Sites Of Double-strand Breaks Research Articles

Structural insights into DNA double-strand break signaling.

Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.

Structure-function analysis of TOPBP1\u2019s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system

The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1’s nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.

BET bromodomain inhibitor JQ1 regulates spermatid development by changing chromatin conformation in mouse spermatogenesis

As a BET bromodomain inhibitor, JQ1 has been proven have efficacy against a number of different cancers. In terms of male reproduction, JQ1 may be used as a new type of contraceptive, since JQ1 treatment in male mice could lead to germ cell defects and a decrease of sperm motility, moreover, this effect is reversible. However, the mechanism of JQ1 acting on gene regulation in spermatogenesis remains unclear. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse testes treated with JQ1 or vehicle control to determine the transcriptional regulatory function of JQ1 in spermatogenesis at the single cell resolution. We confirmed that JQ1 treatment could increase the numbers of somatic cells and spermatocytes and decrease the numbers of spermatid cells. Gene Ontology (GO) analysis demonstrated that differentially expressed genes which were down-regulated after JQ1 injection were mainly enriched in “DNA conformation change” biological process in early developmental germ cells and “spermatid development” biological process in spermatid cells. ATAC-seq data further confirmed that JQ1 injection could change the open state of chromatin. In addition, JQ1 could change the numbers of accessible meiotic DNA double-stranded break sites and the types of transcription factor motif that functioned in pachytene spermatocytes and round spermatids. The multi-omics analysis revealed that JQ1 had the ability to regulate gene transcription by changing chromatin conformation in mouse spermatogenesis, which would potentiate the availability of JQ1 in male contraceptive.

A Review on CRISPR/ Cas9: A Molecular Approach to Genome Editing

CRISPR/Cas9 is an RNA-guided endonuclease that specifically targets a sequence of DNA by selecting bacteria, archaea enabling an organism to respond to with the utilization of nucleotide base pairing and eliminate invading genetic material. The CRISPR-Cas9 framework utilizes a single guide RNA (sgRNA) and the CRISPR-associated endonuclease Cas9 to produce double-strand breaks (DSBs) at the specific DNA site, consequently promoting genetic changes in view of non-homologous end-joining (NHEJ) repair. Random insertion or deletion (indel) transformations caused by the CRISPR/Cas9 system generally happen proximate to the DSB site at 3 bp upstream of the protospacer adjacent motif (PAM). Cas9 based genome altering apparatus for presentation of site explicit double-stranded DNA break (DSB) and resulting mutagenesis in a plant, mouse and human cells It an efficient, fast, easy, and cheap technique. It is involved in the regulation of endogenous gene expression, live-cell labeling of chromosomal loci certain recent versions of it can also be employed in activation or repression of the desired gene in an organism through the gene expression regulation by fusing heterologous domains for modifying epigenetic signatures on histones or transcriptional activators and repressors CRISPR/Cas9 can also be utilized for treating various diseases. CRISPR/Cas9 is also very efficient in enhancing T cell therapy, and with the accumulation of more clinical data; efficacy and safety of CRISPR-based therapy will be utilized in coming years for treating many diseases. CRISPR/Cas9’s requirement of 5 ‘-NGG-3 ‘ PAMjust next to 20 nucleotide DNA target sequence, where it can only recognize NGG PAM site, reduces and limits its the effectiveness of genome editing.

A Paradigm Revolution or Just Better Resolution-Will Newly Emerging Superresolution Techniques Identify Chromatin Architecture as a Key Factor in Radiation-Induced DNA Damage and Repair Regulation?

Simple SummaryRadiation-induced double-strand breaks (DSBs) are the most toxic and most difficult to repair DNA lesions and are very heterogeneous. These characteristics place considerable demands on the selection of the most suitable repair mechanism at each individual damage site. Here, we review the current knowledge on this still enigmatic process and hypothesize that it critically involves the local chromatin architecture at the micro- and nanoscales, later manifested in the architecture of DSB repair foci (i.e., IRIFs).DNA double-strand breaks (DSBs) have been recognized as the most serious lesions in irradiated cells. While several biochemical pathways capable of repairing these lesions have been identified, the mechanisms by which cells select a specific pathway for activation at a given DSB site remain poorly understood. Our knowledge of DSB induction and repair has increased dramatically since the discovery of ionizing radiation-induced foci (IRIFs), initiating the possibility of spatiotemporally monitoring the assembly and disassembly of repair complexes in single cells. IRIF exploration revealed that all post-irradiation processes—DSB formation, repair and misrepair—are strongly dependent on the characteristics of DSB damage and the microarchitecture of the whole affected chromatin domain in addition to the cell status. The microscale features of IRIFs, such as their morphology, mobility, spatiotemporal distribution, and persistence kinetics, have been linked to repair mechanisms. However, the influence of various biochemical and structural factors and their specific combinations on IRIF architecture remains unknown, as does the hierarchy of these factors in the decision-making process for a particular repair mechanism at each individual DSB site. New insights into the relationship between the physical properties of the incident radiation, chromatin architecture, IRIF architecture, and DSB repair mechanisms and repair efficiency are expected from recent developments in optical superresolution microscopy (nanoscopy) techniques that have shifted our ability to analyze chromatin and IRIF architectures towards the nanoscale. In the present review, we discuss this relationship, attempt to correlate still rather isolated nanoscale studies with already better-understood aspects of DSB repair at the microscale, and consider whether newly emerging “correlated multiscale structuromics” can revolutionarily enhance our knowledge in this field.

Abstract PO-43: Targeting DNA repair in EZH2 gain-of-function diffuse large B-cell lymphoma

Abstract An FDA-approved HDAC inhibitor for B-cell lymphoma is currently not available. While pan-HDAC inhibitors (HDIs) hold much promise, they display unwanted toxic side effects, because they hit multiple enzymes belonging to both Class I and/or Class II HDAC families. Class I HDAC (HDAC 1, 2, 3, and 8) are the key targets of many pan-HDIs, including the FDA-approved drugs SAHA and depsipeptide. Diffuse large B-cell lymphoma (DLBCL), a type of non-Hodgkin's lymphoma, is the most common lymphoid malignancy of all adult lymphomas. These lymphoma cells acquire chemoresistance and consequently have a high relapse rate, making it a hard-to-treat disease. About 30% of germinal center (GC)-derived DLBCLs have a recurrent somatic gain-of-function mutation (GOF) in the polycomb-group oncogene EZH2. EZH2 codes for the methyltransferase that catalyzes histone H3 lysine-27 trimethylation (H3K27me3) and promotes lymphomagenesis. Increased levels of EZH2 are purported to also contribute to chemoresistance in EZH2GOF DLBCL cells. EZH2-catalyzed H3K27me3 localizes to double-strand breaks (DSBs) and participates in DNA repair. Our published and recent unpublished results showed that selective inhibition of HDAC1,2 decreases H3K27me3 at DSBs during DSB repair, alters the H3K27me3/H3K27ac switch specifically at DSB sites, and impairs EZH2-mediated downstream repair signaling. In addition to H3K27me3, we found that EZH2GOF DLBCL cells overexpress B-lymphoma and BAL-associated protein (BBAP) enzyme that also confers chemoresistance to EZH2GOF DLBCL cells. BBAP is an E3 ubiquitin ligase and catalyzes H4K91ub1, which is also involved in DNA repair. Our published results showed that selective inhibition of HDAC1,2 decreases H3K27me3 at DSBs and H4K91ub1 during DSB repair by increasing H3K27ac and H4K91ac. This alteration of H3K27me3/H3K27ac and H4K91ub1/H4K91ac switches results in decreased DSB repair, an induction of DNA damage response, and cytotoxic effects, providing a novel mechanism by which HDAC1,2 inhibition can overcome survival advantage in chemoresistant DLBCL cells. The consequences of the altered H3K27me3/H3K27ac switch on DNA repair events are still not understood. How HDAC1,2 acts in concert with enzymes that modulate the chromatin dynamics during DSB repair in EZH2GOF DLBCL is not clear. We hypothesize that HDAC1,2 inhibition specifically impairs DSB repair used by EZH2GOF DLBCL cells at various steps (local transcriptional repression at DSBs during active repair and the chromatin remodeling required for DNA repair). We will present our recent results obtained from the mechanistic studies performed to address the above-mentioned questions. We are also investigating how HDAC1,2 functions in normal germinal center functions to complement our lymphoma studies. Citation Format: Danielle Johnson, Sneha Patel, Srividya Bhaskara. Targeting DNA repair in EZH2 gain-of-function diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-43.

Targeting SMYD3 to Sensitize Homologous Recombination-Proficient Tumors to PARP-Mediated Synthetic Lethality.

SummarySMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.

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.

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.

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. • HBO1 mRNA expression was enhanced after genotoxic stress. • Genotoxic stress did not increase other MYST family protein expression. • HBO1 depletion resulted in hypersensitivity to genotoxic stress.

Kinase Phosphorylation-based Mechanisms of PARP Inhibitor Resistance During Synthetic Lethal Oncotherapy

Background: Poly-(ADP-Ribose) Polymerase (PARP) plays a central role in recovery from single-strand DNA (ssDNA) damage via base excision repair. When PARP activity is inhibited by a NAD+ mimetic analog, ssDNA is converted into a Double-Strand Break (DSB) during the S-phase in a cell cycle. However, the DSB site is repaired in a process of Homologous Recombination (HR) that is derived by genes such as BRCA1/2, PALB2, and RAD51. Under conditions of HR dysfunction, including mutations of BRCA1/2 (called BRCAness), PARP inhibitor (PARPi) induces “synthetic lethality” in BRCAness-specific cancer cells. Indeed, clinical trials using forms of PARPi that include olaparib, veliparib and rucaparib, have revealed that PARP inhibition produces a dramatic effect that actually arrests cancer progression. Its clinical efficiency is limited, however, due to the acquisition of PARPi resistance during long-term use of this inhibitor. Thus, it is important to elucidate the mechanisms of PARPi resistance. Methods: We searched the scientific literature published in PubMed, with a special focus on kinase phosphorylation that is involved in acquiring PARPi resistance. We also summarized the possible molecular events for recovering HR system, a key event for acquiring PARPi resistance. Results: CDK1 is a critical kinase for 5’-3’ DNA end resection, which is important for generating ssDNA for recruiting HR-priming factors. CDK12 is necessary for the transcription of HR-driver genes, such as BRCA1, BRCA2, RAD51 and ATR via the phosphorylation of RNA Pol-II. PLK-1 participates in driving HR via the phosphorylation of RAD51. The PI3K-AKT-mTOR signaling cascade is involved in BRCA1 induction via an ETS1 transcriptional pathway. Even under ATMdeficient conditions, the ATR-CHK1 axis compensates for loss in the DNA damage response, which results in HR recovery. The HGF receptor Met tyrosine kinase is responsible for promoting DNA repair by activating the PARP catalytic domain. Conclusion: These kinase-based signaling pathways are biologically important for understanding the compensatory system of HR, whereas inactivation of these kinases has shown promise for the release of PARPi resistance. Several lines of preclinical studies have demonstrated the potential use of kinase inhibitors to enhance PARPi sensitivity. We emphasize the clinical importance of chemical inhibitors as adjuvant drugs to block critical kinase activities and prevent the possible PARPi resistance.

METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

LRIK interacts with the Ku70-Ku80 heterodimer enhancing the efficiency of NHEJ repair.

Despite recent advances in our understanding of the function of long noncoding RNAs (lncRNAs), their roles and functions in DNA repair pathways remain poorly understood. By screening a panel of uncharacterized lncRNAs to identify those whose transcription is induced by double-strand breaks (DSBs), we identified a novel lncRNA referred to as LRIK that interacts with Ku, which enhances the ability of the Ku heterodimer to detect the presence of DSBs. Here, we show that depletion of LRIK generates significantly enhanced sensitivity to DSB-inducing agents and reduced DSB repair efficiency. In response to DSBs, LRIK enhances the recruitment of repair factors at DSB sites and facilitates γH2AX signaling. Our results demonstrate that LRIK is necessary for efficient repairing DSBs via nonhomologous end-joining pathway.

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.

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.

COM1, a factor of alternative non-homologous end joining, lagging behind the classic non-homologous end joining pathway in rice somatic cells.

The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double-stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation-induced DSBs. Similar retarded growth at the post-germination stage was observed in the com1-2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non-homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ-irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11RNAi plant and in the wortmannin-treated wild-type plant. Under non-irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild-type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11-Rad50-Nbs1 (MRN) complex and facilitated by PI3K-like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells.

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.

The effects of varying DNA flap length from 20 to 50 deoxynucleotides on the mechanism of single‐strand annealing in Saccharomyces cerevisiae

Mutagens constantly threaten the integrity of genetic material by causing damage in the form of double‐strand breaks (DSBs). Such damage is typically repaired through various modes of homologous recombination (HR). This project focuses on a form of HR known as single‐strand annealing (SSA), which is triggered when a DSB is located between two DNA repeats. This pathway generates 3' overhanging single‐stranded DNA “flaps” which are cleaved by the Rad1‐Rad10 endonuclease, aided by the mediator protein Saw1 in S. cerevisiae. Recent literature has demonstrated that Saw1‐dependent recruitment of Rad1‐Rad10 to damage sites, and SSA repair proficiency, are both a function of flap length. In this study, we set out to elucidate the precise flap length threshold requiring Saw1 for optimal SSA repair in vivo through fluorescence microscopy and real‐time quantitative PCR (qPCR).In order to investigate the importance of DNA flap length on Rad1‐Rad10 and Saw1 recruitment, we designed yeast strains generating 20, 30, or 50 nt flaps during SSA. A DSB was induced at a fluorescently labeled site in the yeast genome (DSB‐RFP), allowing us to monitor the recruitment of Rad10‐YFP via fluorescence microscopy. The percentages of cells containing colocalized foci in strains either wild‐type or mutant in Saw1 were quantified, revealing that Saw1‐mediated recruitment of the Rad1‐Rad10 complex increased with flap length. Surprisingly, we found that Saw1 was actively recruited to the DSB site across all flap length substrates via experiments with a Saw1‐CFP strain. Analyses via qPCR were also carried out to quantify SSA repair products formed in WT and saw1Δstrains. The data show diminished repair product formation in the absence of Saw1 as flap lengths increased. Together, these results suggest that Saw1 is required for Rad1‐Rad10 recruitment and efficient SSA repair in longer flap substrates with ~20–30 nt representing the key size triggering Saw1‐dependence for repair. Current efforts are underway to determine the Saw1‐dependence of proficient SSA repair under conditions in which background induction is not amplified by qPCR.Support or Funding InformationThe authors thank our Department and the NIH SC3 program for funding.

Investigating SLX4‐Dependent Recruitment of a DNA Repair Protein to DNA Double Strand Breaks in Dividing Cells

Cancer is a disease that functions as a consequence of abnormal cell growth, generally caused by DNA damage that triggers abnormal cell function. One form of DNA damage is the double‐strand break (DSB) in which both strands of a chromosome are severed in near proximity. In normal cells, several forms of DSB repair, including single strand annealing (SSA) and synthesis dependent strand annealing (SDSA), fix the double strand break (DSB), restoring an intact chromosome, albeit sometimes with an altered DNA sequence. These DNA repair processes require the sequential action of numerous proteins that enable their many steps. In S. cerevisiae these include the Rad1‐Rad10 endonuclease complex, which cleaves nonhomologous DNA flaps in SSA and SDSA as one of the final steps.We have evidence that the Slx4 protein is partially required for the recruitment of Rad1‐Rad10 to DSB sites when cells are dividing (in S, G2 or M phases). Those experiments did not show the basis for the “partial” nature of the recruitment, but our hypothesis is that the Slx4‐dependency is tied to a specific phase of the cell cycle, possibly S phase. A plausible explanation is that when Slx4 exercises its checkpoint signal dampening role in S phase, which promotes homologous recombination, it indirectly recruits Rad1‐Rad10 to double‐strand break repair sites. Since, following DNA damage, checkpoint signal dampening is triggered (in part) by phosphorylation of Slx4 in S phase and the specific phosphorylation sites for this are known, we will test our hypothesis by creating mutant strains which lack these sites and repeating the earlier experiments using synchronized cell cultures. We began creating the mutant strains by cloning DNA plasmids containing the slx4‐7MUT gene, our focus, in which wild‐type SLX4 has been mutated to alanine at the seven phosphorylation sites activating checkpoint signal dampening. Candidate clones were screened by PCR and DNA sequencing. The successful candidate plasmids were transformed into a panel of W303 background yeast strains and integration of the mutant cassettes was confirmed by PCR and DNA sequencing screens. The strains were then crossed to strains bearing the fluorescence microscopy assay markers we will use in the fluorescence microscopy assay being employed. We have carried out preliminary fluorescence microscopy assays in which cultures of the progeny of these crosses were synchronized at the G1/S boundary and released back into the cell cycle that show acceptable arrest but the limited release back into the cell cycle that was observed requires further optimization. Using this separation‐of‐function mutant strain will allow us to compare recruitment of Rad1‐Rad10 in SLX4 wild‐type strains with Slx4 checkpoint signal dampening mutants. Understanding the process of Rad1‐Rad10 recruitment may aid in the design and development of a more effective means of cancer treatment.

Pioneering meiotic recombination.

To induce cell type-specific forms of gene regulation, pioneer factors open tightly packed, inaccessible chromatin sites, enabling the molecular machinery to act on functionally significant information encoded in DNA. While previous studies of pioneer factors have revealed their functions in transcriptional regulation, pioneer factors that open chromatin for other physiological events remain undetermined. In this issue of Genes & Development, Spruce and colleagues (pp. 398-412) report the functional significance of a "pioneer complex" in mouse meiotic recombination. This complex, comprised of the zinc finger DNA-binding protein PRDM9 and the SNF2 family chromatin remodeler HELLS, exposes nucleosomal DNA to designate the sites of DNA double-strand breaks that initiate meiotic recombination. Both HELLS and PRDM9 are required for the determination of these recombination hot spots. Through the identification of a pioneer complex for meiotic recombination, this study broadens the conceptual scope of pioneer factors, indicating their functional significance in biological processes beyond transcriptional regulation.