FUS unveiled in mitochondrial DNA repair and targeted ligase-1 expression rescues repair-defects in FUS-linked motor neuron disease.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.

Correction: The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3'-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis.

[This corrects the article DOI: 10.1371/journal.pgen.1004749.].

Characterizing the Repair of DNA Double-Strand Breaks: A Review of Surrogate Plasmid-Based Reporter Methods.

DNA double-strand breaks (DSBs) are the most lethal genomic lesions that are induced endogenously during physiological reactions as well as by external stimuli and genotoxicants. DSBs are repaired in mammalian cells via one of three well-studied pathways depending on the cell cycle status and/or the nature of the break. First, the homologous recombination (HR) pathway utilizes the duplicated sister chromatid as a template in S/G2 cells. Second, the nonhomologous end-joining (NHEJ) is the predominant DSB repair pathway throughout the cell cycle. The third pathway, microhomology-mediated/alternative end-joining (MMEJ/Alt-EJ), is a specialized backup pathway that works not only in the S phase but also in G0/G1 cells that constitute the bulk of human tissues. In vitro experimental methods to recapitulate the repair of physiologically relevant DSBs pose a challenge. Commonly employed plasmid- or oligonucleotide-based substrates contain restriction enzyme-cleaved DSB mimics, which undoubtedly do not mimic DSB ends generated by ionizing radiation (IR), chemotherapeutics, and reactive oxygen species (ROS). DSBs can also be indirectly generated by reactive oxygen species (ROS). All such DSBs invariably contain blocked termini. In this methodology chapter, we describe a method to recapitulate the DSB repair mechanism using in cellulo and in vitro cell-free systems. This methodology enables researchers to assess the contribution of NHEJ vs. Alt-EJ using a reporter plasmid containing DSB lesions with non-ligatable termini. Limitations and challenges of prevailing methods are also addressed.

FUS Unveiled in Mitochondrial DNA Repair and Targeted Ligase-1 Expression Rescues Repair-Defects in FUS-Linked Neurodegeneration.

This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated neurodegeneration.

Pervasive Genomic Damage in Experimental Intracerebral Hemorrhage: Therapeutic Potential of a Mechanistic-Based Carbon Nanoparticle.

Therapy for intracerebral hemorrhage (ICH) remains elusive, in part dependent on the severity of the hemorrhage itself as well as multiple deleterious effects of blood and its breakdown products such as hemin and free iron. While oxidative injury and genomic damage have been seen following ICH, the details of this injury and implications remain unclear. Here, we discovered that, while free iron produced mostly reactive oxygen species (ROS)-related single-strand DNA breaks, hemin unexpectedly induced rapid and persistent nuclear and mitochondrial double-strand breaks (DSBs) in neuronal and endothelial cell genomes and in mouse brains following experimental ICH comparable to that seen with γ radiation and DNA-complexing chemotherapies. Potentially as a result of persistent DSBs and the DNA damage response, hemin also resulted in senescence phenotype in cultured neurons and endothelial cells. Subsequent resistance to ferroptosis reported in other senescent cell types was also observed here in neurons. While antioxidant therapy prevented senescence, cells became sensitized to ferroptosis. To address both senescence and resistance to ferroptosis, we synthesized a modified, catalytic, and rapidly internalized carbon nanomaterial, poly(ethylene glycol)-conjugated hydrophilic carbon clusters (PEG-HCC) by covalently bonding the iron chelator, deferoxamine (DEF). This multifunctional nanoparticle, DEF-HCC-PEG, protected cells from both senescence and ferroptosis and restored nuclear and mitochondrial genome integrity in vitro and in vivo. We thus describe a potential molecular mechanism of hemin/iron-induced toxicity in ICH that involves a rapid induction of DSBs, senescence, and the consequent resistance to ferroptosis and provide a mechanistic-based combinatorial therapeutic strategy.

A multi-faceted genotoxic network of alpha-synuclein in the nucleus and mitochondria of dopaminergic neurons in Parkinson's disease: Emerging concepts and challenges.

α-Synuclein is a hallmark amyloidogenic protein component of the Lewy bodies (LBs) present in dopaminergic neurons affected by Parkinson's disease (PD). Despite an enormous increase in emerging knowledge, the mechanism(s) of α-synuclein neurobiology and crosstalk among pathological events that are critical for PD progression remains enigmatic, creating a roadblock for effective intervention strategies. One confounding question is about the potential link between α-synuclein toxicity and genome instability in PD. We previously reported that pro-oxidant metal ions, together with reactive oxygen species (ROS), act as a "double whammy" in dopaminergic neurons by not only inducing genome damage but also inhibiting their repair. Our recent studies identified a direct role for chromatin-bound, oxidized α-synuclein in the induction of DNA strand breaks, which raised the question of a paradoxical role for α-synuclein's DNA binding in neuroprotection versus neurotoxicity. Furthermore, recent advances in our understanding of α-synuclein mediated mitochondrial dysfunction warrants revisiting the topics of α-synuclein pathophysiology in order to devise and assess the efficacy of α-synuclein-targeted interventions. In this review article, we discuss the multi-faceted neurotoxic role of α-synuclein in the nucleus and mitochondria with a particular emphasis on the role of α-synuclein in DNA damage/repair defects. We utilized a protein-DNA binding simulation to identify potential residues in α-synuclein that could mediate its binding to DNA and may be critical for its genotoxic functions. These emerging insights and paradigms may guide new drug targets and therapeutic modalities.

Loss of endosomal recycling factor RAB11 coupled with complex regulation of MAPK/ERK/AKT signaling in postmortem spinal cord specimens of sporadic amyotrophic lateral sclerosis patients.

Synaptic abnormalities, perturbed endosomal recycling mediated by loss of the small GTPase RAB11, and neuroinflammatory signaling have been associated with multiple neurodegenerative diseases including the motor neuron disease, amyotrophic lateral sclerosis (ALS). This is consistent with the neuroprotective effect of RAB11 overexpression as well as of anti-inflammatory compounds. However, most studies were in animal models, and this phenomenon has not been demonstrated in human patients. Moreover, crosstalk between endosomal trafficking and inflammatory signaling pathways in ALS remains enigmatic. Here, we investigated RAB11 expression and MAPK/ERK/AKT signaling in 10 post-mortem spinal cord specimens from patients with sporadic ALS and age-matched controls. All 10 ALS patients showed TDP-43 pathology, whereas two specimens showed an overlapping FUS pathology and one had an acquired Q331K mutation in TDP-43. There was consistent RAB11 downregulation in all ALS cases, while p-AKT and phospho-ribosomal S6 kinase (p-p90RSK) were upregulated. Furthermore, competition between AKT and ERK pathways was observed in ALS, suggesting subtle differences among the TDP-43-ALS subtypes, which may influence patient therapeutic responses. Our findings demonstrate a complex regulation/perturbation pattern of signaling cascades involving MAPK/AKT/RAB11 in spinal cord tissue from ALS patients. These results underscore the relationships between ALS pathology, altered neuronal trafficking, and inflammation.

Amyotrophic lateral sclerosis-associated TDP-43 mutation Q331K prevents nuclear translocation of XRCC4-DNA ligase 4 complex and is linked to genome damage-mediated neuronal apoptosis.

Dominant mutations in the RNA/DNA-binding protein TDP-43 have been linked to amyotrophic lateral sclerosis (ALS). Here, we screened genomic DNA extracted from spinal cord specimens of sporadic ALS patients for mutations in the TARDBP gene and identified a patient specimen with previously reported Q331K mutation. The patient spinal cord tissue with Q331K mutation showed accumulation of higher levels of DNA strand breaks and the DNA double-strand break (DSB) marker γH2AX, compared to age-matched controls, suggesting a role of the Q331K mutation in genome-damage accumulation. Using conditional SH-SY5Y lines ectopically expressing wild-type (WT) or Q331K-mutant TDP-43, we confirmed the increased cytosolic sequestration of the poly-ubiquitinated and aggregated form of mutant TDP-43, which correlated with increased genomic DNA strand breaks, activation of the DNA damage response factors phospho-ataxia-telangiectasia mutated (ATM), phospho-53BP1, γH2AX and neuronal apoptosis. We recently reported the involvement of WT TDP-43 in non-homologous end joining (NHEJ)-mediated DSB repair, where it acts as a scaffold for the recruitment of XRCC4-DNA ligase 4 complex. Here, the mutant TDP-43, due to its reduced interaction and enhanced cytosolic mislocalization, prevented the nuclear translocation of XRCC4-DNA ligase 4. Consistently, the mutant cells showed significantly reduced DNA strand break sealing activity and were sensitized to DNA-damaging drugs. In addition, the mutant cells showed elevated levels of reactive oxygen species, suggesting both dominant negative and loss-of-function effects of the mutation. Together, our study uncovered an association of sporadic Q331K mutation with persistent genome damage accumulation due to both damage induction and repair defects.

Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects.

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.

Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in Amyotrophic Lateral Sclerosis.

Genome damage and defective repair are etiologically linked to neurodegeneration. However, the specific mechanisms involved remain enigmatic. Here, we identify defects in DNA nick ligation and oxidative damage repair in a subset of amyotrophic lateral sclerosis (ALS) patients. These defects are caused by mutations in the RNA/DNA-binding protein FUS. In healthy neurons, FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA Ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction. We discover that loss of nuclear FUS caused DNA nick ligation defects in motor neurons due to reduced recruitment of XRCC1/LigIII to DNA strand breaks. Moreover, DNA ligation defects in ALS patient-derived iPSC lines carrying FUS mutations and in motor neurons generated therefrom are rescued by CRISPR/Cas9-mediated correction of mutation. Our findings uncovered a pathway of defective DNA ligation in FUS-linked ALS and suggest that LigIII-targeted therapies may prevent or slow down disease progression.

Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress.

Posttranslational modifications of DNA repair proteins have been linked to their function. However, it is not clear if posttranslational acetylation affects subcellular localization of these enzymes. Here, we show that the human DNA glycosylase NEIL1, which is involved in repair of both endo- and exogenously generated oxidized bases via the base excision repair (BER) pathway, is acetylated by histone acetyltransferase p300. Acetylation occurs predominantly at Lys residues 296, 297 and 298 located in NEIL1's disordered C-terminal domain. NEIL1 mutant having the substitution of Lys 296-298 with neutral Ala loses nuclear localization, whereas Lys > Arg substitution (in 3KR mutant) at the same sites does not affect NEIL1's nuclear localization or chromatin binding, presumably due to retention of the positive charge. Although non-acetylated NEIL1 can bind to chromatin, acetylated NEIL1 is exclusively chromatin-bound. NEIL1 acetylation while dispensable for its glycosylase activity enhances it due to increased product release. The acetylation-defective 3KR mutant forms less stable complexes with various chromatin proteins, including histone chaperones and BER/single-strand break repair partners, than the wild-type (WT) NEIL1. We also showed that the repair complex with WT NEIL1 has significantly higher BER activity than the 3KR mutant complex. This is consistent with reduced resistance of non-acetylable mutant NEIL1 expressing cells to oxidative stress relative to cells expressing the acetylable WT enzyme. We thus conclude that the major role of acetylable Lys residues in NEIL1 is to stabilize the formation of chromatin-bound repair complexes which protect cells from oxidative stress.

Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson's Disease.

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson's disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (∼2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.

Aurora kinase B dependent phosphorylation of 53BP1 is required for resolving merotelic kinetochore-microtubule attachment errors during mitosis.

Defects in resolving kinetochore-microtubule attachment mistakes during mitosis is linked to chromosome instability associated with carcinogenesis as well as resistance to cancer therapy. Here we report for the first time that tumor suppressor p53-binding protein 1 (53BP1) is phosphorylated at serine 1342 (S1342) by Aurora kinase B both in vitro and in human cells, which is required for optimal recruitment of 53BP1 at kinetochores. Furthermore, 53BP1 staining normally localized on the outer kinetochore, extended to the whole kinetochore when it is merotelically-attached, in concert with mitotic centromere-associated kinesin. Kinetochore-binding of pS1342-53BP1 is essential for efficient resolving of merotelic attachment, a spontaneous kinetochore-microtubule connection error that usually causes aneuploidy. Consistently, loss of 53BP1 results in significant increase in lagging chromosome events, micronuclei formation and aneuploidy, due to the unresolved merotely in both cancer and primary cells, which is prevented by ectopic wild type 53BP1 but not by the nonphophorylable S1342A mutant. We thus document a novel DNA damage-independent function of 53BP1 in maintaining faithful chromosome segregation during mitosis.

Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex.

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.

Regulation of oxidized base damage repair by chromatin assembly factor 1 subunit A.

Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.

TDP-43/FUS in motor neuron disease: Complexity and challenges.

Amyotrophic lateral sclerosis (ALS), a common motor neuron disease affecting two per 100,000 people worldwide, encompasses at least five distinct pathological subtypes, including, ALS-SOD1, ALS-C9orf72, ALS-TDP-43, ALS-FUS and Guam-ALS. The etiology of a major subset of ALS involves toxicity of the TAR DNA-binding protein-43 (TDP-43). A second RNA/DNA binding protein, fused in sarcoma/translocated in liposarcoma (FUS/TLS) has been subsequently associated with about 1% of ALS patients. While mutations in TDP-43 and FUS have been linked to ALS, the key contributing molecular mechanism(s) leading to cell death are still unclear. One unique feature of TDP-43 and FUS pathogenesis in ALS is their nuclear clearance and simultaneous cytoplasmic aggregation in affected motor neurons. Since the discoveries in the last decade implicating TDP-43 and FUS toxicity in ALS, a majority of studies have focused on their cytoplasmic aggregation and disruption of their RNA-binding functions. However, TDP-43 and FUS also bind to DNA, although the significance of their DNA binding in disease-affected neurons has been less investigated. A recent observation of accumulated genomic damage in TDP-43 and FUS-linked ALS and association of FUS with neuronal DNA damage repair pathways indicate a possible role of deregulated DNA binding function of TDP-43 and FUS in ALS. In this review, we discuss the different ALS disease subtypes, crosstalk of etiopathologies in disease progression, available animal models and their limitations, and recent advances in understanding the specific involvement of RNA/DNA binding proteins, TDP-43 and FUS, in motor neuron diseases.

Scaffold attachment factor A (SAF-A) and Ku temporally regulate repair of radiation-induced clustered genome lesions.

Ionizing radiation (IR) induces highly cytotoxic double-strand breaks (DSBs) and also clustered oxidized bases in mammalian genomes. Base excision repair (BER) of bi-stranded oxidized bases could generate additional DSBs as repair intermediates in the vicinity of direct DSBs, leading to loss of DNA fragments. This could be avoided if DSB repair via DNA-PK-mediated nonhomologous end joining (NHEJ) precedes BER initiated by NEIL1 and other DNA glycosylases (DGs). Here we show that DNA-PK subunit Ku inhibits DGs via direct interaction. The scaffold attachment factor (SAF)-A, (also called hnRNP-U), phosphorylated at Ser59 by DNA-PK early after IR treatment, is linked to transient release of chromatin-bound NEIL1, thus preventing BER. SAF-A is subsequently dephosphorylated. Ku inhibition of DGs in vitro is relieved by unphosphorylated SAF-A, but not by the phosphomimetic Asp59 mutant. We thus propose that SAF-A, in concert with Ku, temporally regulates base damage repair in irradiated cell genome.

The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD.

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis.

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.