Abstract: The ability of DNA double-strand breaks (DSBs) to cluster in mammalian cells has been a subject of intense debate in recent years. Here we used a high-throughput chromosome conformation capture assay (capture Hi-C) to investigate clustering of DSBs induced at defined loci in the human genome. The results unambiguously demonstrated that DSBs cluster, but only when they are induced within transcriptionally active genes. Clustering of damaged genes occurs primarily during the G1 cell-cycle phase and coincides with delayed repair. Moreover, DSB clustering depends on the MRN complex as well as the Formin 2 (FMN2) nuclear actin organizer and the linker of nuclear and cytoplasmic skeleton (LINC) complex, thus suggesting that active mechanisms promote clustering. This work reveals that, when damaged, active genes, compared with the rest of the genome, exhibit a distinctive behavior, remaining largely unrepaired and clustered in G1, and being repaired via homologous recombination in postreplicative cells.

Abstract: The integrity of genome is a prerequisite for healthy life. Indeed, defects in DNA repair have been associated with several human diseases, including tissue-fibrosis, neurodegeneration and cancer. Despite decades of extensive research, the spatio-mechanical processes of double-strand break (DSB)-repair, especially the auxiliary factor(s) that can stimulate accurate and timely repair, have remained elusive. Here, we report an ATM-kinase dependent, unforeseen function of the nuclear isoform of the Receptor for Advanced Glycation End-products (nRAGE) in DSB-repair. RAGE is phosphorylated at Serine376 and Serine389 by the ATM kinase and is recruited to the site of DNA-DSBs via an early DNA damage response. nRAGE preferentially co-localized with the MRE11 nuclease subunit of the MRN complex and orchestrates its nucleolytic activity to the ATR kinase signaling. This promotes efficient RPA2S4-S8 and CHK1S345 phosphorylation and thereby prevents cellular senescence, IPF and carcinoma formation. Accordingly, loss of RAGE causatively linked to perpetual DSBs signaling, cellular senescence and fibrosis. Importantly, in a mouse model of idiopathic pulmonary fibrosis (RAGE-/-), reconstitution of RAGE efficiently restored DSB-repair and reversed pathological anomalies. Collectively, this study identifies nRAGE as a master regulator of DSB-repair, the absence of which orchestrates persistent DSB signaling to senescence, tissue-fibrosis and oncogenesis.

Abstract: Maintenance of telomere length is the most consistent attribute of cancer cells. Tightly connected to their capacity to overcome replicative mortality, it is achieved either by activation of telomerase or an Alternative mechanism of Lengthening of Telomeres (ALT). Disruption of either of these mechanisms has been shown to induce DNA damage signalling leading to senescence or apoptosis. Telomerase inhibitors are considered as potential anticancer drugs but are ineffective for ALT cancers (~15% of all cancers). Withaferin-A (Wi-A), a major constituent of the medicinal plant, Withania somnifera (Ashwagandha), has been shown to exert anti-tumour activity. However, its effect on either telomerase or ALT mechanisms has not been investigated. Here, by using isogenic cancer cells with/without telomerase, we found that Wi-A caused stronger cytotoxicity to ALT cells. It was associated with inhibition of ALT-associated promyelocytic leukemia nuclear bodies, an established marker of ALT. Comparative analyses of telomerase positive and ALT cells revealed that Wi-A caused stronger telomere dysfunction and upregulation of DNA damage response in ALT cells. Molecular computational and experimental analyses revealed that Wi-A led to Myc-Mad mediated transcriptional suppression of NBS-1, an MRN complex protein that is an essential component of the ALT mechanism. The results suggest that Wi-A could be a new candidate drug for ALT cancers.

Abstract: The MRN (MRE11–RAD50–NBS1) complex is essential for repair of DNA double-strand breaks and stalled replication forks. Mutations of the MRN complex subunit MRE11 cause the hereditary cancer-susceptibility disease ataxia-telangiectasia-like disorder (ATLD). Here we show that MRE11 directly interacts with PIH1D1, a subunit of heat-shock protein 90 cochaperone R2TP complex, which is required for the assembly of large protein complexes, such as RNA polymerase II, small nucleolar ribonucleoproteins and mammalian target of rapamycin complex 1. The MRE11-PIH1D1 interaction is dependent on casein kinase 2 (CK2) phosphorylation of two acidic sequences within the MRE11 C terminus containing serines 558/561 and 688/689. Conversely, the PIH1D1 phospho-binding domain PIH-N is required for association with MRE11 phosphorylated by CK2. Consistent with these findings, depletion of PIH1D1 resulted in MRE11 destabilization and affected DNA-damage repair processes dependent on MRE11. Additionally, mutations of serines 688/689, which abolish PIH1D1 binding, also resulted in decreased MRE11 stability. As depletion of R2TP frequently leads to instability of its substrates and as truncation mutation of MRE11 lacking serines 688/689 leads to decreased levels of the MRN complex both in ATLD patients and an ATLD mouse model, our results suggest that the MRN complex is a novel R2TP complex substrate and that their interaction is regulated by CK2 phosphorylation.

Abstract: Cdc7-Dbf4 kinase plays a key role in the initiation of DNA replication and contributes to the replication stress in cancer. The activity of human Cdc7-Dbf4 kinase remains active and acts as an effector of checkpoint under replication stress. However, the downstream targets of Cdc7-Dbf4 contributed to checkpoint regulation and replication stress-support function in cancer are not fully identified. In this work, we showed that aberrant Cdc7-Dbf4 induces DNA lesions that activate ATM/ATR-mediated checkpoint and homologous recombination (HR) DNA repair. Using a phosphoproteome approach, we identified HSP90-S164 as a target of Cdc7-Dbf4 in vitro and in vivo. The phosphorylation of HSP90-S164 by Cdc7-Dbf4 is required for the stability of HSP90-HCLK2-MRN complex and the function of ATM/ATR signaling cascade and HR DNA repair. In clinically, the phosphorylation of HSP90-S164 indeed is increased in oral cancer patients. Our results indicate that aberrant Cdc7-Dbf4 enhances replication stress tolerance by rewiring ATR/ATM mediated HR repair through HSP90-S164 phosphorylation and by promoting recovery from replication stress. We provide a new solution to a subtyping of cancer patients with dominant ATR/HSP90 expression by combining inhibitors of ATR-Chk1, HSP90, or Cdc7 in cancer combination therapy.

Abstract: Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5' strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5' strand DNA ∼10-20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5' strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP-MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.

Abstract: Background: The MRN complex consisting of MRE11A-RAD50-NBS1 proteins is involved in the repair of double-strand breaks, and mutations in genes coding for the MRN

Abstract: Solid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. Here we used a common MB cell line (D283-MED), to investigate the mechanisms of chemo and radio-resistance in MB, comparing to another MB cell line (MEB-Med8A) and to a widely used glioblastoma cell line (U87MG). In D283-MED and U87MG, chronic hypoxia (5 days), but not acute hypoxia (24 h) induced resistance to etoposide and X-ray irradiation. This acquired resistance upon chronic hypoxia was much less pronounced in MEB-Med8A cells. Using a transcriptomic approach in D283-MED cells, we found a large transcriptional remodelling upon long term hypoxia, in particular the expression of a number of genes involved in detection and repair of double strand breaks (DSB) was altered. The levels of Nibrin (NBN) and MRE11, members of the MRN complex (MRE11/Rad50/NBN) responsible for DSB recognition, were significantly down-regulated. This was associated with a reduction of Ataxia Telangiectasia Mutated (ATM) activation by etoposide, indicating a profound dampening of the DNA damage signalling in hypoxic conditions. As a consequence, p53 activation by etoposide was reduced, and cell survival enhanced. Whilst U87MG shared the same dampened p53 activity, upon chemotherapeutic drug treatment in chronic hypoxic conditions, these cells used a different mechanism, independent of the DNA damage pathway. Together our results demonstrate a new mechanism explaining hypoxia-induced resistance involving the alteration of the response to DSB, but also highlight the cell type to cell type diversity and the necessity to take into account the differing tumour genetic make-up when considering re-sensitisation therapeutic protocols.

Abstract: Pharmaceutical compositions comprising one or more of miR-494-3p, miR-99b-5p, and/or miR-21-3p and a pharmaceutically acceptable carrier as well as methods of using said compositions to treat cancer characterized by aberrant activity of an MRN complex.

Abstract: The Mre11-Rad50-Nbs1 (MRN) protein complex, CtIP/Ctp1/Sae2 and ATM/Tel1 kinase protect genome integrity through their functions in DNA double-strand break (DSB) repair, checkpoint signaling, and telomere maintenance. Nbs1 has a conserved C-terminal motif that binds ATM, but the full extent of ATM interactions with MRN are unknown. Here, we show that Tel1 overexpression in Schizosaccharomyces pombe restores Tel1 activity at DSBs and telomeres in the absence of Nbs1. This activity requires Mre11, indicating that Tel1 overexpression drives low affinity binding to the Mre11-Rad50 subcomplex. Mre11-Rad50 binds DSBs in nbs1Δ cells, and fusing the Tel1-binding motif of Nbs1 to Mre11 fully restores Tel1 signaling in these cells. Tel1 overexpression does not restore Tel1 signaling in cells carrying the rad50-I1192W mutation, which impairs the ability of Mre11-Rad50 to form the ATP-bound closed conformation. From these findings, we propose that Tel1 activation at DNA ends proceeds by a sequential mechanism initiated by high affinity binding to Nbs1 which recruits Tel1, followed by a low affinity interaction with Mre11-Rad50 in the closed conformation to activate Tel1.

Abstract: IN KOREAN 121

Abstract: C-terminal binding protein (CtBP) is responsible for regulating the pathogenesis of a lot of cancer types. However, whether CtBP1/2 is involved in regulating the growth and development of human glioma is still obscure. In the present study presented here, our results firstly reveal that CtBP1/2 deficiency, induced by siRNA interference, disrupts the functional integrity of the MRN complex that is responsible for DNA repair in human glioma cells. The dysfunction of the MRN complex further contributes to the up-regulation of ATM and Rad3-related kinase (ATR) and Chk1 signaling pathway, which inhibits cell cycle progression mediated by CDK2, preparing for the initiation of DNA repair. Under the condition of hypoxia, hypoxia-inducible factor-1α (HIF-1α) can be directly regulated by CDK2 on protein level, playing coordinately regulatory role in the carcinogenesis of human glioma cells. Overall, our findings reveal that CtBP1/2 is essential to promote to human glioma cell growth through maintaining the DNA stability regulated by the MRN/ATR/Chk1/CDK2/HIF-1α signaling pathway.

Abstract: MicroRNAs contribute to biological robustness by buffering cellular processes from external perturbations. Here we report an unexpected link between DNA damage response and angiogenic signaling that is buffered by two distinct microRNAs. We demonstrate that genotoxic stress-induced miR-494 and miR-99b inhibit the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Functionally, miR-494 and miR-99b affect telomerase activity, activate p21 and Rb pathways and diminish angiogenic sprouting in vitro and in vivo. Genetic and pharmacological disruption of VEGFR-2 signaling and the MRN complex reveal a surprising co-dependency of these pathways in regulating endothelial senescence and proliferation. miR-99b diminishes VEGF signaling, transcriptional responses and proliferation. Disruption of the MRN complex induces CD44, a known driver of senescence and regulator of VEGF signaling. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence.

Abstract: B-Myb, a highly conserved member of the Myb transcription factor family, is expressed ubiquitously in proliferating cells and controls the cell cycle dependent transcription of G2/M-phase genes. Deregulation of B-Myb has been implicated in oncogenesis and loss of genomic stability. We have identified B-Myb as a novel interaction partner of the Mre11-Rad50-Nbs1 (MRN) complex, a key player in the repair of DNA double strand breaks. We show that B-Myb directly interacts with the Nbs1 subunit of the MRN complex and is recruited transiently to DNA-damage sites. In response to DNA-damage B-Myb is phosphorylated by protein kinase GSK3β and released from the MRN complex. A B-Myb mutant that cannot be phosphorylated by GSK3β disturbs the regulation of pro-mitotic B-Myb target genes and leads to inappropriate mitotic entry in response to DNA-damage. Overall, our work suggests a novel function of B-Myb in the cellular DNA-damage signalling.

Abstract: Nijmegen breakage syndrome (NBS), a condition similar to Ataxia-Telangiectasia (A-T), is a radiation-hypersensitive genetic disorder showing chromosomal instability, radio-resistant DNA synthesis, immunodeficiency, and predisposition to malignances. The product of the responsible gene, NBS1, forms a complex with MRE11 and RAD50 (MRN complex). The MRN complex is necessary for the DNA damage-induced activation of ATM. However, the regulation of MRN complex formation is still unclear. Here, we investigated the regulatory mechanisms of MRN complex formation. We used an immunoprecipitation assay to determine whether levels of the MRN complex were increased by radiation-induced DNA damage and found that the levels of these proteins and their mRNAs did not increase. ATM-dependent phosphorylation of NBS1 contributed to the DNA damage-induced MRN complex formation. However, pre-treatment of cells with an ATM-specific inhibitor did not affect homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. G0 phase cells, decreasing NBS1 and HR activity but not NHEJ, gained HR-related chromatin association of RAD51 by overexpression of NBS1, suggesting that the amount of NBS1 may be important for repressing accidental activation of HR. These evidences suggest that NBS1 is regulated by two kind of mechanisms: complex formation dependent on ATM, and protein degradation mediated by an unknown MG132-resistant pathway. Such regulation of NBS1 may contribute to cellular responses to double-strand breaks.

Abstract: While the Mre11-Rad50-Nbs1 (MRN) complex has known roles in repair processes like homologous recombination and microhomology-mediated end-joining, its role in nonhomologous end-joining (NHEJ) is unclear as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mammals have different requirements for repairing cut DNA ends. Most double-strand breaks (DSBs) require nucleolytic processing prior to DNA ligation. Therefore, we studied repair using the Hermes transposon, whose excision leaves a DSB capped by hairpin ends similar to structures generated by palindromes and trinucleotide repeats. We generated single Hermes insertions using a novel S. pombe transient transfection system, and used Hermes excision to show a requirement for MRN in the NHEJ of nonligatable ends. NHEJ repair was indicated by the >1000-fold decrease in excision in cells lacking Ku or DNA ligase 4. Most repaired excision sites had 1000-fold in cells lacking each MRN subunit, and loss of MRN-associated Ctp1 caused a 30-fold reduction. An Mre11 dimer is thought to hold DNA ends together for repair, and Mre11 dimerization domain mutations reduced repair 300-fold. In contrast, a mre11 mutant defective in endonucleolytic activity, the same mutant lacking Ctp1, or the triple mutant also lacking the putative hairpin nuclease Pso2 showed wild-type levels of repair. Thus, MRN may act to recruit the hairpin opening activity that allows subsequent repair.

Abstract: The mitotic kinase Plk1 contributes to the DNA damage response (DDR) by targeting multiple factors downstream of the core responder kinase ATM/ATR In this study, we show that Plk1 also phosphorylates key factors upstream of ATM/ATR and regulates their DDR-related functions Plk1 phosphorylated Mre11, a component of the Mre11/Rad50/Nbs1 (MRN) complex, at serine 649 (S649) during DDR Phosphorylation of Mre11-S649 by Plk1 primed subsequent CK2-mediated phosphorylation at Mre11-serine 688 (S688) Phosphorylation of Mre11 at S649/S688 inhibited loading of the MRN complex to damaged DNA, leading to both premature DNA damage checkpoint termination and inhibition of DNA repair Tumors expressing phosphomimetic Mre11 were more sensitive to the PARP inhibitor olaparib, compared to those expressing unphosphorylatable Mre11, suggesting that patients with elevated Plk1 expression might benefit from olaparib treatment

Abstract: Oncolytic viruses and radiotherapy represent two diverse areas of cancer therapy, utilising quite different treatment modalities and with non-overlapping cytotoxicity profile. It is therefore an intriguing possibiltiy to consider that oncolytic ('cancer-killing') viruses may act as cancer-selective radiosensitisers, enhancing the therapeutic consequences of radiation treatment on tumours whilst exerting minimal effects on normal tissue. There is a solid mechanistic basis for this potential synergy, with many viruses having developed strategies to inhibit cellular DNA repair pathways in order to protect themselves, during genome replication, from unwanted interference by cell processes that are normally triggered by DNA damage. Exploiting these abilities to inhibit cellular DNA repair following damage by therapeutic irradiation may well augment the anticancer potency of the approach. In this review we focus on oncolytic adenovirus, the most widely developed and best understood oncolytic virus, and explore its various mechanisms for modulating cellular DNA repair pathways. The most obvious effects of the various adenovirus serotypes are to interfere with activity of the MRN complex, temporally one of the first sensors of double stranded DNA damage, and inhibition of DNA ligase IV, a central repair enzyme for healing double stranded breaks by non-homologous end-joining (NHEJ). There have been several preclinical and clinical studies of this approach and we assess the current state of progress. In addition, oncolytic viruses provide the option to promote a localised proinflammatory response, both by mediating immunogenic death of cancer cells by oncosis and also by encoding and expressing proinflammatory biologics within the tumour microenvironment. Both of these approaches provide exciting potential to augment the known immunological consequences of radiotherapy, aiming to develop systems capable of creating a systemic anticancer immune response following localised tumour treatment.

Abstract: Double-strand breaks (DSBs) in DNA are recognized by the Ku70/80 heterodimer and the MRE11-RAD50-NBS1 (MRN) complex and result in activation of the DNA-PK and ATM kinases, which play key roles in regulating the cellular DNA damage response (DDR). DNA tumor viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) are known to interact extensively with the DDR during the course of their replicative cycles. Here we show that during lytic amplification of KSHV DNA, the Ku70/80 heterodimer and the MRN complex consistently colocalize with viral genomes in replication compartments (RCs), whereas other DSB repair proteins form foci outside RCs. Depletion of MRE11 and abrogation of its exonuclease activity negatively impact viral replication, while in contrast, knockdown of Ku80 and inhibition of the DNA-PK enzyme, which are involved in nonhomologous end joining (NHEJ) repair, enhance amplification of viral DNA. Although the recruitment of DSB-sensing proteins to KSHV RCs is a consistent occurrence across multiple cell types, activation of the ATM-CHK2 pathway during viral replication is a cell line-specific event, indicating that recognition of viral DNA by the DDR does not necessarily result in activation of downstream signaling pathways. We have also observed that newly replicated viral DNA is not associated with cellular histones. Since the presence and modification of these DNA-packaging proteins provide a scaffold for docking of multiple DNA repair factors, the absence of histone deposition may allow the virus to evade localization of DSB repair proteins that would otherwise have a detrimental effect on viral replication.IMPORTANCE Tumor viruses are known to interact with machinery responsible for detection and repair of double-strand breaks (DSBs) in DNA, although detail concerning how Kaposi's sarcoma-associated herpesvirus (KSHV) modulates these cellular pathways during its lytic replication phase was previously lacking. By undertaking a comprehensive assessment of the localization of DSB repair proteins during KSHV replication, we have determined that a DNA damage response (DDR) is directed to viral genomes but is distinct from the response to cellular DNA damage. We also demonstrate that although recruitment of the MRE11-RAD50-NBS1 (MRN) DSB-sensing complex to viral genomes and activation of the ATM kinase can promote KSHV replication, proteins involved in nonhomologous end joining (NHEJ) repair restrict amplification of viral DNA. Overall, this study extends our understanding of the virus-host interactions that occur during lytic replication of KSHV and provides a deeper insight into how the DDR is manipulated during viral infection.

Abstract: BRCA1/2-deficient ovarian carcinomas are recognized as target for Poly (ADP-ribose) polymerase (PARP) inhibitors. BRCA1 and BRCA2 proteins are involved in homologous recombination repair of double-strand DNA breaks. The relevance of other homologous recombination repair proteins, e.g. MRE11, RAD50, NBS1 (MRN complex) in ovarian carcinomas is unclear. The objective of this study was to investigate the prevalence of lack of MRE11, RAD50, NBS1 protein detection in epithelial ovarian cancer (EOC). A tissue microarray (TMA) with 134 EOC was immunohistochemically evaluated for MRE11, RAD50 and NBS1. Data was analysed for associations with clinicopathological parameters, histological subtype, patient overall survival and mismatch repair (MMR) protein status. Sensitivity towards the PARP inhibitor BMN673 was tested in two ovarian cancer cell lines (TOV-21 and OVTOKO) using colony formation assays. Lack of MRN complex protein detection was seen in 41% (55/134) of EOC and was more frequent in low-grade (57.6%; 19/33) than in high-grade EOC (18.8%; 36/101; n = 134; p = 0.04). There was an association with the ovarian carcinoma subtype (60.3%; 35/58 lack of detection in type I versus 26.3%; 20/76 in type II; n = 134; p < 0.001) as well as undetectable DNA mismatch repair proteins MLH1 and MSH2 (89.3%; 25/28; n = 131; p < 0.001). MRE11 knockdown led to moderately increased sensitivity towards the PARP inhibitor BMN673 in one ovarian carcinoma cell line in vitro. Frequent lack of MRE11, RAD50, NBS1 protein detection in type I human ovarian carcinomas is observed in EOC and our data suggests further investigation regarding sensitivity to PARP-inhibition in tumours lacking MRE11 expression.

Abstract: Kaposi Sarcoma Herpesvirus (KSHV), a γ2-herpesvirus and class 1 carcinogen, is responsible for at least three human malignancies: Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman's Disease (MCD). Its major nuclear latency protein, LANA, is indispensable for the maintenance and replication of latent viral DNA in infected cells. Although LANA is mainly a nuclear protein, cytoplasmic isoforms of LANA exist and can act as antagonists of the cytoplasmic DNA sensor, cGAS. Here, we show that cytosolic LANA also recruits members of the MRN (Mre11-Rad50-NBS1) repair complex in the cytosol and thereby inhibits their recently reported role in the sensing of cytoplasmic DNA and activation of the NF-κB pathway. Inhibition of NF-κB activation by cytoplasmic LANA is accompanied by increased lytic replication in KSHV-infected cells, suggesting that MRN-dependent NF-κB activation contributes to KSHV latency. Cytoplasmic LANA may therefore support the activation of KSHV lytic replication in part by counteracting the activation of NF-κB in response to cytoplasmic DNA. This would complement the recently described role of cytoplasmic LANA in blocking an interferon response triggered by cGAS and thereby promoting lytic reactivation. Our findings highlight a second point at which cytoplasmic LANA interferes with the innate immune response, as well as the importance of the recently discovered role of cytoplasmic MRN complex members as innate sensors of cytoplasmic DNA for the control of KSHV replication.

Abstract: Cancer cells require telomere maintenance to enable uncontrolled growth. Most often telomerase is activated, although a subset of human cancers are telomerase-negative and depend on recombination-based mechanisms known as ALT (Alternative Lengthening of Telomeres). ALT depends on proteins that are essential for homologous recombination, including BLM and the MRN complex, to extend telomeres. This study surveyed the requirement for requisite homologous recombination proteins, yet to be studied in human ALT cell lines, by protein depletion using RNA interference. Effects on ALT were evaluated by measuring C-circle abundance, a marker of ALT. Surprisingly, several proteins essential for homologous recombination, BARD1, BRCA2, and WRN, were dispensable for C-circle production, while PALB2 had varying effects on C-circles among ALT cell lines. Depletion of homologous recombination proteins BRCA1 and BLM, which have been previously studied in ALT, decreased C-circles in all ALT cell lines. Depletion of the non-homologous end joining proteins 53BP1 and LIG4 had no effect on C-circles in any ALT cell line. Proteins such as chromatin modifiers that recruit double-strand break proteins, RNF8 and RNF168, and other proteins loosely grouped into excision DNA repair processes, XPA, MSH2, and MPG, reduced C-circles in some ALT cell lines. MSH2 depletion also reduced recombination at telomeres as measured by intertelomeric exchanges. Collectively, the requirement for DNA repair proteins varied between the ALT cell lines compared. In sum, our study suggests that ALT proceeds by multiple mechanisms that differ between cell lines and that some of these depend on DNA repair proteins not associated with homologous recombination pathways.