Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

Abstract

DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.Oxidative DNA damage, which occurs frequently in all cells, is linked to aging and human disease, such as cancer and various degenerative disorders (6,13,45,81,83). Reactive oxygen species (ROS) are a by-product of normal cellular metabolic processes that can cause oxidative damage to DNA, lipids, and proteins (79). Unrepaired oxidative DNA lesions can result in mutations and lead to arrest of both DNA replication and transcription (34). In order to combat such continuous insults to the genome, cells have evolved DNA repair and DNA damage tolerance pathways (2).Base excision repair (BER) is the primary process by which oxidative DNA damage is repaired (74,90). BER is initiated by the recognition and excision of a base lesion by an N-glycosylase, resulting in an apurinic/apyrimidinic (AP) site (47, 48). The resulting AP site is processed by an AP endonuclease or an AP lyase, which cleaves the sugar-phosphate DNA backbone on the 5Ј side or 3Ј side of the AP site, respectively (5). Subsequent processing involving DNA repair polymerases replaces the excised nucleotides, and DNA ligase completes the repair process (8).Very little is known about how eukaryotic cells regulate events that initiate BER in response to oxidative stress. Deleterious oxidative DNA damage can occur in both nuclear and mitochondrial genomes, adding a level of complexity to this cellular response. In this case, the intracellular localization of BER proteins would be regulated dynamically in response to the introduction of either nuclear or mitochondrial DNA damage. Controlled protein localization has been implicate...

Repair of persistent strand breaks in the mitochondrial genome

“…Indeed, the localization of many of the above described mitochondrial repair proteins could be dependent on the balance of damage between the nucleus and the mitochondria, similar to the translocation of CSB to the mitochondria after stress (Aamann et al, 2010; Stevnsner et al, 2008). In support, the cellular localization of the glycoslyase protein Ntg1, could be modulated using damage inducing agents (Griffiths et al, 2009). Stress induced re-localization of proteins including PARP and potentially FEN-1 could offer an explanation for the disparate mitochondrial localization reported for these proteins.…”

Direct visualization of repair of oxidative damage by OGG1 in the nuclei of live cells

“…We hypothesize that this observation reflects the fact that mitochondria are a major source of ROS and that mitochondrial DNA is therefore particularly prone to oxidative damage. Interestingly, Griffiths et al [27] have also recently reported that in yeast, the intracellular location of the base excision repair protein Ntg1 is regulated by oxidative stress and that Ntg1 is able to localize to either nuclear or mitochondrial compartments in response to specific induction of oxidative stress in these two organelles. These findings show that regulation of base excision repair (BER) protein activity and location is complex and cells are able to rapidly adapt to meet specific oxidative challenges.…”

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

“…Upon genotoxic insults a dynamic redistribution of DNA repair proteins to the site of damage is frequently observed; this phenomenon leads to the accumulation of DNA repair factors, often into spatially restricted foci. Relocalization events usually occur through cytoplasmic-mitochondrial, cytoplasmic-nucleoplasmic, and nucleolar-nucleoplasmic shuttling regulated by post-translational modifications (PTMs) and triggered by the damage itself (51).…”