Chromosomal DNA Double-strand Breaks Research Articles

Distinct functions of PAXX and MRI during chromosomal end joining.

A key step of Canonical Nonhomologous End Joining (C-NHEJ) is synapsis of DNA double strand break (DSB) ends for ligation. The DNA-PKcs dimer mediates synapsis in a long-range complex with DSB ends remaining apart, whereas the XLF homodimer can mediate synapsis in both long-range and short-range complexes. Recent structural studies found the PAXX homodimer may also facilitate synapsis in long-range complexes with DNA-PKcs via its interactions with Ku70. Thus, we examined the influence of PAXX in C-NHEJ of chromosomal DSBs, which we compared to another Ku-binding factor, MRI. Using EJ of blunt DSBs with Cas9 reporters as a readout for C-NHEJ, we found that PAXX and/or MRI are dispensable. However, when combined with disruption of DNA-PKcs, particularly with DNA-PKcs kinase inhibition, PAXX becomes important for blunt DSB EJ. In contrast, while DNA-PKcs is also important to suppress short deletion mutations with microhomology, this effect is not magnified with PAXX loss. MRI loss had no effect combined with DNA-PKcs disruption, but becomes important for blunt DSB EJ when combined with disruption of XLF, as is PAXX. Finally, XLF loss causes an increase in larger deletions compared to DNA-PKcs inhibition, which is magnified with combined loss of MRI. Altogether, we suggest that PAXX promotes DSB end synapsis during C-NHEJ in a manner that is partially redundant with DNA-PKcs and XLF, whereas MRI appears to be mainly important in the context of XLF disruption.

Arsenic affects homologous recombination and single-strand annealing but not end-joining pathways during DNA double-strand break repair.

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double-strand breaks (DSBs) have been implicated in arsenic-induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2 O3 ) on four different DSB repair pathways using the human pre-B cell line Nalm-6. Random integration analysis showed that As2 O3 does not negatively affect non-homologous end joining or polymerase theta-mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2 O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single-strand annealing. Consistent with this finding, As2 O3 decreased gene-targeting efficiency, owing to a significant reduction in the frequency of HR-mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one-ended DSBs requiring HR for precise repair. Intriguingly, we found that As2 O3 significantly enhances sensitivity to those anticancer agents in HR-proficient cells. Our results suggest that arsenic-induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic-associated carcinogenesis and therapeutic options.

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11β and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Protein Arginine Methyltransferase 5 (PRMT5) Mutations in Cancer Cells.

Arginine methylation is a form of posttranslational modification that regulates many cellular functions such as development, DNA damage repair, inflammatory response, splicing, and signal transduction, among others. Protein arginine methyltransferase 5 (PRMT5) is one of nine identified methyltransferases, and it can methylate both histone and non-histone targets. It has pleiotropic functions, including recruitment of repair machinery to a chromosomal DNA double strand break (DSB) and coordinating the interplay between repair and checkpoint activation. Thus, PRMT5 has been actively studied as a cancer treatment target, and small molecule inhibitors of its enzymatic activity have already been developed. In this report, we analyzed all reported PRMT5 mutations appearing in cancer cells using data from the Catalogue of Somatic Mutations in Cancers (COSMIC). Our goal is to classify mutations as either drivers or passengers to understand which ones are likely to promote cellular transformation. Using gold standard artificial intelligence algorithms, we uncovered several key driver mutations in the active site of the enzyme (D306H, L315P, and N318K). In silico protein modeling shows that these mutations may affect the affinity of PRMT5 for S-adenosylmethionine (SAM), which is required as a methyl donor. Electrostatic analysis of the enzyme active site shows that one of these mutations creates a tunnel in the vicinity of the SAM binding site, which may allow interfering molecules to enter the enzyme active site and decrease its activity. We also identified several non-coding mutations that appear to affect PRMT5 splicing. Our analyses provide insights into the role of PRMT5 mutations in cancer cells. Additionally, since PRMT5 single molecule inhibitors have already been developed, this work may uncover future directions in how mutations can affect targeted inhibition.

To indel or not to indel: Factors influencing mutagenesis during chromosomal break end joining

Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many approaches to gene editing. Ligation of the DSBs by end joining (EJ) pathways can restore the broken chromosome, but the repair junctions can have insertion/deletion (indel) mutations. The indel patterns resulting from DSB EJ are likely defined by the initial structure of the DNA ends, how the ends are processed and synapsed prior to ligation, and the factors that mediate the ligation step. In this review, we describe key factors that influence these steps of DSB EJ in mammalian cells, which is significant both for understanding mutagenesis resulting from clastogenic cancer therapeutics, and for developing approaches to manipulating gene editing outcomes.

The importance of DNAPKcs for blunt DNA end joining is magnified when XLF is weakened

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF for such EJ. However, weakening XLF via disrupting interaction interfaces causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

DNA Double-Strand Breaks as Pathogenic Lesions in Neurological Disorders.

The damage and repair of DNA is a continuous process required to maintain genomic integrity. DNA double-strand breaks (DSBs) are the most lethal type of DNA damage and require timely repair by dedicated machinery. DSB repair is uniquely important to nondividing, post-mitotic cells of the central nervous system (CNS). These long-lived cells must rely on the intact genome for a lifetime while maintaining high metabolic activity. When these mechanisms fail, the loss of certain neuronal populations upset delicate neural networks required for higher cognition and disrupt vital motor functions. Mammalian cells engage with several different strategies to recognize and repair chromosomal DSBs based on the cellular context and cell cycle phase, including homologous recombination (HR)/homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the classic non-homologous end-joining (NHEJ). In addition to these repair pathways, a growing body of evidence has emphasized the importance of DNA damage response (DDR) signaling, and the involvement of heterogeneous nuclear ribonucleoprotein (hnRNP) family proteins in the repair of neuronal DSBs, many of which are linked to age-associated neurological disorders. In this review, we describe contemporary research characterizing the mechanistic roles of these non-canonical proteins in neuronal DSB repair, as well as their contributions to the etiopathogenesis of selected common neurological diseases.

Elp1 facilitates RAD51-mediated homologous recombination repair via translational regulation

BackgroundRAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear.MethodsTo investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS) proteomics approach.ResultsHere, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion.ConclusionsOur study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

Bloom Helicase Along with Recombinase Rad51 Repairs the Mitochondrial Genome of the Malaria Parasite.

ABSTRACTThe homologous recombination (HR) pathway has been implicated as the predominant mechanism for the repair of chromosomal DNA double-strand breaks (DSBs) of the malarial parasite. Although the extrachromosomal mitochondrial genome of this parasite experiences a greater number of DSBs due to its close proximity to the electron transport chain, nothing is known about the proteins involved in the repair of the mitochondrial genome. We investigated the involvement of nucleus-encoded HR proteins in the repair of the mitochondrial genome, as this genome does not code for any DNA repair proteins. Here, we provide evidence that the nucleus-encoded “recombinosome” of the parasite is also involved in mitochondrial genome repair. First, two crucial HR proteins, namely, Plasmodium falciparum Rad51 (PfRad51) and P. falciparum Bloom helicase (PfBlm) are located in the mitochondria. They are recruited to the mitochondrial genome at the schizont stage, a stage that is prone to DSBs due to exposure to various endogenous and physiologic DNA-damaging agents. Second, the recruitment of these two proteins to the damaged mitochondrial genome coincides with the DNA repair kinetics. Moreover, both the proteins exit the mitochondrial DNA (mtDNA) once the genome is repaired. Most importantly, the specific chemical inhibitors of PfRad51 and PfBlm block the repair of UV-induced DSBs of the mitochondrial genome. Additionally, overexpression of these two proteins resulted in a kinetically faster repair. Given the essentiality of the mitochondrial genome, blocking its repair by inhibiting the HR pathway could offer a novel strategy for curbing malaria.IMPORTANCE The impact of malaria on global public health and the world economy continues to surge despite decades of vaccine research and drug development efforts. An alarming rise in resistance toward all the commercially available antimalarial drugs and the lack of an effective malaria vaccine brings us to the urge to identify novel intervention strategies for curbing malaria. Here, we uncover the molecular mechanism behind the repair of the most deleterious form of DNA lesions on the parasitic mitochondrial genome. Given that the single-copy mitochondrion is an indispensable organelle of the malaria parasite, we propose that targeting the mitochondrial DNA repair pathways should be exploited as a potential malaria control strategy. The establishment of the parasitic homologous recombination machinery as the predominant repair mechanism of the mitochondrial DNA double-strand breaks underscores the importance of this pathway as a novel druggable target.

Effects of combined exposure to modeled radiation and gravitation factors of the interplanetary flight: Monkeys' cognitive functions and the content of monoamines and their metabolites; cytogenetic changes in peripheral blood lymphocytes

In a study on primates (Macaca mulatta), neurobiological and radiobiological effects have been studied of the synchronous combined action of 7-day antiorthostatic hypokinesia and exposure of the monkeys' head first to γ-rays during 24 h and then to accelerated 12C ions. The neurobiological effects were evaluated by the cognitive functions which model the basic elements of operator activity and the concentration of monoamines and their metabolites in peripheral blood. The radiobiological effects were evaluated by the chromosomal aberration and DNA double-strand break (DSB) yield in peripheral blood lymphocytes. The results of the cognitive function research show that the typological features of the animals' higher nervous activity are the prevailing factor that determines changes in these functions. The monkey of the strong balanced type effectively retained its cognitive functions after the exposures, while in the weak unbalanced type animals these functions were impaired. These changes went along with a decrease in the concentration of monoamines and their metabolites and an increase in the DNA DSB and chromosomal aberration yield in lymphocytes.

Ku70 suppresses alternative end joining in G1-arrested progenitor B cells

Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition

Persistent R-loops (RNA–DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2’s association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.

Surveillance of cohesin-supported chromosome structure controls meiotic progression

Chromosome movements and programmed DNA double-strand breaks (DSBs) promote homologue pairing and initiate recombination at meiosis onset. Meiotic progression involves checkpoint-controlled termination of these events when all homologue pairs achieve synapsis and form crossover precursors. Exploiting the temporo-spatial organisation of the C. elegans germline and time-resolved methods of protein removal, we show that surveillance of the synaptonemal complex (SC) controls meiotic progression. In nuclei with fully synapsed homologues and crossover precursors, removing different meiosis-specific cohesin complexes, which are individually required for SC stability, or a SC central region component causes functional redeployment of the chromosome movement and DSB machinery, triggering whole-nucleus reorganisation. This apparent reversal of the meiotic programme requires CHK-2 kinase reactivation via signalling from chromosome axes containing HORMA proteins, but occurs in the absence of transcriptional changes. Our results uncover an unexpected plasticity of the meiotic programme and show how chromosome signalling orchestrates nuclear organisation and meiotic progression.

Some mutations in the xeroderma pigmentosum D gene may lead to moderate but significant radiosensitivity associated with a delayed radiation-induced ATM nuclear localization

Purpose: Xeroderma Pigmentosum (XP) is a rare, recessive genetic disease associated with photosensitivity, skin cancer proneness, neurological abnormalities and impaired nucleotide excision repair of the UV-induced DNA damage. Less frequently, XP can be associated with sensitivity to ionizing radiation (IR). Here, a complete radiobiological characterization was performed on a panel of fibroblasts derived from XP-group D patients (XPD).Materials and methods: Cellular radiosensitivity and the functionality of the recognition and repair of chromosome breaks and DNA double-strand breaks (DSB) was evaluated by different techniques including clonogenic cell survival, micronuclei, premature chromosome condensation, pulsed-field gel electrophoresis, chromatin decondensation and immunofluorescence assays. Quantitative correlations between each endpoint were analyzed systematically.Results: Among the seven fibroblast cell lines tested, those derived from three non-relative patients holding the p.[Arg683Trp];[Arg616Pro] XPD mutations showed significant cellular radiosensitivity, high yield of residual micronuclei, incomplete DSB recognition, DSB and chromosome repair defects, impaired ATM, MRE11 relocalization, significant chromatin decondensation. Interestingly, XPD transduction and treatment with statins and bisphosphonates known to accelerate the radiation-induced ATM nucleoshuttling led to significant complementation of these impairments.Conclusions: Our findings suggest that some subsets of XPD patients may be at risk of radiosensitivity reactions and treatment with statins and bisphosphonates may be an interesting approach of radioprotection countermeasure. Different mechanistic models were discussed to better understand the potential specificity of the p.[Arg683Trp];[Arg616Pro] XPD mutations.

The phage T4 DNA ligase mediates bacterial chromosome DSBs repair as single component non-homologous end joining

DNA double-strand breaks (DSBs) are one of the most lethal forms of DNA damage that is not efficiently repaired in prokaryotes. Certain microorganisms can handle chromosomal DSBs using the error-prone non-homologous end joining (NHEJ) system and ultimately cause genome mutagenesis. Here, we demonstrated that Enterobacteria phage T4 DNA ligase alone is capable of mediating in vivo chromosome DSBs repair in Escherichia coli. The ligation efficiency of DSBs with T4 DNA ligase is one order of magnitude higher than the NHEJ system from Mycobacterium tuberculosis. This process introduces chromosome DNA excision with different sizes, which can be manipulated by regulating the activity of host-exonuclease RecBCD. The DNA deletion length reduced either by inactivating recB or expressing the RecBCD inhibitor Gam protein from λ phage. Furthermore, we also found single nucleotide substitutions at the DNA junction, suggesting that T4 DNA ligase, as a single component non-homologous end joining system, has great potential in genome mutagenesis, genome reduction and genome editing.

Pharmacological boost of DNA damage response and repair by enhanced biogenesis of DNA damage response RNAs

A novel class of small non-coding RNAs called DNA damage response RNAs (DDRNAs) generated at DNA double-strand breaks (DSBs) in a DROSHA- and DICER-dependent manner has been shown to regulate the DNA damage response (DDR). Similar molecules were also reported to guide DNA repair. Here, we show that DDR activation and DNA repair can be pharmacologically boosted by acting on such non-coding RNAs. Cells treated with enoxacin, a compound previously demonstrated to augment DICER activity, show stronger DDR signalling and faster DNA repair upon exposure to ionizing radiations compared to vehicle-only treated cells. Enoxacin stimulates DDRNA production at chromosomal DSBs and at dysfunctional telomeres, which in turn promotes 53BP1 accumulation at damaged sites, therefore in a miRNA-independent manner. Increased 53BP1 occupancy at DNA lesions induced by enoxacin ultimately suppresses homologous recombination, channelling DNA repair towards faster and more accurate non-homologous end-joining, including in post-mitotic primary neurons. Notably, augmented DNA repair stimulated by enoxacin increases the survival also of cancer cells treated with chemotherapeutic agents.

DSB structure impacts DNA recombination leading to class switching and chromosomal translocations in human B cells.

Class switch recombination (CSR) requires activation-induced cytidine deaminase (AID) to trigger DNA double strand breaks (DSBs) at the immunoglobulin heavy chain (IGH) in B cells. Joining of AID-dependent DSBs within IGH facilitate CSR and effective humoral immunity, but ligation to DSBs in non-IGH chromosomes leads to chromosomal translocations. Thus, the mechanism by which AID-dependent DSBs are repaired requires careful examination. The random activity of AID in IGH leads to a spectrum of DSB structures. In this report, we investigated how DSB structure impacts end-joining leading to CSR and chromosomal translocations in human B cells, for which models of CSR are inefficient and not readily available. Using CRISPR/Cas9 to model AID-dependent DSBs in IGH and non-IGH genes, we found that DSBs with 5’ and 3’ overhangs led to increased processing during end-joining compared to blunt DSBs. We observed that 5’ overhangs were removed and 3’ overhangs were filled in at recombination junctions, suggesting that different subsets of enzymes are required for repair based on DSB polarity. Surprisingly, while Cas9-mediated switching preferentially utilized NHEJ regardless of DSB structure, A-EJ strongly preferred repairing blunt DSBs leading to translocations in the absence of NHEJ. We found that DSB polarity influenced frequency of Cas9-mediated switching and translocations more than overhang length. Lastly, recombination junctions from staggered DSBs exhibited templated insertions, suggesting iterative resection and filling in during repair. Our results demonstrate that DSB structure biases repair towards NHEJ or A-EJ to complete recombination leading to CSR and translocations, thus helping to elucidate the mechanism of genome rearrangements in human B cells.

XRN2 Depletion is Synthetic Lethal with PARP1 Inhibition

Persistent R‐loops (RNA‐DNA hybrids with a displaced single‐stranded DNA) create DNA damage and lead to genomic instability. The 5’‐3’‐exoribonuclease 2 (XRN2) degrades RNA to resolve R‐loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. XRN2 function in DSB repair is not fully understood. We hypothesize that XRN2 plays a critical role in multiple DNA repair pathways. Here, using tandem affinity purification‐mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70‐Ku80, DNA‐PKcs, PARP1, MCM2‐7, PCNA, RPA1), and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non‐homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2‐deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2‐deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. The XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 loss and PARP1 inhibition.Support or Funding InformationThis work was largely supported by the National Institutes of Health/National Cancer Institute (NCI/NIH) [R01 CA139217] to D.A.B, a minority supplement [R01 CA139217‐05S1] and the Cancer Biology Training Grant T32CA124334‐06 to E.A.M. (PI: Dr. Jerry Shay), Simmons Comprehensive Cancer Center, UT Southwestern. This work was also supported by grant 5P30CA142543, NCI/NIH to the UT Southwestern Proteomics Core. The UT Southwestern Proteomics Core was also supported, in part, by a grant from the Cancer Prevention and Research Institute of Texas [CPRIT, RP1206130] to Dr. Hamid Mirzaei. Funding for open access charges was from NCI/NIH CA139217 to D.A.B. Research reported in this study was also supported by the UT Southwestern CCSG grant 5P30CA142543 from the NCI/NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4365: Targeting the RhoA guanine nucleotide-exchange factor Syx for the treatment of glioblastoma

Abstract The RhoA GTPase is thought to play a crucial role in cancer development. Its activation is tightly controlled by numerous guanine nucleotide-exchange factors (GEFs). We previously showed that Syx, a RhoA-GEF, promotes junctional integrity in endothelial cells and governs cell polarity and directed migration in glioblastoma (GBM) cells. GBM is a grade IV glioma with a median survival of approximately 14 months. Importantly, the gene locus of Syx (Plekhg5) is located within chromosome 1p36, a region deleted in oligodendroglioma (a type II/III glioma) and associated with better prognosis, suggesting a role of Syx in GBM aggressiveness. Here we show that Syx is a potential target for the treatment of GBM. Knockdown of Syx resulted in altered expression of cell cycle regulators, including cyclins A, B, and E, cyclin-dependent kinase inhibitors (CDIs), namely p21 and p27, and mitosis modulators CDC20 and survivin. These defects resulted in G2/M arrest and dysfunction of cell division, leading to improper chromosome segregation and DNA double-strand breaks. However, DNA damage response could not be activated effectively for DNA repair, leading to cellular apoptosis. Consistent with these findings, we used an orthotopic patient-derived xenograft GBM model to show that Syx silencing results in decreased tumor growth accompanied by increased animal survival. Agents that cause DNA damage and/or disruption of DNA repair pathways are often used for cancer therapy. We thus tested the possibility of targeting Syx for GBM treatment, and showed that Syx knockdown in combination with temozolomide, a first-line drug for GBM, synergistically inhibit cell growth. In summary, this is the first study showing the role of Syx in GBM cell growth and that targeting this signaling pathway can be beneficial for GBM therapy. Citation Format: Wan-Hsin Lin, Ryan Feathers, Laura Lewis-Tuffin, Panos Anastasiadis. Targeting the RhoA guanine nucleotide-exchange factor Syx for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4365.