce significant increases in respiration loss; furthermore, not all mtDNA point mutations give rise to respiratory deficient cells.
Because the rad27
-Δ strains have reduced growth rates relative to the wild-type strains, we tested whether the number of ER
colonies continued to increase after increased incubation times up to 21 days. We found that deletion of RAD27
causes a significant increase in mtDNA point mutations that continue to accumulate over time, and this phenomenon is not evident for nuclear point mutations. These experiments are performed on solid medium, so the additional mutants that arise are independently-derived. In addition, because nuclear mutations do not increase during these extended incubations, the ER
mutants cannot arise as an indirect consequence of nuclear mutations. How these mutations continue to accumulate is not clear, however, mtDNA replication is not tied to cell cycle regulation [44
] and mtDNA replication has been demonstrated in post-mitotic eukaryotic cells [45
]. In the rad27
-Δ yeast strain there may be increased turnover of mtDNA even in cells that are no longer dividing, resulting from a failure to complete BER.
We have developed a powerful reporter system that allows us to measure the rates of nuclear and mitochondrial DRMD within the same cells. Using these reporters, we show that deletion of RAD27 results in a nuclear hyper-recombination phenotype, and simultaneously causes a reduction in mitochondrial DRMD. This suggests that the mechanism of nuclear and mitochondrial DRMD may differ significantly. In addition, Rad27p acts to promote mitochondrial DRMD, although this effect may result from direct action of the enzyme on recombination intermediates, indirect effect of interaction with mitochondrial recombinational repair proteins, or competition for mtDNA substrates.
Nuclear repeat tract expansion in RAD27
mutants is attributed to the role of Rad27p in Okazaki fragment maturation, and it is thought that the expansion occurs on the lagging strand during DNA replication [46
]. Using our mitochondrial microsatellite reporter system, we observed a decrease in microsatellite instability in RAD27
deficient strains. However, the mechanism by which Rad27p may affect these types of mutations in the nucleus and mitochondria is likely to differ due to the different modes of DNA replication in these compartments, or because Rad27p may not play a critical role in mtDNA replication.
We have employed several different techniques to investigate the mitochondrial subcellular localization of Rad27p/FEN1. In yeast, we found that Rad27p is localized to mitochondria using western blotting and fluorescence microscopy. Furthermore, we have observed FEN1 in mitochondria purified from mouse brain, heart, and kidney tissue, and we show that in yeast and mouse brain mitochondria FEN1 is protected from protease digestion indicative of its localization within the mitochondrial compartment. These data support our hypothesis that Rad27p impacts mtDNA mutation frequencies via direct interaction with mitochondrial substrates.
For some time, nuclear and mitochondrial BER have been known to share protein components. Most mammalian mitochondrial N-glycosylases are products of alternative splicing of the same transcript that encodes the nuclear enzymes [47
]. Similarly, the yeast mitochondrial N-glycosylase proteins Ogg1p, Ung1p, and Ntg1p are also isoforms of nuclear proteins [51
]. The identification of a role for Rad27p in mitochondrial LP BER is perhaps more significant, however, given its important role in other pathways of nuclear DNA maintenance, and has raised the possibility of its action in similar mitochondrial pathways. Our results are consistent with those of Liu et al.
], and suggest that the endo/exonuclease activity of Rad27p may be important in mitochondrial LP BER, however, further studies need to be conducted to determine if Rad27p performs additional functions.