BER is a critical process for the maintenance of both nuclear and mitochondrial genomic stability, which, in humans is significant for the prevention of disease. Very little is known about the mechanisms that regulate BER in eukaryotes. The data in this study provide new insight into the regulation of BER by defining key components required for dynamic localization of Ntg1 that may function as part of a general mechanism for the regulation of BER. Importantly, we demonstrated that in the absence of dynamic localization of Ntg1, nuclear and mitochondrial mutation rates increase. Given the role of BER in the prevention of human disease, investigating this novel mode of BER regulation reveals how the cell counters deleterious consequences that result from oxidative DNA damage. Taken together, these data support a new paradigm for the processes involved in the regulation of BER.
We propose a model where the nuclear localization of Ntg1 is driven by nuclear oxidative DNA damage signals (NODDS), and mitochondrial localization is driven by mitochondrial oxidative DNA damage signals (MODDS) (15
). This mode of BER regulation can be further delineated as our data suggest that NODDS and MODDS compete for the recruitment of Ntg1 to DNA damage loci (). This concept is illustrated by the lack of dynamic localization of the MTS mutant Ntg1 to the nucleus during conditions of nuclear oxidative stress and of the cNLS mutant Ntg1 to mitochondria following mitochondrial oxidative stress (). The inability to mobilize additional Ntg1 into damage-containing organelles indicates that NODDS and MODDS compete with one another to recruit Ntg1, and the capacity for recruitment is exceeded under conditions where Ntg1 is already localized to one compartment or another. Following the production of NODDS or MODDS, certain cellular components (responders) which are likely to include nuclear import factors, post translational modifications and chaperones become activated to interact with or modify Ntg1 in order to recruit it to the appropriate organelle to repair oxidative DNA damage (15
). As a means of competing for Ntg1, we hypothesize that NODDS and MODDS activate DNA damage responders capable of not only facilitating recruitment, but also preventing localization to the opposing organelle by interfering with crucial localization signals (). The results from this study suggest that the classical nuclear protein import proteins, importin α/β, are novel DNA damage responders. As DNA damage responders, the classical nuclear protein import machinery associates with the bipartite cNLS of Ntg1 in order to facilitate recruitment of Ntg1 to nuclei in response to nuclear oxidative DNA damage.
Figure 7. Model of Ntg1 dynamic localization in response to nuclear and mitochondrial oxidative DNA damage. Nuclear oxidative DNA damage signals (NODDS) and mitochondrial oxidative DNA damage signals (MODDS) compete for the recruitment of Ntg1 to sites of oxidative (more ...)
We speculate that dynamic localization in response to DNA damage signals may be a general mode of regulation for BER. Several BER proteins localize to both mitochondria and nuclei (60
), all of which are candidates for regulation by dynamic localization. One particularly interesting candidate is the multifunctional human AP endonuclease 1 (hAPE1), which relocalizes from the cytoplasm to nuclei and mitochondria following exposure to oxidative stress (H2
). Other potential candidates can be identified by sequence homology to Ntg1, such as the S. cerevisiae
BER protein uracil DNA glycosylase, Ung1, which contains predicted bipartite cNLS and MTS signals tantalizingly similar to that of Ntg1 (62
). In addition, both human hNTH1 and mouse mNTH1 contain bipartite cNLS and MTS signals that target these proteins to both nuclei and mitochondria (16
), signifying that the mode for regulating eukaryotic Nth-like proteins is conserved from yeast to humans. These findings suggest that dynamic localization of BER proteins in response to DNA damage signals may be a general mechanism for regulating BER.
The discovery of mechanisms underlying the stress-induced relocalization of an initiating protein for BER has implications not only for general regulation of BER but also for regulation of other stress components. It is conceivable that specialized, stress-activated nuclear import factors could orchestrate the mobilization and delivery of components that mediate responses such as changes in transcription programs and activation of checkpoints. A key example of such a responder is Yap1, a transcription factor that continuously cycles between the nucleus and cytoplasm under nonstress conditions, but whose nuclear export is blocked under oxidative stress conditions allowing it to upregulate genes that protect against cell stress-induced damage (64
). The activation of a nuclear transport ‘stressome’ could provide a central clearinghouse to mount a coordinated stress response that synchronizes multiple distinct nuclear activities. Our findings provide the first direct evidence for this type of integrated response to cellular stress, with strong implications regarding the DNA damage response.
Dynamic localization of Ntg1 protects nuclear and mitochondrial DNA from mutation (), suggesting that dynamic localization plays an important role in the regulation of genomic stability. As the accumulation of DNA mutations is associated with nuclear genomic stability (18
), these results indicate that regulation of BER through dynamic localization of Ntg1 plays a key role in maintaining the integrity of the nuclear and mitochondrial genomes. Interestingly, nuclear dynamic localization of Ntg1 guards against cytotoxicity induced by MMS, but not H2
. These two agents generate distinct DNA lesions, which are repaired by Ntg1 with differing efficiencies (11
). Altering key localization signals may not preclude Ntg1 from entering nuclei via alternate import pathways. In the case of H2
-induced DNA base damage, residual Ntg1 levels are sufficient to repair cytotoxic lesions (A). However, for MMS-induced DNA damage a large fraction of base lesions would be expected to be converted into AP sites, exceeding the capacity of the remaining Ntg1 to mediate repair of such toxic damage (B).
The fact that dynamic localization is important for thwarting mutagenesis and DNA damage induced cytotoxicity emphasizes the impact that this mode of regulation may have on disease etiology. In this regard, the human homolog of Ntg1 (hNth1) predominantly displays cytoplasmic localization in a significant percentage of gastric and colorectal cancer tissues, suggesting that corruption of dynamic organelle targeting of BER proteins may be a characteristic of certain tumors (66