DNA Ends Research Articles

Abstract 5612: MRE11 function in DNA damage response is regulated by SIRT2 deacetylation

Abstract MRE11 is known to play a role in DNA damage repair through the MRE11-RAD50-NBS1 (MRN) complex and homologous recombination. However, the upstream signaling mechanisms regulating MRE11 in DNA repair are unclear. Here, we aimed to determine the extent to which MRE11 is regulated by deacetylation by the SIRT2 sirtuin deacetylase and tumor suppressor protein. We overexpressed MRE11 in HEK293T cells, induced DNA damage through radiation treatment, and performed a series of co-immunoprecipitation (co-IP) studies with and without ionizing radiation (IR). Additionally, we evaluated the acetylation of MRE11 with and without the presence of SIRT2. In a series of functional experiments, immunofluorescence staining assays were performed in U2OS-265 Fok1 cells with SIRT2 knockdown to determine the effect of on MRE11 recruitment to DNA damage sites. Co-IP studies and in vitro DNA pulldown assays were performed to determine the role of SIRT2 in impacting MRE11 interaction with RAD50 and NBS1 and binding to DNA damage. We also examined for IR-induced RPA70 foci formation and downstream ATM-dependent signaling. We found that SIRT2 interacts with and deacetylates MRE11. Furthermore, SIRT2 deacetylation facilitated MRE11 binding to DNA but not interaction with RAD50 and NBS1. Finally, SIRT2 deacetylation facilitated DNA end resection and ATM-dependent phosphorylation of KAP1. In conclusion, our results define a role for SIRT2 in directing MRE11 localization and binding to DNA to promote DNA end resection and ATM-dependent signaling through deacetylation. Citation Format: Mark Essien, Fatmata Sesay, Hui Zhang, Andrew Jung, David Yu. MRE11 function in DNA damage response is regulated by SIRT2 deacetylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5612.

Abstract 2290: Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies

Abstract Multiple studies have shown that the plasma cell-free DNA (cfDNA) from cancer patients differs from healthy individuals in both fragment length and fragment end motifs (FEMs). Yet, there is a lack of understanding regarding how the two factors combined can be associated with cancer and gene transcription. In this study, we evaluated cfDNA fragmentomics in plasma from lung cancer patients (n = 17) and healthy individuals (n = 7) using targeted sequencing (lung cancer: n = 12, healthy: n = 7) and whole-genome sequencing (WGS) (lung cancer: n = 5). A personal gene expression profile was established with H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). cfChIP-seq isolates nucleosomes with histone marks of active transcription directly from plasma, meaning cfDNA enrichment with cfChIP reflects gene activity in the cells of origin. Short cfDNA fragments were isolated using in vitro size-selection. Somatic mutations were identified from input cfDNA through the AVENIO pipeline. The findings were validated in an external public dataset comprising of WGS-cfDNA data from healthy individuals. Cancer patients contain a larger fraction of short (<150bp) cfDNA fragments compared to healthy individuals. Cancer patients also demonstrate an enrichment of distinct FEMs compared to healthy individuals. Investigating the association between gene activity and fragment lengths revealed that genes with high expression display an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94% - 27.13%, P < 0.0001) compared to genes with low expression. Furthermore, distinct GC-rich FEMs are enriched in active genes. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs results in a further enrichment of the highest expressed genes (median = 37.85%, IQR: 30.10% - 39.49%, P < 0.0001). In vitro size selection of short cfDNA also results in enrichment of GC-rich FEMs. In vitro size-selection can isolate cfDNA representing active genes and in vitro size-selection enrichment correlates with the cfChIP-seq enrichment (Spearman’s ρ range: 0.499-0.882). This study expands the knowledge regarding cfDNA fragmentomics. We document how cancer patients and healthy individuals have different fragmentomic features. In addition, we shed new light on how gene activity in the cells of origin is associated with cfDNA fragment lengths and FEMs. Together, the results can increase the utility of liquid biopsies by determining gene activity in cancer patients. Citation Format: Christoffer Trier Maansson, Louise Skov Thomsen, Peter Meldgaard, Anders Lade Nielsen, Boe Sandahl Sorensen. Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2290.

Mutant huntingtin protein induces MLH1 degradation, DNA hyperexcision, and cGAS–STING-dependent apoptosis

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.

Effects of manganese glycine on eggshell quality, eggshell ultrastructure, and elemental deposition in aged laying hens

Poor eggshell quality of eggs laid by aged laying hens is the major problem affecting the length of the rearing period in the laying hen industry. Trace elements are required and play vital roles in the eggshell quality of laying hens. Appropriate dose of organic microelements is environmentally friendly and sufficient to satisfy the needs of hens because of their greater bioavailability and lower excretion than inorganic forms. The aim of this experiment was to investigate the effects of manganese (Mn) glycine (MG) on eggshell quality, elemental deposition, and eggshell ultrastructure in aged laying hens. A total of 720 Hy-Line Brown hens 70 weeks old were assigned equally to four groups with six replicates of 30 birds each. The hens were fed basal diets (without Mn supplementation) supplemented with 120 mg/kg of Mn from manganese sulfate monohydrate (MSM), or 40, 80, or 120 mg/kg Mn from MG for 12 weeks. Dietary supplementation with 80 mg/kg Mn from MG resulted in the greatest eggshell strength after 6 weeks of treatment (P = 0.047), and in greater eggshell strength than observed in the MSM control after 12 weeks of treatment (P = 0.025). After 12 weeks of treatment, the eggs of hens in the MG groups showed lower mammillary layer thickness in the blunt end, equator, and acute end than observed in the MSM control group (P < 0.001). With the exception of the blunt ends of eggs from hens in the 120 mg/kg MG group, the eggs of hens in the MG groups, compared with the MSM control group, exhibited a lower mammillary layer ratio, and greater palisade layer ratio and effective layer ratio in the blunt end, equator, and acute end (P < 0.001). Dietary supplementation with 80 mg/kg Mn from MG, compared with the MSM control and 40 and 120 mg/kg MG, resulted in the greatest palisade layer thickness and effective layer thickness, and the lowest mammillary layer thickness in the equator (P < 0.001, P = 0.001, P < 0.001, respectively). Furthermore, supplementation with 80 mg/kg Mn from MG exhibited the greatest ratio of the palisade layer and effective layer, and the lowest mammillary layer ratio in the blunt end and equator (all P < 0.001). The Mn content of eggshells in hens-fed diets supplemented with 80 and 120 mg/kg Mn from MG was greater than that in the MSM control and 40 mg/kg MG groups (P = 0.035). Dietary supplementation with 80 or 120 mg/kg Mn from MG resulted in greater tibia Mn content than observed in the 40 mg/kg MG group (P = 0.019), and greater yolk Mn content than observed in the 40 mg/kg MG and MSM control groups (P = 0.018). In conclusion, dietary supplementation with 80 mg/kg Mn from MG, compared with the MSM control (120 mg/kg Mn), may increase the deposition efficiency of Mn, alter eggshell elemental composition, improve eggshell ultrastructure, and enhance eggshell strength in aged laying hens.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Pulley Effect in the Capture of DNA Translocation through Solid-State Nanopores.

Nanopores are powerful single-molecule sensors for analyzing biomolecules such as DNA and proteins. Understanding the dynamics of DNA capture and translocation through nanopores is essential for optimizing their performance. In this study, we examine the effects of applied voltage and pore diameter on current blockage, translocation time, collision, and capture location by translocating λ-DNA through 5.7 and 16 nm solid-state nanopores. Ionic current changes are used to infer DNA conformations during translocation. We find that translocation time increases with pore diameter, which can be attributed to the decrease of the stall force. Linear and exponential decreases of collision frequency with voltage are observed in the 16 and 5.7 nm pores, respectively, indicating a free energy barrier in the small pore. Moreover, the results reveal a voltage-dependent bias in the capture location toward the DNA ends, which is explained by a "pulley effect" deforming the DNA as it approaches the pore. This study provides insights into the physics governing DNA capture and translocation, which can be useful for promoting single-file translocation to enhance nanopore sensing.

Dominant roles of BRCA1 in cellular tolerance to a chain-terminating nucleoside analog, alovudine

Alovudine is a chain-terminating nucleoside analog (CTNA) that is frequently used as an antiviral and anticancer agent. Generally, CTNAs inhibit DNA replication after their incorporation into nascent DNA during DNA synthesis by suppressing subsequent polymerization, which restricts the proliferation of viruses and cancer cells. Alovudine is a thymidine analog used as an antiviral drug. However, the mechanisms underlying the removal of alovudine and DNA damage tolerance pathways involved in cellular resistance to alovudine remain unclear. Here, we explored the DNA damage tolerance pathways responsible for cellular tolerance to alovudine and found that BRCA1-deficient cells exhibited the highest sensitivity to alovudine. Moreover, alovudine interfered with DNA replication in two distinct mechanisms: first: alovudine incorporated at the end of nascent DNA interfered with subsequent DNA synthesis; second: DNA replication stalled on the alovudine-incorporated template strand. Additionally, BRCA1 facilitated the removal of the incorporated alovudine from nascent DNA, and BRCA1-mediated homologous recombination (HR) contributed to the progressive replication on the alovudine-incorporated template. Thus, we have elucidated the previously unappreciated mechanism of alovudine-mediated inhibition of DNA replication and the role of BRCA1 in cellular tolerance to alovudine.

Absence of both MGME1 and POLG EXO abolishes mtDNA whereas absence of either creates unique mtDNA duplications

Both POLG and MGME1 are needed for mitochondrial DNA (mtDNA) maintenance in animal cells. POLG, the primary replicative polymerase of the mitochondria, has an exonuclease activity (3’→5’) that corrects for the misincorporation of bases. MGME1 serves as an exonuclease (5’→3’), producing ligatable DNA ends. Although both have a critical role in mtDNA replication and elimination of linear fragments, these mechanisms are still not fully understood. Using digital PCR to evaluate and compare mtDNA integrity, we show that Mgme1 knock out (Mgme1 KK) tissue mtDNA is more fragmented than POLG exonuclease deficient “Mutator” (Polg MM) or WT tissue. In addition, next generation sequencing of mutant hearts showed abundant duplications in/nearby the D-loop region and unique 100bp duplications evenly spaced throughout the genome only in Mgme1 KK hearts. However, despite these unique mtDNA features at steady-state, we observed a similar delay in the degradation of mtDNA after an induced double strand DNA break in both Mgme1 KK and Polg MM models. Lastly, we characterized double mutant (Polg MM/Mgme1 KK) cells and show that mtDNA cannot be maintained without at least one of these enzymatic activities. We propose a model for the generation of these genomic abnormalities which suggests a role for MGME1 outside of nascent mtDNA end ligation. Our results highlight the role of MGME1 in and outside of the D-loop region during replication, support the involvement of MGME1 in dsDNA degradation and demonstrate that POLG EXO and MGME1 can partially compensate for each other in maintaining mtDNA.

A di-acetyl-decorated chromatin signature couples liquid condensation to suppress DNA end synapsis

Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.

DNA-PK controls Apollo's access to leading-end telomeres.

The complex formed by Ku70/80 and DNA-PKcs (DNA-PK) promotes the synapsis and the joining of double strand breaks (DSBs) during canonical non-homologous end joining (c-NHEJ). In c-NHEJ during V(D)J recombination, DNA-PK promotes the processing of the ends and the opening of the DNA hairpins by recruiting and/or activating the nuclease Artemis/DCLRE1C/SNM1C. Paradoxically, DNA-PK is also required to prevent the fusions of newly replicated leading-end telomeres. Here, we describe the role for DNA-PK in controlling Apollo/DCLRE1B/SNM1B, the nuclease that resects leading-end telomeres. We show that the telomeric function of Apollo requires DNA-PKcs's kinase activity and the binding of Apollo to DNA-PK. Furthermore, AlphaFold-Multimer predicts that Apollo's nuclease domain has extensive additional interactions with DNA-PKcs, and comparison to the cryo-EM structure of Artemis bound to DNA-PK phosphorylated on the ABCDE/Thr2609 cluster suggests that DNA-PK can similarly grant Apollo access to the DNA end. In agreement, the telomeric function of DNA-PK requires the ABCDE/Thr2609 cluster. These data reveal that resection of leading-end telomeres is regulated by DNA-PK through its binding to Apollo and its (auto)phosphorylation-dependent positioning of Apollo at the DNA end, analogous but not identical to DNA-PK dependent regulation of Artemis at hairpins.

Activating the abscission checkpoint: Top2α senses chromatin bridges in cytokinesis: Top2α binds to DNA knots on chromatin bridges to activate the abscission checkpoint in human cells.

How chromatin bridges are detected by the abscission checkpoint during mammalian cell division is unknown. Here, we discuss recent findings from our lab showing that the DNA topoisomerase IIα (Top2α) enzyme binds to catenated ("knotted") DNA next to the midbody and forms abortive Top2-DNA cleavage complexes (Top2ccs) on chromatin bridges. Top2ccs are then processed by the proteasome to promote localization of the DNA damage sensor protein Rad17 to Top2-generated double-strand DNA ends on DNA knots. In turn, Rad17 promotes local recruitment of the MRN protein complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin bridge breakage in cytokinesis.

Anthracnose of Macleaya cordata Caused by Colletotrichum aenigma in China.

Macleaya cordata (Willd.) R. Br. is a perennial herbaceous medicinal plant (Papaveraceae) commercially cultivated in China which has been studied for detumescence, detoxification, and insecticidal effect (Lin et al. 2018). In August 2021, anthracnose was observed in 2-year-old M. cordata plants in Benxi county, northeast China (41°45'48″N, 123°69'15″E). Dozens of irregular reddish-brown spots (3-11 mm) were observed on each diseased leaf. The lesions were covered with a layer of gray-white mycelia. As the disease progressed, the spots became necrosis and perforation or they would merged into large lesions, ultimately resulting in wilted leaves (Fig. 1). More than 33% of the plants in a 16-ha field were infected in 2021. The diseased leaves were collected and cut into 3-8 mm pieces, surface-disinfested by immersing them into 1% NaOCl for 2 min, and rinsed three times with sterile distilled water. They were then dried with sterilized absorbent paper, placed on PDA medium amended with chloramphenicol (40 mg/L), and incubated in darkness at 25°C with a 12-h photoperiod. Twenty isolates (BLH1 to 20) were obtained and purified using a single-spore method. Isolate BLH12 was identified and used for the pathogenicity test. Colonies were sparsely fluffy with smooth edges, and gradually became gray to pale orange from the initial white. The underside of the colonies was pale orange towards the center. Conidia were single-celled, cylindrical, and transparent with broadly blunt ends, measuring (15.13 ± 1.14) × (5.80 ± 0.60) μm (n=50). Appressoria were single-celled, brown-to-dark brown, usually elliptical or irregular, and sometimes lobed. Setae were not observed. The isolate was initially identified as Colletotrichum gloeosporioides complex (Prihastuti et al. 2009). The identification was confirmed as described previously (Weir et al. 2012). The rDNA internal transcribed spacer region (OP415560), the glyceraldehyde-3-phosphate dehydrogenase (OP433642), chitin synthase (OP433643), calmodulin (OP433644), actin (OP433645), glutamine synthetase (OP433646), β-tubulin (OP433647), and superoxide dismutase (OP433648) gene sequences were obtained (Carbone & Kohn 1999; Weir et al. 2012), and BLAST searches revealed 99-100% homology with the type culture ICMP 18608 (JX010244, JX010044, JX009683, JX009443, JX009744, JX010078, JX010389, and JX010311). A phylogenetic analysis of combining all loci indicated BLH12 and the type strain of C. aenigma were clustered in one group (Fig. 2). Based on the basis of morphological characteristics and phylogenetic relationships, BLH12 was identified as C. aenigma. For the pathogenicity test, healthy 2-year-old plants were sprayed with a BLH12 spore suspension (1 × 105/mL). Control plants were sprayed with sterile water.There were three replicates (five plants each) per treatment. All plants were incubated at 25°C (12-h photoperiod and 86% relative humidity) and examined after 7 days. The experiment was repeated twice. The inoculated plants showed lesions on the leaf surface, similar to those in the field, whereas the control plants were asymptomatic. The pathogen was successfully reisolated and identified as the methods mentioned above. This fungus reportedly infects the leaves of many woody plants in China (Wang et al. 2020; Zhang et al. 2021). This is the first report of C. aenigma causing anthracnose on M. cordata, which will provide an guideline for developing effective field control practices for the disease.

Henneguya (Cnidaria: Myxobolidae) species infecting Oligosarcus jenynsii (Characiformes: Characidae) in a Neotropical shallow lake from Argentina: morphological and molecular characterisation.

Two previously undescribed myxozoan species, Henneguya sardellae sp. n. and H. margaritae sp. n., found infecting connective tissues of the Neotropical characid fish Oligosarcus jenynsii (Günther) from Argentina are morphologically and molecularly characterised. Mature spores of H. sardellae sp. n. are ellipsoid, with two, straight and visibly fused caudal appendages cleaved at its blunt terminal end; measuring 33.5 ± 1.2 (30.9-35.5) μm in total length, spore body 17.5 ± 0.6 (16.3-18.6) µm, 7.8 ± 0.4 (7.0-8.8) µm wide and 6.9 ± 0.2 (6.6-7.2) µm thick, with two elongated, unequally-sized polar capsules situated at anterior end, and 11-13 turns of polar tubules. Mature spores of H. margaritae sp. n. are pyriform, with two caudal appendages visible fused together and much longer than spore body, with unequal endings; measuring 35.9 ± 2.8 (29.2-40.7) µm in total length, spore body 11.5 ± 0.9 (9.2-13.0) µm long, 5.8 ± 0.4 (5.1-6.7) µm wide and 5.5 ± 0.2 (5.1-5.8) µm thick, with two polar capsules similar in size, pyriform polar capsules containing polar tubules with 4-5 coils. Both species showed a membraneous sheath surrounding the spore body and caudal appendages; in H. sardellae sp. n. this feature can deploy laterally. Phylogenetic analyses based on SSU rDNA sequences showed that H. sardellae sp. n. and H. margaritae sp. n. clustered with other myxobolids parasitising Characiformes in Brazil, Cichliformes in Mexico and Cyprinodontiformes in Mexico and the United States. The description of these two new species of Henneguya as the first described species of the genus that parasitise freshwater fish in Argentina highlights the importance of further research on the diversity and distribution of myxozoans in this region.

Bisphosphonate's effect on the tongue in adult male albino rats and the possible protective role of rutin: light and scanning electron microscopic study.

Alendronate sodium (ALS) is a nitrogen-containing bisphosphonate used for the treatment of different bone disorders. However, its adverse effect on oral soft tissue has been detected. Rutin (RUT) is natural flavonoid with antioxidant and anti-inflammatory properties. This work aimed to investigate the possible effect of ALS on the tongue of adult male albino rats and to evaluate the possible protective role of RUT. Forty adult male albino rats were equally divided into four groups: group I (control), group II (RUT): Received RUT 50 mg/kg, group III (ALS): Received ALS 1 mg/kg, group IV (ALS+RUT): Received ALS and RUT with the same doses as pervious groups. The drugs were given once daily for 5 weeks. Tongue specimens were taken and processed for light and scanning electron microscopic inspection. ALS treated group revealed structural changes in the tongue in the form of decrease in the height of the filiform papillae with blunt ends, marked atrophy in some papillae with areas of focal loss, loss of some epithelial cells, pyknotic nuclei and cytoplasmic vacuoles in some epithelial cells. The lamina propria showed inflammatory cellular infiltration with congested blood vessels. Statistically, there were highly significant decrease in the number of proliferating cell nuclear antigen immunopositive cells, area percentage of Bcl-2 immunoexpression and highly significant increase in the collagen content compared to control group. Administration of RUT with ALS minimizes these changes. RUT protected the rat tongue against the histological and immunohistochemical changes induced by ALS through its antioxidant and anti-inflammatory properties.

Structural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining

Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.

NUDT16 regulates CtIP PARylation to dictate homologous recombination repair.

CtIP initiates DNA end resection and mediates homologous recombination (HR) repair. However, the underlying mechanisms of CtIP regulation and how the control of its regulation affects DNA repair remain incompletely characterized. In this study, NUDT16 loss decreases CtIP protein levels and impairs CtIP recruitment to double-strand breaks (DSBs). Furthermore, overexpression of a catalytically inactive NUDT16 mutant is unable to rescue decreased CtIP protein and impaired CtIP recruitment to DSBs. In addition, we identified a novel posttranslational modification of CtIP by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to CtIP ubiquitination and degradation. These data suggest that the hydrolase activity of NUDT16 plays a major role in controlling CtIP protein levels. Notably, ADP-ribosylation of CtIP is required for its interaction with NUDT16, its localization at DSBs, and for HR repair. Interestingly, NUDT16 can also be ADP-ribosylated. The ADP-ribosylated NUDT16 is critical for CtIP protein stability, CtIP recruitment to DSBs, and HR repair in response to DNA damage. In summary, we demonstrate that NUDT16 and its PARylation regulate CtIP stability and CtIP recruitment to DSBs, providing new insights into our understanding of the regulation of CtIP-mediated DNA end resection in the HR repair pathway.

Ccq1 restrains Mre11-mediated degradation to distinguish short telomeres from double-strand breaks.

Telomeres protect chromosome ends and are distinguished from DNA double-strand breaks (DSBs) by means of a specialized chromatin composed of DNA repeats bound by a multiprotein complex called shelterin. We investigated the role of telomere-associated proteins in establishing end-protection by studying viable mutants lacking these proteins. Mutants were studied using a Schizosaccharomyces pombe model system that induces cutting of a 'proto-telomere' bearing telomere repeats to rapidly form a new stable chromosomal end, in contrast to the rapid degradation of a control DSB. Cells lacking the telomere-associated proteins Taz1, Rap1, Poz1 or Rif1 formed a chromosome end that was stable. Surprisingly, cells lacking Ccq1, or impaired for recruiting Ccq1 to the telomere, converted the cleaved proto-telomere to a rapidly degraded DSB. Ccq1 recruits telomerase, establishes heterochromatin and affects DNA damage checkpoint activation; however, these functions were separable from protection of the new telomere by Ccq1. In cells lacking Ccq1, telomere degradation was greatly reduced by eliminating the nuclease activity of Mre11 (part of the Mre11-Rad50-Nbs1/Xrs2 DSB processing complex), and higher amounts of nuclease-deficient Mre11 associated with the new telomere. These results demonstrate a novel function for S. pombe Ccq1 to effect end-protection by restraining Mre11-dependent degradation of the DNA end.

PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

Regulation of DNA end resection by the Werner syndrome helicase

Regulation of DNA end resection by the Werner syndrome helicase

The role of RNF138 in DNA end resection is regulated by ubiquitylation and CDK phosphorylation

Double-strand breaks (DSBs) are DNA lesions that pose a significant threat to genomic stability. The repair of DSBs by the homologous recombination (HR) pathway is preceded by DNA end resection, the 5' to 3' nucleolytic degradation of DNA away from the DSB. We and others previously identified a role for RNF138, a really interesting new gene finger E3 ubiquitin ligase, in stimulating DNA end resection and HR. Yet, little is known about how RNF138's function is regulated in the context of DSB repair. Here, we show that RNF138 is phosphorylated at residue T27 by cyclin-dependent kinase (CDK) activity during the S and G2 phases of the cell cycle. We also observe that RNF138 is ubiquitylated constitutively, with ubiquitylation occurring in part on residue K158 and rising during the S/G2 phases. Interestingly, RNF138 ubiquitylation decreases upon genotoxic stress. By mutating RNF138 at residues T27, K158, and the previously identified S124 ataxia telangiectasia mutated phosphorylation site (Han etal., 2016, ref. 22), we find that post-translational modifications at all three positions mediate DSB repair. Cells expressing the T27A, K158R, and S124A variants of RNF138 are impaired in DNA end resection, HR activity, and are more sensitive to ionizing radiation compared to those expressing wildtype RNF138. Our findings shed more light on how RNF138 activity is controlled by the cell during HR.