Repair Of Double-strand DNA Breaks Research Articles

Integrated Molecular Analysis Reveals 2 Distinct Subtypes of Pure Seminoma of the Testis.

Testicular germ cell tumors (TGCT) are the most common solid malignancy in adolescent and young men, with a rising incidence over the past 20 years. Overall, TGCTs are second in terms of the average life years lost per person dying of cancer, and clinical therapeutics without adverse long-term side effects are lacking. Platinum-based regimens for TGCTs have heterogeneous outcomes even within the same histotype that frequently leads to under- and over-treatment. Understanding of molecular differences that lead to diverse outcomes of TGCT patients may improve current treatment approaches. Seminoma is the most common subtype of TGCTs, which can either be pure or present in combination with other histotypes. Here we conducted a computational study of 64 pure seminoma samples from The Cancer Genome Atlas, applied consensus clustering approach to their transcriptomic data and revealed 2 clinically relevant seminoma subtypes: seminoma subtype 1 and 2. Our analysis identified significant differences in pluripotency stage, activity of double stranded DNA breaks repair mechanisms, rates of loss of heterozygosity, and expression of lncRNA responsible for cisplatin resistance between the subtypes. Seminoma subtype 1 is characterized by higher pluripotency state, while subtype 2 showed attributes of reprograming into non-seminomatous TGCT. The seminoma subtypes we identified may provide a molecular underpinning for variable responses to chemotherapy and radiation. Translating our findings into clinical care may help improve risk stratification of seminoma, decrease overtreatment rates, and increase long-term quality of life for TGCT survivors.

A new partial loss of function allele of rad-54.L.

RAD-54.L is required for the repair of meiotic double-strand DNA breaks (DSBs), playing an essential role in promoting removal of recombinase RAD-51 and normal completion of meiotic recombination. Failure to complete meiotic DSB repair leads to 100% lethality of embryos produced by rad-54.L null mutant mothers. Here we report a new partial loss of function allele, rad-54.L(me139) , that may prove useful for investigating meiotic mechanisms by providing a sensitized genetic background that reduces but does not eliminate the essential functions of RAD-54.L.

Single-strand annealing: Molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing.

Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated. Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing. The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.

Putative role of uncoupling proteins in mitochondria-nucleus communications and DNA damage response

Mitochondria-nucleus communications and DNA damage response (DDR) play roles in cellular stress and closely associate with a range of diseases. Mitochondrial uncoupling proteins (UCPs) are capable of uncoupling mitochondrial oxidative phosphorylation and protecting against oxidative stress. However, the potential role of UCPs in DDR and DDR-related mitochondria-nucleus communications remains unknown. The review deduces UCPs functions in mitochondria-nucleus communications implicated in metabolite regulation (e.g., reactive oxygen species) and Ca2+ signaling, and in DDR (e.g., base excision repair, double-strand DNA break repair, mitophagy and nuclear DNA degradation). Represented are shared microRNAs that regulate UCPs and DDR. It would provide novel insight into UCPs-mediated mitochondria-nucleus communications and DDR, and potentially promote drug target identification, drug discovery and clinical therapy of DDR-related diseases.

Toward CRISPR Therapies for Cardiomyopathies.

Toward CRISPR Therapies for Cardiomyopathies.

HMGN1 expression Predisposes Down Syndrome Patients to Develop P2RY8-CRLF2 acute Lymphoblastic Leukemia

IntroductionChildren with Down Syndrome (DS) frequently develop hematological malignancies including acute lymphoblastic leukemia (ALL), however, the genomic basis for the predisposition to DS-ALL remains unknown. DS-ALL is associated with increased rates of relapse, chemotherapy related toxicity and poor overall survival compared to non-DS patients. The P2RY8-CRLF2 (CRLF2r) gene fusion, the result of deletion of the pseudoautosomal region 1 (PAR1) on the X/Y chromosome (chr), has been identified in 60% of DS-ALL (+21) patients and 30% of iAMP21 (intrachromosomal amplification of chr21) ALL patients compared to only 5-16% of other B-ALL patients. The high mobility group nucleosome binding protein 1, encoded by HMGN1 on chr21 may play a role in DS-ALL leukemogenesis, due to its demethylase activity associated with transcriptional activation.MethodsmRNA seq was performed on the blast cells of 76 ALL patients using the Universal Plus mRNA-seq with NuQuant kit. FusionCatcher, SOAPfuse and JAFFA were used to identify fusions and featureCounts used for mRNA expression of STAR (v2.7.3a) aligned BAM files. CRISPR/Cas9 gRNAs targeting P2RY8 intron 1 or CRLF2 5' UTR created the 320 KB (PAR1) deletion. Cas9/gRNAs were transduced into Jurkat cells ± HMGN1 overexpression, activated with doxycycline and stained with TSLPR (CRLF2/IL-7Rα dimer) for single cell sort and clonal expansion. Phosphorylated proteins were measured by intracellular flow cytometry and SYBR Green RQ-PCR was used for mRNA expression.ResultsSignificantly higher HMGN1 expression was identified in CRLF2r ALL patients, compared to the control (BCR-ABL1+ patients. Fig. 1A; p<0.0001). There was no significant difference in HMGN2 (p=0.7881) or JAK2 (p=0.1171) control genes between groups. Of the CRLF2r ALL patients, 26% harbored +21, and 5% iAMP21, strongly suggesting a relationship between chr21, HMGN1 and the CRLF2r fusion.To further understand the role of HMGN1 in CRLF2r development, HMGN1 was overexpressed (1.5-fold; HMGN1H) in Jurkat Cas9 cells to represent a trisomy level of expression. gRNAs targeting P2RY8 and CRLF2 were activated in cells ± HMGN1H with undirected repair. TSLPR surface and CRLF2 mRNA expression were higher in CRLF2r + HMGN1H cells compared to CRLF2r only cells (Fig. 1B,C; p=0.034 and <0.001 respectively). This suggests higher HMGN1 expression significantly predisposed to PAR1 deletion and formation of CRLF2r fusion after a double stranded DNA break (DSB) versus repair where HMGN1 expression is normal. This is consistent with higher HMGN1 expression observed in patients with CRLF2r. There was also increased gene editing activity in CRLF2r + HMGN1H cells, identified through T7 gene editing assay, compared to CRLF2r only cells.CRLF2r + HMGN1H cells also demonstrated significantly increased pSTAT5 (MFI: 2359 ± 1; Fig. 1D), pAKT (MFI: 2339 ± 6; Fig. 1E) and pERK (MFI: 2478 ± 48; Fig. 1F) compared to CRLF2r only cells (MFI pSTAT5: 1910 ± 10; pAKT: 1727 ± 14; pERK: 1946 ± 6; all p<0.001), consistent with the data observed in CRLF2r patients, and confirming cooperation between CRLF2 and HMGN1. CRLF2r + HMGN1H cells demonstrated reduced H3K27me3 (MFI: 1 ± 0.07) compared to CRLF2r only cells (MFI: 1.6 ± 0.03; p=0.003) and higher expression levels of STAT5 target genes (BCL2, CDKN1,MCL1and MYC).ConclusionThe P2RY8-CRLF2 (CRLF2r) gene fusionis prevalent in DS, +21 and iAMP21 ALL patients. Here, we demonstrate that CRLF2r is associated with high expression of HMGN1 (chr21)in ALL patient cells . Using CRISPR/Cas9 in an in vitro model, we demonstrate that enforced high expression of HMGN1 alters DSB repair mechanism, favoring PAR1 deletion and the subsequent formation of the P2RY8-CRLF2 gene fusion, with attendant higher expression of STAT5 target genes. Understanding the role of HMGN1 in the disproportionate number of DS-ALL patients who are diagnosed with CRLF2r has the potential to lead to novel therapeutic interventions, in this high-risk group of patients where efficacious therapeutic options are currently poor. [Display omitted] DisclosuresYeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. White: Novartis: Research Funding; BMS: Honoraria, Research Funding.

MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage.

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.

Setting a diagnostic benchmark for tumor BRCA testing: detection of BRCA1 and BRCA2 large genomic rearrangements in FFPE tissue – A pilot study

PARP inhibitors are used for treatment of tumors lacking function of the double-strand DNA break repair proteins BRCA1 or BRCA2 and are already approved for several cancer types. Thus, it is clinically crucial to determine germline as well as somatic BRCA1/2 mutations in those patients. The amplicon-based Oncomine BRCA1 and BRCA2 Assay is a test routinely used in diagnostics with FFPE specimens. The assay is validated for the detection of mutations, however, data on its performance in detecting large genomic rearrangements in FFPE tissue, is scarce.We cross-validated Oncomine BRCA1 and BRCA2 Assay in blood samples and/or FFPE tissue with multiplex ligation-dependent probe amplification (MLPA) for exon deletions and with OncoScan and an in-house hybridization-based target capture assay (MelArray) with a customized pipeline for the detection of loss of heterozygosity (LOH) and heterozygous versus complete gene loss.The Oncomine BRCA1 and BRCA2 Assay could detect both exon deletion and mono- and bi-allelic losses of the BRCA1/2 genes.We show that the therapeutically relevant large genomic rearrangements are reliably detected with the amplicon-based Oncomine BRCA1 and BRCA2 Assay in FFPE tumor tissue. Based on our data, we suggest tumor BRCA testing as standard diagnostic prescreening prior to germline BRCA testing.

Determinants of Homologous Recombination Deficiency in Pancreatic Cancer.

Simple SummaryA subset of patients with pancreatic cancer demonstrate heightened response rates and prolonged survival to platinum chemotherapy and PARP inhibitors. Deficient homologous recombination (HR), a critical DNA repair program, is a major driver of this susceptibility. Furthermore, the clinical impact of mutations in distinct HR genes is variable and is modified by diverse tumor intrinsic and extrinsic factors. In this review, we discuss the determinants of homologous recombination deficiency (HRD) in pancreatic cancer. We also highlight emerging methods for identifying and inducing HRD in cancer.Pancreatic cancer is a treatment-resistant malignancy associated with high mortality. However, defective homologous recombination (HR), a DNA repair mechanism required for high-fidelity repair of double-strand DNA breaks, is a therapeutic vulnerability. Consistent with this, a subset of patients with pancreatic cancer show unique tumor responsiveness to HR-dependent DNA damage triggered by certain treatments (platinum chemotherapy and PARP inhibitors). While pathogenic mutations in HR genes are a major driver of this sensitivity, another layer of diverse tumor intrinsic and extrinsic factors regulate the HR deficiency (HRD) phenotype. Defining the mechanisms that drive HRD may guide the development of novel strategies and therapeutics to induce treatment sensitivity in non-HRD tumors. Here, we discuss the complexity underlying HRD in pancreatic cancer and highlight implications for identifying and treating this distinct subset of patients.

RecA finds homologous DNA by reduced dimensionality search

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3′ single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.

1145P Reliable detection of BRCA1 and BRCA2 large genomic rearrangements in FFPE tissue: A new diagnostic benchmark for somatic BRCA testing

PARP inhibitors are used for treatment of tumors lacking function of the double-strand DNA break repair proteins BRCA1 or BRCA2 and are already approved for several cancer types. Thus, it is clinically crucial to determine germline as well as somatic BRCA1/2 mutations in those patients. The amplicon-based Oncomine BRCA1 and BRCA2 Assay is a test routinely used in diagnostics with FFPE specimens. The assay is validated for the detection of mutations, however, data on its performance in detecting large genomic rearrangements in FFPE tissue, is scarce.

Crystal structure of the INTS3/INTS6 complex reveals the functional importance of INTS3 dimerization in DSB repair

SOSS1 is a single-stranded DNA (ssDNA)-binding protein complex that plays a critical role in double-strand DNA break (DSB) repair. SOSS1 consists of three subunits: INTS3, SOSSC, and hSSB1, with INTS3 serving as a scaffold to stabilize this complex. Moreover, the integrator complex subunit 6 (INTS6) participates in the DNA damage response through direct binding to INTS3, but how INTS3 interacts with INTS6, thereby impacting DSB repair, is not clear. Here, we determined the crystal structure of the C-terminus of INTS3 (INTS3c) in complex with the C-terminus of INTS6 (INTS6c) at a resolution of 2.4 Å. Structural analysis revealed that two INTS3c subunits dimerize and interact with INTS6c via conserved residues. Subsequent biochemical analyses confirmed that INTS3c forms a stable dimer and INTS3 dimerization is important for recognizing the longer ssDNA. Perturbation of INTS3c dimerization and disruption of the INTS3c/INTS6c interaction impair the DSB repair process. Altogether, these results unravel the underappreciated role of INTS3 dimerization and the molecular basis of INTS3/INTS6 interaction in DSB repair.

Global detection of DNA repair outcomes induced by CRISPR-Cas9.

CRISPR–Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR–Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR–Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

Mutational Analysis of Redβ Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo.

Redβ is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redβ, models of its DNA binding domain (residues 1–188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redβ were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.

Abstract LB130: Combination of IGF-1R targeted alpha therapy with Olaparib results in synergistic efficacy against colorectal and lung cancer xenografts

Abstract Objectives: FPI-1434 is a targeted alpha-particle therapeutic consisting of an IGF-1R targeting antibody radiolabeled with Actinium-225. The primary mechanism of action for FPI-1434 is induction of double strand DNA breaks (DSB) in targeted tumors resulting in cell death. Poly (ADP-ribose) polymerase (PARP) is part of the cellular mechanism that repairs DSB. In cancer patients with genetic defects in DSB repair (eg. BRCA1/2), the PARP pathway becomes a primary repair system and its inhibition results in cell death. This mechanism has been leveraged as a therapy against DNA-repair deficient tumors leading to FDA-approval of PARP inhibitors (PARPi) including Olaparib. Treatment with PARPi to block repair of DNA damage driven by FPI-1434 may act synergistically to increase the lethal DNA damage load. To that end, Fusion has performed studies to combine FPI-1434 and Olaparib against tumor models with no pre-disposing defects in DNA repair. Methods: Colorectal (Colo-205) or lung (A549) cancer xenografts were established in Balb/c nude mice. For efficacy studies, a dose combination matrix for FPI-1434 and Olaparib was tested. FPI-1434 was dosed (i.v.) once at 20-200 nCi followed with Olaparib (i.p.) at doses of 0-50 mg/kg. Olaparib was dosed 24h after FPI-1434 using a 5 day on/2 day off schedule (5 mice/group). DSB formation (γH2AX) and apoptosis (cleaved caspase 3) were evaluated in treated tumors by IHC staining. Results: DSB formation and induction of apoptosis were detected in FPI-1434-treated tumors in a time and dose dependent manner. DSB formation was observed in all areas of tumor containing intact or apoptotic cancer cells confirming FPI-1434 mechanism. In tumor efficacy studies, Olaparib had no single-agent efficacy in the Colo-205 or A549 models. Single doses of FPI-1434 at 20 nCi had no effect on Colo-205 tumors, suppressed growth at 50 nCi and caused regression at 100 nCi. Combination efficacy was seen in the 20 and 50 nCi dose groups, including Olaparib. Doses of 20 nCi FPI-1434 and 25 mg/kg Olaparib had the strongest combined effect in this model. In the A549 model, single doses of FPI-1434 had no effect at 20 or 50 nCi, caused growth suppression at 100 nCi and regression at 200 nCi. Combination efficacy was seen in the 20, 50 and 100 nCi dose groups, including Olaparib. Doses of 50 nCi FPI-1434 and 25 mg/kg Olaparib had the strongest combined effect in this model. In general, the strongest efficacy was observed by combining ineffective single-agent doses of the two compounds. Strong efficacy of high dose FPI-1434 masked observable combination effects suggesting that FPI-1434 was the therapeutic driver. Conclusions: Olaparib co-dosing enhanced FPI-1434 efficacy which supports consideration of this combination for clinical use. Ineffective single-agent doses, when combined, resulted in synergistic efficacy in both Colo-205 and A549 models suggesting that the mechanism can be applied to multiple cancer types where predisposing mutations in DNA repair are lacking. The strongest combination effect appeared to occur at the low single-agent doses suggesting that PARP inhibition may potentiate efficacy at lower clinical doses of FPI-1434. Citation Format: Meiduo Hu, John Forbes, Ryan Simms, Yaryna Storozhuk, Eric Burak, John Valliant. Combination of IGF-1R targeted alpha therapy with Olaparib results in synergistic efficacy against colorectal and lung cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB130.

Inhibition of MRN activity by a telomere protein motif

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles’ heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.

A Role for VCP/p97 in the Processing of Drug-Stabilized TOP2-DNA Covalent Complexes.

DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzyme-induced double-strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct, which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2. VCP/p97 is a AAA ATPase that utilizes energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A and TOP2B complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2A.X, indicative of fewer DSBs. This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. SIGNIFICANCE STATEMENT: TOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers. A better understanding of how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97.

Complex Mechanisms of Antimony Genotoxicity in Budding Yeast Involves Replication and Topoisomerase I-Associated DNA Lesions, Telomere Dysfunction and Inhibition of DNA Repair.

Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.

The dystonia gene THAP1 controls DNA double-strand break repair choice

The Shieldin complex shields double-strand DNA breaks (DSBs) from nucleolytic resection. Curiously, the penultimate Shieldin component, SHLD1, is one of the least abundant mammalian proteins. Here, we report that the transcription factors THAP1, YY1, and HCF1 bind directly to the SHLD1 promoter, where they cooperatively maintain the low basal expression of SHLD1, thereby ensuring a proper balance between end protection and resection during DSB repair. The loss of THAP1-dependent SHLD1 expression confers cross-resistance to poly (ADP-ribose) polymerase (PARP) inhibitor and cisplatin in BRCA1-deficient cells and shorter progression-free survival in ovarian cancer patients. Moreover, the embryonic lethality and PARPi sensitivity of BRCA1-deficient mice is rescued by ablation of SHLD1. Our study uncovers a transcriptional network that directly controls DSB repair choice and suggests a potential link between DNA damage and pathogenic THAP1 mutations, found in patients with the neurodevelopmental movement disorder adult-onset torsion dystonia type 6.

Repair of DNA Breaks by Break-Induced Replication.

Double-strand DNA breaks (DSBs) are the most lethal type of DNA damage, making DSB repair critical for cell survival. However, some DSB repair pathways are mutagenic and promote genome rearrangements, leading to genome destabilization. One such pathway is break-induced replication (BIR), which repairs primarily one-ended DSBs, similar to those formed by collapsed replication forks or telomere erosion. BIR is initiated by the invasion of a broken DNA end into a homologous template, synthesizes new DNA within the context of a migrating bubble, and is associated with conservative inheritance of new genetic material. This mode of synthesis is responsible for a high level of genetic instability associated with BIR. Eukaryotic BIR was initially investigated in yeast, but now it is also actively studied in mammalian systems. Additionally, a significant breakthrough has been made regarding the role of microhomology-mediated BIR in the formation of complex genomic rearrangements that underly various human pathologies.