Sites Of Breaks Research Articles

Measuring Chromosome Pairing During Homologous Recombination in Yeast.

The precise organization of the genome inside the cell nucleus is vital to many cell functions including gene expression, cell division, and DNA repair. Here we describe a method to measure pairing of DNA loci during homologous recombination (HR) at a site-specific double-strand break (DSB) in Saccharomyces cerevisiae. This method utilizes a chromosome tagging system in diploid yeast cells to visualize both the DNA at the break site and the homologous DNA that serves as a repair template. DNA repair products are confirmed in parallel by genomic blot. This visualization method provides insight into the physical contact that occurs between homologous loci during HR and correlates physical interaction with the timing of DNA repair.

A versatile system to introduce clusters of genomic double-strand breaks in large cell populations.

In vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks (DSBs). We present a novel method generating localized DNA DSBs using UV irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this setup with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells, and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by 5-bromo-2-deoxyuridine incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

Metastasis Suppressor NME1 Modulates Choice of Double-Strand Break Repair Pathways in Melanoma Cells by Enhancing Alternative NHEJ while Inhibiting NHEJ and HR.

Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.

Abstract 6164: Small-molecule targeting of the NPM1/Rad51 repair pathway for the radiosensitization of cancer

Abstract Nucleophosmin1 (NPM1) is rapidly recruited to sites of DNA double-strand breaks and is required for radiation-induced Rad51 foci formation. Inhibition of NPM1 or Rad51 is a radiosensitizing event. YTR107 is a small molecule, identified in a forward chemical genetics functional structure/activity screening, that binds to the amino terminus of NPM1 and prevents NPM1 recruitment to damaged chromatin. In silico docking studies of YTR107 and the crystal structure of the NPM1 N-terminal oligomerization domain revealed that YTR107 docked to monomeric NPM1 in a pocket crucial for forming an interface between neighboring subunits of the NPM1 pentamer. YTR107 inhibited radiation-induced Rad51 foci formation and repair of DNA DSBs, as measured by a neutral comet assay. Colony formation assays revealed that YTR107 radiosensitized 7 NSCLC lines, HT29 colorectal cancer cells, D54 glioblastoma cells, PANC1 pancreatic cancer cells, and 2 breast cancer cell lines (dose modifying factors of > 1.5; cells irradiated with 300 kVp/10mA X-rays @ 2 Gy/min). Radiosensitization was found to be NPM1-dependent as YTR107 did not radiosensitize an NPM1 null mouse embryo fibroblast cell line. YTR107 produced a 4.7 fold increase in HT29 xenograft growth delay (time required for the tumor to increase 4-fold in volume (p = 0.001) following 10 mg/kg YTR107 administered 30 min prior to administering 3 Gy to the tumor for 7 consecutive days). YTR107 increased the survival of A549 tumor-bearing mice determined 70 days after the last treatment. Survival of tumor-bearing mice was 60% for mice treated with YTR107 and 3 Gy vs 20% for mice treated with 3 Gy alone, p = 0.0012 (0 or 20 mg/kg YTR107 administered 1 hr prior to 0 or 2.2 Gy being administered to the tumor for 7 consecutive days). Female C57BL/6J mice were administered 0 or 10 mg/kg i.p. YTR107 for 5 consecutive days. Thirty five days after injection mice were euthanized and subjected to necropsy by a veterinarian trained in veterinary pathology. Gross and histological examination of liver, lung, thymus, heart, spleen, cerebellum, pancreas, small intestine, kidney, and adrenal gland did not reveal evidence of toxicity. Both a complete blood count and a white blood cell differential count were performed. No significant differences were noted. Mice were subjected to five daily injections of 30 mg/kg YTR107. This dose level was also tolerated without overt toxicity after 35 days. Mice were injected with 0 or 30 mg/kg YTR107 (i.p.) and 30 min later administered 0 or 16-Gy to the thorax. Pulmonary fibrotic lesions were quantified using respiratory-gated micro CT 136 days later. YTR107 did not increase the severity of radiation-induced lung fibrosis and did not affect body weights or life span of unirradiated or irradiated mice (p > 0.05) over the 136-day study. Thus, this study provides support for development of YTR107 as a therapeutic radiation sensitizer and was supported in part by R44CA228756-02 Citation Format: Konjeti R. Sekhar, Geri Traver, Peter A. Crooks, Michael L. Freeman. Small-molecule targeting of the NPM1/Rad51 repair pathway for the radiosensitization of cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6164.

Engineering designer beta cells with a CRISPR-Cas9 conjugation platform

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a β-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.

Enhanced transcription activity of histone acetyltransferase HBO1 after genotoxic stress

Chromatin remodeling is induced at sites of DNA damage as part of the damage response to allow DNA repair protein accumulation and stability of function. Histone posttranslational modifications are required to orchestrate DNA damage response, and ubiquitination of histone H2A-K15 at DNA double-strand break sites is involved in the recruitment of DNA repair protein 53BP1 to DNA damage sites. Acetylation of histone H4K16 promotes homologous recombination repair by inducing dissociation of 53BP1, while deacetylation inhibits the homologous recombination repair by promoting accumulation of 53BP1 at DNA damage sites. Chromatin remodeling is indispensable for accumulation of numerous DNA damage repair proteins and their subsequent dissociation, and we investigated the possibility of new DNA damage response pathway. In this study, we used qPCR, western blotting and survival assays to demonstrate that genotoxic stress, both biochemical and radiological, specifically upregulated the expression of histone acetyltransferase binding to ORC (HBO1) protein in the histone acetyltransferase MYST family, which colocalized with the DNA damage response protein, Fanconi anemia group D2 (FANCD2). HBO1 expression was upregulated via phosphoinositide-3-kinase-related kinase protein, which plays a crucial role as a DNA damage response signal. These results suggest that HBO1induced by genotoxic stress has a critical role in DNA damage responses.

RNF168-Mediated Ubiquitin Signaling Inhibits the Viability of BRCA1-Null Cancers.

BRCA1 gene mutations impair homologous recombination (HR) DNA repair, resulting in cellular senescence and embryonic lethality in mice. Therefore, BRCA1-deficient cancers require adaptations that prevent excessive genomic alterations from triggering cell death. RNF168-mediated ubiquitination of γH2AX at K13/15 (ub-H2AX) serves as a recruitment module for the localization of 53BP1 to DNA break sites. Here, we found multiple BRCA1-mutant cancer cell lines and primary tumors with low levels of RNF168 protein expression. Overexpression of ectopic RNF168 or a ub-H2AX fusion protein induced cell death and delayed BRCA1-mutant tumor formation. Cell death resulted from the recruitment of 53BP1 to DNA break sites and inhibition of DNA end resection. Strikingly, reintroduction of BRCA1 or 53BP1 depletion restored HR and rescued the ability of cells to maintain RNF168 and ub-H2AX overexpression. Thus, downregulation of RNF168 protein expression is a mechanism for providing BRCA1-null cancer cell lines with a residual level of HR that is essential for viability. Overall, our work identifies loss of RNF168 ubiquitin signaling as a proteomic alteration that supports BRCA1-mutant carcinogenesis. We propose that restoring RNF168-ub-H2AX signaling, potentially through inhibition of deubiquitinases, could represent a new therapeutic approach. SIGNIFICANCE: This study explores the concept that homologous recombination DNA repair is not an all-or-nothing concept, but a spectrum, and that where a tumor stands on this spectrum may have therapeutic relevance.See related commentary by Wang and Wulf, p. 2720.

The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates

SummaryMeiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

Syndesmotic Screw Breakage May Be More Problematic Than Previously Reported: Increased Rates of Hardware Removal Secondary to Pain With Intraosseous Screw Breakage.

The majority of retained syndesmotic screws will either loosen or break once the patient resumes weight-bearing. While evidence is limited, anecdotal experience suggests that intraosseous screw breakage may be problematic for some patients due to painful bony erosion. This study seeks to identify the incidence of intraosseous screw breakage, variables that may predict intraosseous screw breakage, and whether intraosseous screw breakage is associated with higher rates of implant removal secondary to pain. Five hundred thirty-one patients undergoing syndesmotic stabilization were screened, of which 43 patients (with 58 screws) experiencing postoperative screw breakage met inclusion criteria. Patient charts were retrospectively reviewed for demographic data, comorbidities, time to screw breakage, location of screw breakage, and implant removal. Several radiographic parameters were evaluated for their potential to influence the site of screw breakage. Intraosseous screw breakage occurred in 32 patients (74.4%). Screw breakage occurred exclusively in the tibiofibular clear space in the remaining 11 instances (25.6%). Intraosseous screw breakage was significantly associated with eventual implant removal after breakage (P = .034). Screws placed further from the tibiotalar joint were at less risk for intraosseous breakage (odds ratio 0.818, P = .002). Screws placed at a threshold height of 20 mm or greater were more likely to break in the clear space (odds ratio 12.1, P = .002). Syndesmotic screw breakage may be more problematic than previously described. Intraosseous breakage was associated with higher rates of implant removal secondary to pain in this study. Placement of screws 20 mm or higher from the tibiotalar joint may decrease risk of intraosseous breakage.Levels of Evidence: Level III: Retrospective study.

Super-resolution imaging of RAD51 and DMC1 in DNA repair foci reveals dynamic distribution patterns in meiotic prophase.

The recombinase RAD51, and its meiosis-specific paralog DMC1 localize at DNA double-strand break (DSB) sites in meiotic prophase. While both proteins are required during meiotic prophase, their spatial organization during meiotic DSB repair is not fully understood. Using super-resolution microscopy on mouse spermatocyte nuclei, we aimed to define their relative position at DSB foci, and how these vary in time. We show that a large fraction of meiotic DSB repair foci (38%) consisted of a single RAD51 nanofocus and a single DMC1 nanofocus (D1R1 configuration) that were partially overlapping with each other (average center-center distance around 70 nm). The vast majority of the rest of the foci had a similar large RAD51 and DMC1 nanofocus, but in combination with additional smaller nanofoci (D2R1, D1R2, D2R2, or DxRy configuration) at an average distance of around 250 nm. As prophase progressed, less D1R1 and more D2R1 foci were observed, where the large RAD51 nanofocus in the D2R1 foci elongated and gradually oriented towards the distant small DMC1 nanofocus. D1R2 foci frequency was relatively constant, and the single DMC1 nanofocus did not elongate, but was frequently observed between the two RAD51 nanofoci in early stages. D2R2 foci were rare (<10%) and nearest neighbour analyses also did not reveal cofoci formation between D1R1 foci. However, overall, foci localized nonrandomly along the SC, and the frequency of the distance distributions peaked at 800 nm, indicating interference and/or a preferred distance between two ends of a DSB. DMC1 nanofoci where somewhat further away from the axial or lateral elements of the synaptonemal complex (SC, connecting the chromosomal axes of homologs) compared to RAD51 nanofoci. In the absence of the transverse filament of the SC, early configurations were more prominent, and RAD51 nanofocus elongation occurred only transiently. This in-depth analysis of single cell landscapes of RAD51 and DMC1 accumulation patterns at DSB repair sites at super-resolution revealed the variability of foci composition, and defined functional consensus configurations that change over time.

Cytoskeletal Repair: Microtubule Orthopaedics to the Rescue

While the dynamics of microtubule ends are well characterized, the mechanism that repairs breaks in the lattice interior is poorly understood. A new invitro study finds that the microtubule-associated protein CLASP repairs lattice damage by regulating GTP-tubulin incorporation into the break site.

The histone modification reader ZCWPW1 links histone methylation to PRDM9-induced double-strand break repair.

The histone modification writer Prdm9 has been shown to deposit H3K4me3 and H3K36me3 at future double-strand break (DSB) sites during the very early stages of meiosis, but the reader of these marks remains unclear. Here, we demonstrate that Zcwpw1 is an H3K4me3 reader that is required for DSB repair and synapsis in mouse testes. We generated H3K4me3 reader-dead Zcwpw1 mutant mice and found that their spermatocytes were arrested at the pachytene-like stage, which phenocopies the Zcwpw1 knock-out mice. Based on various ChIP-seq and immunofluorescence analyses using several mutants, we found that Zcwpw1's occupancy on chromatin is strongly promoted by the histone-modification activity of PRDM9. Zcwpw1 localizes to DMC1-labelled hotspots in a largely Prdm9-dependent manner, where it facilitates completion of synapsis by mediating the DSB repair process. In sum, our study demonstrates the function of ZCWPW1 that acts as part of the selection system for epigenetics-based recombination hotspots in mammals.

Senataxin Ortholog Sen1 Limits DNA:RNA Hybrid Accumulation at DNA Double-Strand Breaks to Control End Resection and Repair Fidelity

An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity.

A reporter mouse for in vivo detection of DNA damage in embryonic germ cells.

Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1-mCherry fusion protein under the control of the Oct4ΔPE embryonic germ cell-specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA DSB-inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose- and time-dependent manner. The dynamic time-sensitive and dose-sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells.

"Mitotic Slippage" and Extranuclear DNA in Cancer Chemoresistance: A Focus on Telomeres.

Mitotic slippage (MS), the incomplete mitosis that results in a doubled genome in interphase, is a typical response of TP53-mutant tumors resistant to genotoxic therapy. These polyploidized cells display premature senescence and sort the damaged DNA into the cytoplasm. In this study, we explored MS in the MDA-MB-231 cell line treated with doxorubicin (DOX). We found selective release into the cytoplasm of telomere fragments enriched in telomerase reverse transcriptase (hTERT), telomere capping protein TRF2, and DNA double-strand breaks marked by γH2AX, in association with ubiquitin-binding protein SQSTM1/p62. This occurs along with the alternative lengthening of telomeres (ALT) and DNA repair by homologous recombination (HR) in the nuclear promyelocytic leukemia (PML) bodies. The cells in repeated MS cycles activate meiotic genes and display holocentric chromosomes characteristic for inverted meiosis (IM). These giant cells acquire an amoeboid phenotype and finally bud the depolyploidized progeny, restarting the mitotic cycling. We suggest the reversible conversion of the telomerase-driven telomere maintenance into ALT coupled with IM at the sub-telomere breakage sites introduced by meiotic nuclease SPO11. All three MS mechanisms converging at telomeres recapitulate the amoeba-like agamic life-cycle, decreasing the mutagenic load and enabling the recovery of recombined, reduced progeny for return into the mitotic cycle.

DNA:RNA hybrids form at DNA double-strand breaks in transcriptionally active loci

The recent discovery of DNA:RNA hybrids, or R-loops, actively forming at DNA double-strand breaks (DSBs) has unlocked fresh insight into how RNA participates in DNA repair. However, the manner of DSB-induced R-loop formation is vital in determining its mechanism of action and is currently under debate. Here, we analyse published DNA:RNA-hybrid sequencing to elucidate the features that determine DSB-induced R-loop formation. We found that pre-existing transcriptional activity was critical for R-loop generation at break sites, suggesting that these RNAs are transcribed prior to break induction. In addition, this appeared to be a specific DSB response at the break, distinct from traditional, co-transcriptionally formed R-loops. We hypothesise that R-loop formation is orchestrated by the damage response at transcriptionally active DSB loci to specifically maintain these genomic regions. Further investigation is required to fully understand how canonical repair processes regulate R-loops at breaks and how they participate in the repair process.

Exploring Rad1‐Rad10 Recruitment by Slx4 in Relation to Cell Cycle

DNA damage, caused by various exogenous and endogenous agents such as ionizing radiation and replication stress, gives rise to mutations in DNA that can cause cancer. To combat this, eukaryotic cells use evolutionarily conserved repair mechanisms that mend the DNA. The choice of repair pathway depends primarily on the form of damage, its location within the genome, and the cell cycle phase. For example, single‐strand annealing (SSA), a form of DNA double‐strand break repair (DSBR), is favored in regions between DNA repeats while synthesis‐dependent strand annealing (SDSA), another DSBR mode, needs sister chromatids which act as the template DNA for repair. In order for SSA and SDSA to be completed, the Rad1‐Rad10 endonuclease cleaves overhanging flaps that result from the annealing step before the final ligation can proceed.We have preliminary data indicating that Slx4, a protein present in S. cerevisiae (baker’s yeast), is partially required in the recruitment of Rad1‐Rad10 to repair sites during cell division (S, G2 and M phases). However, cells characterized as being in cell division were not differentiated during data analysis due to the size of the population within those phases. Our research seeks to elucidate the phase in which Slx4 exerts its recruitment properties by repeating our earlier fluorescence microscopy recruitment assay using yeast strains that contain fluorescently‐tagged proteins for monitoring both the colocalization of Rad1‐Rad10 to an induced double‐strand break site and the phase of the cell cycle in cell cycle‐synchronized cultures.To help us clearly distinguish between cell cycle phases during fluorescence microscopy, we have integrated a fluorescently labeled spindle pole body protein (Spc110‐CFP) into our strains because of its dramatic morphology shifts during cell division. Yeast genetic crosses were conducted to combine Spc110‐CFP with SLX4 mutant genes as well as other features that will allow us to perform fluorescence microscopy and flow cytometry analysis in a single strain. Following cell cycle arrest by alpha‐factor, &gt; 90% of cells showed a single Spc110‐CFP focus, indicating that most cells were successfully arrested at the G1/S phase boundary, but flow cytometry analysis showed that only about 60% of cells contained a DNA content consistent with G1/S arrest. These data indicate that cell cycle arrest and its monitoring by the appearance of Spc110‐CFP foci are effective but further efforts are needed to optimize the protocol. Along these lines, we began creating a new SLX4 deletion mutant using Polymerase Chain Reaction (PCR) to amplify a transplacement mutant gene in which the SLX4 coding region has been swapped with the antibiotic marker NATMX4. The final PCR product was A‐tailed and then ligated into the pGEMT‐Easy vector. E. coli transformants were screened by PCR for the insert and will be sequenced to verify the SLX4 region is in the plasmid before moving forward with yeast genetic crosses.Support or Funding InformationNational Institute of Health (NIH) (Grant SC1GM127204), NIH MARC Program (NIH Grant GM008395), California State University, Northridge College of Science and Math, California State University, Northridge Department of Chemistry and Biochemistry

The Impact of Chromatin Structure on the Formation of Double Strand Breaks by CRISPR‐CAS9 in the Yeast Genome

DNA is persistently at risk for being damaged by endogenous and exogenous factors. One type of DNA damage is a double‐strand break (DSB), which occurs when both strands of the helix become severed. DSBs are important because upon repair, they could lead to mutations or genomic alteration. The packaging of the DNA into chromatin may affect the susceptibility of an area to DSBs and thus could make the area more prone to mutation. The purpose of this study is to look at how the chromatin environment affects the susceptibility of genes to DSBs in Saccharomyces cerevisiae. A new system, tetracycline‐inducible CRISPR‐Cas9, was used in this experiment to induce DSBs at different sites in the yeast genome Histone modification levels were measured at each site to correlate these chromatin marks with DSB formation frequency. DSB frequency at each genomic locus was measured using quantitative PCR and directly compared to those same sites in unchromatinized DNA to see how chromatin environment and the surrounding histone modifications affects susceptibility to DSBs.To measure the frequency of DSBs at different genomic sites using qPCR, the break site was amplified. DSBs would be indicated through a decrease in product when compared to an uncut control site NFS1 on Chromosome III. While correlation analysis of histone modifications and DSB frequency is still ongoing, preliminary data suggests that repressed genes have higher levels of cutting than active genes after three hours of DSB induction. Of the repressed genes investigated, PPE1 had 82.06% DSB formation and RRT8 had 66.67% after three hours of induction. Active genes RPL22A, TDH2, and TDH3 had on average about 15%, 9.5%, and 56% DSB formation after three hours of DSB induction, respectively. The level of DSB formation in these sites will be directly compared to DSB formation of the same sites on an unchromatinized plasmid, which is currently underway.This research is important because it shows which areas of the genome may be at higher risk for DSBs and whether or that is affected by chromatin structure. Understanding this could help deepen the knowledge of how the genome evolves through spontaneous DSB formation and repair.

Role of MCM8/9 in DNA Fork Protection

Efficiency of DNA replication is crucial to cell survival. Recognition of DNA damage by replication complexes results in impedance of this process until the damage is repaired. Fork stalling and reversal are important for the repair of DNA lesions. Stalled forks must be resolved quickly in order to prevent fork collapse resulting in double‐strand breaks (DSBs). Several proteins are recruited to mitigate damage to DNA prior to DSB formation. MCM8/9 is believed to play a role in both protection of DNA at stalled forks and in repair of DSBs through a helicase activity. Mutation of MCM8 or MCM9 has been attributed to development of cancer and infertility. The MCM8/9 complex recruits Rad51 to double stranded break sites, but its role in fork stalling remains unknown.A CRISPR‐Cas9 generated MCM9 knockout cell line was used to assess MCM8/9‐mediated protection of stalled or reversed forks from nucleolytic degradation using DNA fiber assays. Two halogenated thymine analogs, CldU and IdU, are added to the nucleotide pool to monitor discrete DNA replication tracks within HEK293T cells. After CldU and IdU pulses, DNA replication is stalled through the addition of hydroxyurea (HU) to allow for fork reversal. The IdU/CldU length ratio measured from DNA fiber analysis can reveal fork protection status. MCM9 knockout cells had a significant reduction in IdU/CldU track ratios demonstrating its importance in maintaining replication fork stability following stalling by HU. These results suggest that MCM8/9 plays a crucial but unidentified role in maintenance of replication fork integrity and protection of stalled forks during replication. Current and future experiments will focus on the direct interactions and effects of knockdowns of other known fork protection proteins with MCM9 to more definitely determine MCM8/9 function in maintaining the human genome.Support or Funding InformationThis work is supported by the National Institutes of Health (NIH) GM135791.

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

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