TDP43 interacts with MLH1 and MSH6 proteins in a DNA damage-inducible manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double-strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that the TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complementary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 h treatment of 10 µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS-affected neurons.

TDP43 Interacts with MLH1 and MSH6 Proteins in A DNA Damage-Inducible Manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complimentary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 hr treatment of 10µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS affected neurons.

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

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.

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.

Enhancement of Photoacoustic Signal Strength with Continuous Wave Optical Pre-Illumination: A Non-Invasive Technique.

Use of portable and affordable pulse light sources (light emitting diodes (LED) and laser diodes) for tissue illumination offers an opportunity to accelerate the clinical translation of photoacoustic imaging (PAI) technology. However, imaging depth in this case is limited because of low output (optical) power of these light sources. In this work, we developed a noninvasive technique for enhancing strength (amplitude) of photoacoustic (PA) signal. This is a photothermal-based technique in which a continuous wave (CW) optical beam, in addition to short-pulse ~ nsec laser beam, is employed to irradiate and, thus, raise the temperature of sample material selectively over a pre-specified region of interest (we call the process as pre-illumination). The increase in temperature, in turn enhances the PA-signal strength. Experiments were conducted in methylene blue, which is one of the commonly used contrast agents in laboratory research studies, to validate change in temperature and subsequent enhancement of PA-signal strength for the following cases: (1) concentration or optical absorption coefficient of sample, (2) optical power of CW-optical beam, and (3) time duration of pre-illumination. A theoretical hypothesis, being validated by numerical simulation, is presented. To validate the proposed technique for clinical and/or pre-clinical applications (diagnosis and treatments of cancer, pressure ulcers, and minimally invasive procedures including vascular access and fetal surgery), experiments were conducted in tissue-mimicking Agar phantom and ex-vivo animal tissue (chicken breast). Results demonstrate that pre-illumination significantly enhances PA-signal strength (up to ~70% (methylene blue), ~48% (Agar phantom), and ~40% (chicken tissue)). The proposed technique addresses one of the primary challenges in the clinical translation of LED-based PAI systems (more specifically, to obtain a detectable PA-signal from deep-seated tissue targets).

Heritable pattern of oxidized DNA base repair coincides with pre-targeting of repair complexes to open chromatin.

Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the buildup of free atmospheric oxygen.

Deficiency in classical nonhomologous end-joining-mediated repair of transcribed genes is linked to SCA3 pathogenesis.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.

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.

RT2 PCR array screening reveals distinct perturbations in DNA damage response signaling in FUS-associated motor neuron disease.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease that has been linked to defective DNA repair. Many familial ALS patients harbor autosomal dominant mutations in the gene encoding the RNA/DNA binding protein 'fused in sarcoma' (FUS) commonly inducing its cytoplasmic mislocalization. Recent reports from our group and others demonstrate a role of FUS in maintaining genome integrity and the DNA damage response (DDR). FUS interacts with many DDR proteins and may regulate their recruitment at damage sites. Given the role of FUS in RNA transactions, here we explore whether FUS also regulates the expression of DDR factors. We performed RT2 PCR arrays for DNA repair and DDR signaling pathways in CRISPR/Cas9 FUS knockout (KO) and shRNA mediated FUS knockdown (KD) cells, which revealed significant (> 2-fold) downregulation of BRCA1, DNA ligase 4, MSH complex and RAD23B. Importantly, similar perturbations in these factors were also consistent in motor neurons differentiated from an ALS patient-derived induced pluripotent stem cell (iPSC) line with a FUS-P525L mutation, as well as in postmortem spinal cord tissue of sporadic ALS patients with FUS pathology. BRCA1 depletion has been linked to neuronal DNA double-strand breaks (DSBs) accumulation and cognitive defects. The ubiquitin receptor RAD23 functions both in nucleotide excision repair and proteasomal protein clearance pathway and is thus linked to neurodegeneration. Together, our study suggests that the FUS pathology perturbs DDR signaling via both its direct role and the effect on the expression of DDR genes. This underscors an intricate connections between FUS, genome instability, and neurodegeneration.

An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson's Disease1.

Altered expression of α-synuclein is linked to Parkinson's disease (PD). A major challenge to explore how the increased α-synuclein affect neurotoxicity is the lack of a suitable human neuronal cell model that mimics this scenario. Its expression in neural precursors affects their differentiation process, in addition to the neuronal adaptability and variability in maintaining a constant level of expression across passages. Here, we describe an SH-SY5Y line harboring Tet-ON SNCA cDNA cassette that allows for induction of controlled α-synuclein expression after neuronal differentiation, which can be an important tool for PD research.

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.

Heterocyclic Analogs of Sulforaphane Trigger DNA Damage and Impede DNA Repair in Colon Cancer Cells: Interplay of HATs and HDACs.

SCOPE: DNA repair inhibitors have broad clinical applications in tumor types with DNA repair defects, including colorectal cancer (CRC). Structural analogs of the anticancer agent sulforaphane (SFN) were investigated as modifiers of histone deacetylase (HDAC) and histone acetyltransferase (HAT) activity, and for effects on DNA damage/repair pertinent to human CRC. METHODS AND RESULTS: In the polyposis in rat colon (Pirc) model, single oral administration of SFN and structurally related long-chain isothiocyanates (ITCs) decreased histone deacetylase 3 (HDAC3) expression and increased pH2AX levels markedly in adenomatous colon polyps, extending prior observations on HDAC3 inhibition/turnover in cell-based assays. Colon cancer cells at a high initial plating density had diminished cytotoxicity from SFN, whereas novel tetrazole-containing heterocyclic analogs of SFN retained their efficacy. The potent SFN analogs triggered DNA damage, cell cycle arrest, apoptosis, and loss of a key DNA repair regulator, C-terminal binding protein (CtBP) interacting protein (CtIP). These SFN analogs also altered HAT/HDAC activities and histone acetylation status, lowered the expression of HDAC3, P300/CBP-associated factor (PCAF) and lysine acetyltransferase 2A (KAT2A/GCN5), and attenuated homologous recombination (HR)/non-homologous end joining (NHEJ) repair activities in colon cancer cells. CONCLUSION: Novel tetrazole-containing heterocyclic analogs of SFN provide a new avenue for chemosensitization in colon cancer cells via modulation of HAT/HDAC activities and associated DNA damage/repair signaling pathways.

Tissue specific expression and in-silico characterization of a putative cysteine synthase gene from Lathyrus sativus L.

Grass pea (Lathyrus sativus L.) is a worldwide popular pulse crop especially for its protein rich seeds with least production cost. However, the use of the crop became controversial due to the presence of non-protein amino acid, ß-N-oxalyl-L-α, ß-diaminopropionic acid (ß-ODAP) in its seed and leaf, which is known as the principle neurotoxin to cause neurolathyrism (a motor neurodegenerative disease of humans and animals) during prolonged consumption as regular diet. Till date, the knowledge on ß-ODAP biosynthesis in Lathyrus sp. is limited only to a small part of the complex bio-chemical steps involved including a few known sulfur-containing enzymes (viz. cysteine synthase, ODAP synthase etc.). In Lathyrus sativus, biosynthesis of ß-ODAP varies differentially in a tissue-specific manner as well as in response to several environmental stresses viz. zinc deficiency, iron over-exposure, moisture stress etc. In the present study, a novel cysteine synthase gene (LsCSase) from Lathyrus sativus L was identified and characterized through bioinformatics approaches. The bioinformatic analysis revealed that LsCSase showed maximum similarity with the O-acetyl serine (thiol) lyase of Medicago truncatula with respect to several significant sequence-specific conserved motifs (cysK, CBS like, ADH_zinc_N, PALP), sub-cellular localization (chloroplast or cytoplasm) etc., similar to other members of cysteine synthase protein family. Moreover, the tissue-specific regulation of the LsCSase as well as its transcriptional activation under certain previously reported stressed conditions (low Zn+2-high Fe+2, PEG induced osmotic stress) were also documented through quantitative real-time PCR analyses, suggesting a possible link between the LsCSase gene activation and ß-ODAP biosynthesis to manage external stresses in grass pea. This preliminary study offers a probable way towards the development of less toxic consumer-safe grass pea by down-regulation or deactivation of such gene/s (cysteine synthase) through genetic manipulations.

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.

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.

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.