DNA Ends Research Articles

HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro

Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.

Dbf4-dependent kinase promotes cell cycle controlled resection of DNA double-strand breaks and repair by homologous recombination

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.

First report of Dactylonectria pauciseptata causing root rot on Eucalyptus cinerea in China.

Eucalyptus cinerea is an evergreen tree in the Myrtaceae. It is native to southern and eastern New South Wales and northern and eastern Victoria, Australia. It was introduced into China in the 1980s (Silva et al. 2011). Because of its unique shape, flexible stems, and rapid growth characteristics, it is widely used in the pulp industry and in decorative materials such as flower bouquets. In July 2022, 5- to 10-year-old E. cinerea showing symptoms of dehydration, withering and yellowing leaves, were found in forests and nurseries in Kunming and Songming, China. More than 37% of the trees showed these symptoms at each location, and disease severity was about 30%. Sixty symptomatic plants were collected from five tree nurseries. Diseased roots with 2-cm-long lesions were soaked in 75% ethanol for 15 s, 0.1% mercuric chloride for 2 min, rinsed with sterilized water, and placed on potato dextrose agar (PDA) at 25℃ for 3 days. Thirty samples were plated, and 21 isolates (YJLGF01 to YJLGF21) obtained, 11 strains with similar colony morphology (including representative strains YJLGF03 to YJLGF05). Three isolates (YJLGF03 to YJLGF05) were obtained by single-spore purification. On PDA, the colonies were circular with fluffy white to light yellow mycelium; the underside was yellowish brown. Conidiophores were bifurcated, with macroconidia borne terminally. The macroconidia were cylindrical with rounded, blunt ends, yellow to transparent, 1 to 3 septate (22.5 to 47.6 × 4.5 to 7.1 µm); microconidia were 0 to 1 septate (12.5 to 19.6 × 4.7 to 6.4 µm). Chlamydospores were spherical, rosary-like, and light yellow. Morphological characteristics were consistent with published descriptions of Dactylonectria pauciseptata (Piperkova et al. 2017). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1- alpha (ef1-α) gene, and the beta-tubulin 2 (β-tub2) gene were amplified and sequenced (ITS accessions OR735053, OR735054, OR735055; β-tub2 accessios OR757447, OR757448, OR757449; ef1-α accessions OR757450, OR757451, OR757451) using published primers (White et al. 1990; Carbone et al. 1999). A phylogenetic tree was developed by Maximum Parsimony (MP) and Maximum Likelihood (ML) methods. These three isolates fell into the D. pauciseptata clade and were distinguished clearly from other species. Pathogenicity tests were performed using the same three isolates. Each isolate was cultured on PDA, and then subcultured in V8 juice broth on an orbital shaker at 180 RPM for 5 days. Conidia were collected by centrifugation at 6,000 RPM for 5 min, and then resuspended in sterilized distilled water (1×106 conidia/ml). Injured roots of one-year-old E. cinerea were soaked in the spore suspension for 1 h before being transplanted in sterile vermiculite. The plants were incubated at 25℃ with a 12 h photoperiod and 90% humidity. Five plants were inoculated as a group for each treatment and the entire experiment was completed three times. Among the inoculated plants, the incidence of disease development was 100%. A small sot appeared after 4 days, with a water-soaked lesion appearing and gradually expanding during days 5 to 7. After 10 days symptoms of root necrosis were similar to the those observed in the nursery, and aboveground plant parts had yellow, withering leaves and defoliation after 10 to 15 days. Control plants treated with sterile water showed no disease symptoms. The three strains were successfully reisolated from inoculated seedlings and confirmed them using DNA sequencing. No isolates were obtained from the control plants, thus fulfilling Koch's postulates. Dactylonectria pauciseptata was first reported from necrotic tissue of infected grape roots (Schroers et al. 2008). So far, it has been reported in Turkey, Canada, Brazil, Italy, and other countries (Erper et al. 2013; Úrbez-Torres et al. 2014; Santos et al. 2014). Based on our results, E. cinerea is a new host plant of D. pauciseptata in China. This disease is a threat to the nursery production of E. cinerea, potentially leading to a reduction in yields and economic losses.

Monitoring Anthracycline Cancer Drug-Nucleosome Interaction by NMR Using a Specific Isotope Labeling Approach for Nucleosomal DNA.

Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions. We describe the synthesis of segmental one-strand 13C-thymidine labeled 601-DNA, the assignment of the methyl signals, and demonstrate its use to observe site-specific binding to the nucleosome by aclarubicin, an anthracycline cancer drug that intercalates into the DNA minor grooves. Our results highlight intrinsic conformational heterogeneity in the 601 DNA sequence and show that aclarubicin binds an exposed AT-rich region near the DNA end. Overall, our data point to a model where the drug invades the nucleosome from the terminal ends inward, eventually resulting in histone eviction and nucleosome disruption.

Structures of LIG1 provide a mechanistic basis for understanding a lack of sugar discrimination against a ribonucleotide at the 3'-end of nick DNA

Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three-steps of ligation reaction. LIG1 can tolerate the RNA strand at the upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with preinserted 3'-ribonucleotides as efficient as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.

Abstract 329: NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology

Abstract Background: Telomeres are specialized nucleoprotein structures located at the end of linear chromosomes in eukaryotic cells, and protect the ends of chromosomal DNA from degradation, end-to-end fusion and abnormal recombination. Changes in telomere length have become an important biomarker, which is closely related to cellular aging, all-cause mortality and a variety of diseases. However, there are still challenges in quantifying telomere absolute length, due to repeats of nucleotide sequence (5’-TTAGGG-3’)n in single-stranded G-rich telomeric region, and lack of a method for nucleotide separation and enrichment of individual telomeres. Methods: We developed NanoTelo, a workflow for detecting telomere absolute length based on nanopore sequencing technology. For experimental steps, a self-designed telomere adapter was first connected to the C-rich strand at the end of the chromosome by utilizing the 3' end overhang. And the 5' end of the telomere adapter was complementary to the sequencing adapter, allowing the antisense telomere chain to perform single-strand extension sequencing in the 5' to 3' direction, thus into the sub-telomeric region from the telomere end. The sequencing library was nanopore-sequenced on the QPursue-6k according to the manufacturer’s instructions. For data analysis, reads were strictly extracted (dataset B) with the telomere adapter and library structure in raw sequencing data (dataset A). The reads of dataset A and dataset B were corrected, aligned to the human reference genome (T2T-CHM13v2.0), and calculated the telomere length distribution of each chromosomal (autosomes and sex chromosomes) p/q-arm, respectively. Defined the mean telomere length of all chromosomes represented the overall telomere length, the estimated results of dataset A and dataset B were weighted to obtain the final telomere absolute length. Results: We selected 5 cell lines, which was HCT116, 293T, T24, IMR90 and WI38, and performed 2 experimental replicates for each one. We found that the estimated telomere lengths for each cell line were 3,528/3,512 bp, 4,298/4,311 bp, 2,745/2,685 bp, 4,926/4,915 bp and 4,143/4,096 bp, respectively. A comparative analysis with results from the Pacbio sequencing platform for each cell line in the Tham et al.'s paper, showed that there was no significant statistical difference (two-tailed T-test: p-value=0.98), and the mean absolute difference for estimated telomere length between the two platforms was 7.7 bp (95% CI: 5.4-10.0 bp). Conclusions: NanoTelo demonstrated the possibility of measuring telomere absolute length with a single chromosomal p/q-arm at nucleotide-level resolution. We hope that NanoTelo can not only be used as an important tool for estimating telomere length to dynamically monitor aging, but also provide strong technical support for scientific research and/or clinical application related to human anti-aging and disease intervention. Citation Format: Jidong Lang, Yanmei Chen, Zhi Yang, Beibei Huo. NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology [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 329.

Abstract 325: Validation of Aventa FusionPlus: An LDT NGS assay for comprehensive cancer genomic profiling

Abstract Next-generation sequencing (NGS) assays play a pivotal role in illuminating the genomic landscape of cancer by providing insights into gene regulation and biomarker detection for the purpose of guiding therapeutic decisions to advance personalized medicine. We validated the Aventa FusionPlus Test, a Laboratory-Developed Test (LDT) that integrates HiC technology to enhance detection of actionable gene fusions and rearrangements in solid tumors, including those that are missed by current standard of care tests. This is a DNA-based test that is robust to DNA degradation in FFPE specimens, in contrast with RNA-seq and long-read technologies. The test utilizes the Element Biosciences AVITI sequencing platform to achieve high quality sequencing data at low cost per sample. The Aventa FusionPlus test enables gene-level detection of structural variants near or within 361 medically actionable or diagnostically relevant cancer genes. The 3D structure and long-range linkages captured by the test amplify the signal counts for each rearrangement. The test integrates the Arima-HiC+ FFPE sample prep and the Arima Library Prep Kit workflow. After dewaxing and rehydrating the FFPE tissue, the chromatinized DNA is fragmented and the fragmented ends are filled in with a biotinylated nucleotide. Next, spatially proximal fragmented ends of DNA are ligated, capturing the 3D structure of the genome. The proximally-ligated DNA is then purified and further fragmented, and the biotinylated ligation fragments are enriched. DNA libraries are prepared from these enriched fragments and sequenced in “paired-end” mode. Genes involved in genomic rearrangements were detected using the Arima-SV pipeline along with manual curation. The test achieved robust detection of structural variants, with accuracy of 98.8% relative to reference methods. FFPE tumor specimens from 10 different tissue types and a wide range of tumor content (20%-100%) were included. No correlation between tumor cellularity and sensitivity was observed, suggesting that samples with as low as 20% tumor cellularity can be tested. When comparing to RNA-based and other targeted reference methods, we found additional fusions nearby oncogenes missed by these reference methods. As an example, in a breast cancer case, a fusion of genomic regions neighboring BRAF and MYC oncogenes on chromosomes 7 and 8 respectively, was missed by the reference method but was successfully detected by the Aventa FusionPlus test. The Aventa FusionPlus test is a highly sensitive, specific, and accurate method for comprehensive cancer genomic profiling. The enhanced understanding of gene fusions and rearrangements positions the test as a valuable tool for clinical decision-making in oncology. Citation Format: Shadi Melnyk, Jon Belton, Sofia Nomikou, Katelyn Powell, Daniel Heiney, Roy Khalife, Damien Jones, Anthony Schmitt, Catherine Wilson, Melissa Won, Anthony Magliocco, Chris Roberts. Validation of Aventa FusionPlus: An LDT NGS assay for comprehensive cancer genomic profiling [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 325.

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.