Biology subjects

Gartner, A.

Publications and source records attributed to Gartner, A..

14 recordsLinked to original sources

bioRxiv2026

Multiple ShKT domain-containing MUL-1 proteins act as redox-responsive modulators of oxidative stress signaling in C. elegans

Organismal survival depends on coordinated responses to oxidative stress and DNA damage. Using Caenorhabditis elegans, we investigate mul-1, a robust transcriptional target of ionizing radiation and reactive oxygen species. Although annotated as a mucin, MUL-1 is a small ShKT domain-containing protein belonging to an invertebrate expanded family of cysteine-rich proteins. mul-1 is selectively induced by oxidative stress, including IR, hydrogen peroxide (H2O2), Pseudomonas aeruginosa infection, or loss of the peroxiredoxin PRDX-2, via the p38 MAPK-ATF-7 pathway in intestinal cells. Loss of mul-1 and its paralogs increases ROS accumulation, oxidative stress sensitivity, and CEP-1/p53 dependent germ cell apoptosis. Combined deletion of mul-1 paralogs causes constitutive apoptosis, reduced fecundity, and compensatory activation of DAF-16/Foxo and SKN-1/Nrf2 stress response pathways. Together with genetic analysis of SYSM-1, these findings suggest MUL-1-like ShKT proteins buffer oxidative stress.

genetics↗

bioRxiv2024

Functional dissection of the conserved C. elegans LEM-3/ANKLE1 nuclease reveals a crucial requirement for the LEM-like and GIY-YIG domains for DNA bridges processing

Faithful chromosome segregation requires the removal of all DNA bridges physically linking chromatids before the completion of cell division. While several redundant safeguard mechanisms to process these DNA bridges exist from S-phase to late anaphase, the conserved LEM-3/ANKLE1 nuclease has been proposed to be part of a last chance mechanism that acts at the midbody to eliminate DNA bridges that persist until late cytokinesis. We show that LEM-3 can cleave a wide range of branched DNA substrates, including flaps, forks, nicked and intact Holliday Junctions. AlphaFold modeling data suggest that the catalytic mechanism of LEM-3/ANKLE1 is conserved, mirroring the mechanism observed in bacterial GIY-YIG nucleases. We also present evidence that LEM-3 may form a homodimeric complex on the Holliday Junction DNA. LEM-3 DNA binding capacity requires both the LEM-like and the GIY-YIG nuclease domains; both are also essential for LEM-3 recruitment to the midbody and its nuclease activity. Finally, we show that preventing LEM-3 nuclear access is important to avoid toxicity, likely caused by branched DNAs cleavage during normal DNA metabolism. Our data suggest that C. elegans LEM-3 acts as a last chance catch-all enzyme that processes DNA bridges caused by various perturbations of DNA metabolism just before cells divide.

biochemistry↗

bioRxiv2024

Comprehensive Whole Genome Sequencing Reveals Origins of Mutational Signatures Associated with Aging and Temozolomide Chemotherapy

In a comprehensive study to decipher the multi-layered response to the chemotherapeutic agent temozolomide (TMZ), we analyzed 427 genomes and determined mutational patterns in a collection of [~]40 isogenic DNA repair-deficient human TK6 lymphoblast cell lines. We demonstrate that the spontaneous mutational background is very similar to the aging-associated mutational signature SBS40 and mainly caused by polymerase zeta-mediated translesion synthesis (TLS). MSH2-/- mismatch repair knockout in conjunction with additional repair deficiencies uncovers cryptic mutational patterns. We report how distinct mutational signatures are induced by TMZ upon sequential inactivation of DNA repair pathways, mirroring the acquisition of chemotherapy resistance by glioblastomas. The most toxic adduct induced by TMZ, O6-meG, is directly repaired by the O6-methylguanine-DNA methyltransferase (MGMT). In MGMT-/- cells, mismatch repair (MMR) leads to cell death and limits mutagenesis. MMR deficiency results in TMZ resistance, allowing the accumulation of [~]105 C>T substitutions corresponding to signature SBS11. Under these conditions, N-alkylated bases, processed by base excision repair (BER), limit cell survival. Without BER, 3-meA is read through via error-prone TLS, causing T>A substitutions but not affecting survival. Blocking BER after abasic site formation results in large deletions and TMZ hypersensitization. Our findings reveal potential vulnerabilities of TMZ-resistant tumors.

genomics↗

bioRxiv2023

The histone chaperone activity of SPT2 controls chromatin structure and function in Metazoa

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans orthologue of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at these loci. We also show that human SPT2 controls the incorporation of new H3.3 into chromatin. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

molecular biology↗

bioRxiv2022

Disparate roles for C. elegans DNA translocase paralogs RAD-54.L and RAD-54.B in meiotic prophase germ cells

RAD54 family DNA translocases partner with RAD51 recombinases to ensure stable genome inheritance, exhibiting biochemical activities both in promoting recombinase removal and in stabilizing recombinase association with DNA. Understanding how such disparate activities of RAD54 paralogs align with their biological roles is an ongoing challenge. Here we investigate the in vivo functions of C. elegans RAD54 paralogs RAD-54.L and RAD-54.B during meiotic prophase, revealing distinct contributions to the dynamics of RAD-51 association with DNA and to the progression of meiotic double-strand break repair (DSBR). While RAD-54.L is essential for RAD-51 removal from meiotic DSBR sites to enable recombination progression, RAD-54.B is largely dispensable for meiotic DSBR. However, RAD-54.B is required to prevent hyperaccumulation of RAD-51 on unbroken DNA during a key meiotic sub-stage when protein kinase CHK-2 is active. Moreover, DSB-independent hyperaccumulation of RAD-51 foci in the absence of RAD-54.B is RAD-54.L-dependent, revealing a hidden activity of RAD-54.L in promoting promiscuous RAD-51 association that is antagonized by RAD-54.B. We propose a model wherein a division of labor among RAD-54 paralogs allows germ cells to ramp up their capacity for efficient homologous recombination that is crucial to successful meiosis while counteracting potentially deleterious effects of unproductive RAD-51 association with unbroken DNA.

genetics↗

bioRxiv2020

Human ANKLE1 is a nuclease specific for branched DNA

ABSTRACTAll physical connections between sister chromatids must be broken before cells can divide, and eukaryotic cells have evolved multiple ways in which to process branchpoints connecting DNA molecules separated both spatially and temporally. A single DNA link between chromatids has the potential to disrupt cell cycle progression and genome integrity, so it is highly likely that cells require a nuclease that can process remaining unresolved and hemi-resolved DNA junctions and other branched species at the very late stages of mitosis. We argue that ANKLE1 probably serves this function in human cells (LEM-3 in C. elegans). LEM-3 has previously been shown to be located at the cell mid-body, and we show here that human ANKLE1 is a nuclease that cleaves a range of branched DNA species. It thus has the substrate selectivity consistent with an enzyme required to process a variety of unresolved and hemi-resolved branchpoints in DNA. Our results imply that ANKLE1 acts as a catch-all enzyme of last resort that allows faithful chromosome segregation and cell division to occur.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Systematic analysis of mutational spectra associated with DNA repair deficiency in C. elegans

Genome integrity is particularly important in germ cells to faithfully preserve genetic information across generations. As yet little is known about the contribution of various DNA repair pathways to prevent mutagenesis. Using the C. elegans model we analyse mutational spectra that arise in wild-type and 61 DNA repair and DNA damage response mutants cultivated over multiple generations. Overall, 44% of lines show >2-fold increased mutagenesis with a broad spectrum of mutational outcomes including changes in single or multiple types of base substitutions induced by defects in base excision or nucleotide excision repair, or elevated levels of 50-400 bp deletions in translesion polymerase mutants rev-3(pol {zeta}) and polh-1(pol {eta}). Mutational signatures associated with defective homologous recombination fall into two classes: 1) mutants lacking brc-1/BRCA1 or rad-51/RAD51 paralogs show elevated base substitutions, indels and structural variants, while 2) deficiency for MUS-81/MUS81 and SLX-1/SLX1 nucleases, and HIM-6/BLM, HELQ-1/HELQ and RTEL-1/RTEL1 helicases primarily cause structural variants. Genome-wide investigation of mutagenesis patterns identified elevated rates of tandem duplications often associated with inverted repeats in helq-1 mutants, and a unique pattern of translocation events involving homeologous sequences in rip-1 paralog mutants. atm-1/ATM DNA damage checkpoint mutants harboured complex structural variants enriched in subtelomeric regions, and chromosome end-to-end fusions. Finally, while inactivation of the p53-like gene cep-1 did not affect mutagenesis, combined brc-1 cep-1 deficiency displayed increased, locally clustered mutagenesis. In summary, we provide a global view of how DNA repair pathways prevent germ cell mutagenesis.

genomics↗

bioRxiv2019

Mutational signatures are jointly shaped by DNA damage and repair

Mutations arise when DNA lesions escape DNA repair. To delineate the contributions of DNA damage and DNA repair deficiency to mutagenesis we sequenced 2,717 genomes of wild-type and 53 DNA repair defective C. elegans strains propagated through several generations or exposed to 11 genotoxins at multiple doses. Combining genotoxin exposure and DNA repair deficiency alters mutation rates or leads to unexpected mutation spectra in nearly 40% of all experimental conditions involving 9/11 of genotoxins tested and 32/53 genotypes. For 8/11 genotoxins, signatures change in response to more than one DNA repair deficiency, indicating that multiple genes and pathways are involved in repairing DNA lesions induced by one genotoxin. For many genotoxins, the majority of observed single nucleotide variants results from error-prone translesion synthesis, rather than primary mutagenicity of altered nucleotides. Nucleotide excision repair mends the vast majority of genotoxic lesions, preventing up to 99% of mutations. Analogous mutagenic DNA damage-repair interactions can also be found in cancers, but, except for rare cases, effects are weak owing to the unknown histories of genotoxic exposures and DNA repair status. Overall, our data underscore that mutation spectra are joint products of DNA damage and DNA repair and imply that mutational signatures computationally derived from cancer genomes are more variable than currently anticipated.

genomics↗

bioRxiv2019

The NEDD8 cycle controlled by NEDP1 upon DNA damage is a regulatory module of the HSP70 ATPase activity

Ubiquitin and ubiquitin-like chains are finely balanced by the action of conjugating and de-conjugating enzymes. Alterations in this balance trigger signalling events required for the response to stress conditions and are often observed in pathologies. How such changes are detected is not well-understood. We show that upon DNA damage the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of poly-NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerisation and apoptosis induction, a step that requires the HSP70 ATPase activity. We found that HSP70 binds to NEDD8 and in vitro, mono-NEDD8 stimulates the ATPase activity of HSP70, counteracted upon poly-NEDDylation. This effect is independent of NEDD8 conjugation onto substrates. The studies identify the HSP70 chaperone as sensor of changes in the NEDD8 cycle, providing mechanistic insights for a cytoplasmic role of NEDD8 in the DNA damage induced apoptosis. They also indicate that the balance between mono- versus poly-NEDDylation is a regulatory module of HSP70 function. The above findings may be important in tumorigenesis, as we find that NEDP1 levels are downregulated in Hepatocellular Carcinoma with concomitant accumulation of NEDD8 conjugates.

molecular biology↗

bioRxiv2017

The conserved LEM-3/Ankle1 nuclease is involved in the combinatorial regulation of meiotic recombination repair and chromosome segregation in Caenorhabditis elegans

Homologous recombination is essential for crossover (CO) formation and accurate chromosome segregation during meiosis. It is of considerable importance to work out how recombination intermediates are processed leading to CO and non-crossover (NCO) outcome. Genetic analysis in budding yeast and Caenorhabditis elegans indicates that the processing of meiotic recombination intermediates involves a combination of nucleases and DNA repair enzymes. We previously reported that in C. elegans meiotic Holiday junction resolution is mediated by two redundant pathways, conferred by the SLX-1 and MUS-81 nucleases, and by the HIM-6 Blooms helicase in conjunction with the XPF-1 endonucleases, respectively. Both pathways require the scaffold protein SLX-4. However, in the absence of all these enzymes residual processing of meiotic recombination intermediates still occurs and CO formation is reduced but not abolished. Here we show that the LEM-3 nuclease, mutation of which by itself does not have an overt meiotic phenotype, genetically interacts with slx-1 and mus-81 mutants, the respective double mutants leading to 100% embryonic lethality. LEM-3 and MUS-81 act redundantly, their combined loss leading to a reduced number of early meiotic recombination intermediates, to a delayed disassembly of foci associated with CO designated sites, and to the formation of univalents linked by SPO-11 dependent chromatin bridges (dissociated bivalents). However, LEM-3 foci do not co-localize with ZHP-3 a marker that congresses into CO designated sites. In addition, neither CO frequency nor distribution is altered in lem-3 single mutants or in combination with mus-81 or slx-4 mutations, indicating that LEM-3 drives NCO outcome. Finally, we found persistent chromatin bridges during meiotic divisions in lem-3; slx-4 double mutants. Supported by the localization of LEM-3 between dividing meiotic nuclei, this data suggests that LEM-3 is able to process erroneous recombination intermediates that persist into the second meiotic divisions.\n\nAuthor SummaryMeiotic recombination is required for genetic diversity and for the proper chromosome segregation. Recombination intermediates, such as Holliday junctions (HJs), are generated and eventually resolved to produce crossover (CO) and non-crossover (NCO). While an excess of meiotic double-strand breaks is generated, most breaks are repaired without leading to a CO outcome and usually only one break for each chromosome pair matures into a CO-designated site in Caenorhabditis elegans. Resolution of meiotic recombination intermediates and CO formation have been reported to be highly regulated by several structure-specific endonucleases and the Bloom helicase. However, little is known about enzymes involved in the NCO recombination intermediate resolution. In this study, we found that a conserved nuclease LEM-3/Ankle1 acts in parallel to the SLX-1/MUS-81 pathway to process meiotic recombination intermediates. Mutation of lem-3 has no effect on CO frequency and distribution, indicating LEM-3 functions as a nuclease promoting NCO outcome. Interestingly, a prominent localization of LEM-3 is found between dividing meiotic nuclei. We provide evidence that LEM-3 is also involved in processing remaining, erroneous recombination intermediates during meiotic divisions.

cell biology↗

bioRxiv2017

LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during cytokinesis

Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a new genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 locally processes chromatin bridges that arise from incomplete DNA replication, unresolved recombination intermediates or the perturbance of chromosome structure. Proper LEM-3 midbody localization and function is regulated by AIR-2/Aurora B kinase. Strikingly, LEM-3 act cooperatively with the BRC-1/BRCA1 homologous recombination factor to promote genome integrity. These findings provide a molecular basis for the suspected role of the LEM-3 orthologue Ankle1 in human breast cancer.

cell biology↗

bioRxiv2017

6-OHDA-induced dopaminergic neurodegeneration in C. elegans is promoted by the engulfment pathway and inhibited by the transthyretin-related protein TTR-33

Oxidative stress is linked to many pathological conditions including the loss of dopaminergic neurons in Parkinsons disease. The vast majority of disease cases appear to be caused by a combination of genetic mutations and environmental factors. We screened for genes protecting Caenorhabditis elegans dopaminergic neurons from oxidative stress induced by the neurotoxin 6-hydroxydopamine (6-OHDA) and identified the transthyretin-related gene ttr-33. The only described C. elegans transthyretin-related protein to date, TTR-52, has been shown to mediate corpse engulfment as well as axon repair. We demonstrate that TTR-52 and TTR-33 have distinct roles. TTR-33 is likely produced in the posterior arcade cells in the head of C. elegans larvae and is predicted to be a secreted protein. TTR-33 protects C. elegans from oxidative stress induced by paraquat or H2O2 at an organismal level. The increased oxidative stress sensitivity of ttr-33 mutants is alleviated by mutations affecting the KGB-1 MAPK kinase pathway, whereas it is enhanced by mutation of the JNK-1 MAPK kinase. Finally, we provide genetic evidence that the C. elegans cell corpse engulfment pathway is required for the degeneration of dopaminergic neurons after exposure to 6-OHDA. In summary, we describe a new neuroprotective mechanism and demonstrate that TTR-33 normally functions to protect dopaminergic neurons from oxidative stress-induced degeneration, potentially by acting as a secreted sensor or scavenger of oxidative stress.\n\nAuthor summaryAnimals employ multiple mechanisms to prevent their cells from damage by reactive oxygen species, chemically reactive molecules containing oxygen. Oxidative stress, caused by the overabundance of reactive oxygen species or a decreased cellular defence against these chemicals, is linked to a variety of neurodegenerative conditions, including the loss of dopaminergic neurons in Parkinsons disease. In this study, we discovered a novel protective molecule that functions to prevent dopaminergic neurodegeneration caused by oxidative stress induced by the neurotoxin 6-hydroxydopamine (6-OHDA). We used the nematode C. elegans, a well-characterised model in which mechanisms can be studied on an organismal level. When C. elegans is exposed to 6-OHDA, its dopaminergic neurons gradually die. Our major findings include (i) mutations of the transthyretin-related gene ttr-33 causes highly increased dopaminergic neurodegeneration after 6-OHDA exposure; (ii) TTR-33 is likely produced and secreted by several cells in the head of the animal; (iii) TTR-33 protects against oxidative stress induced by other compounds; (iv) mutations in the KGB-1 MAP kinase stress pathway alleviate dopaminergic neuron loss in the ttr-33 mutant; and (v) the cell corpse engulfment pathway is required for dopaminergic neurodegeneration. We hypothesise that TTR-33 protects dopaminergic neurons against 6-OHDA-induced oxidative stress by acting as an oxygen sensor or scavenger.

genetics↗

bioRxiv2017

Mutations in C. elegans neuroligin-like glit-1, the apoptosis pathway and the calcium chaperone crt-1 increase dopaminergic neurodegeneration after 6-OHDA treatment

The loss of dopaminergic neurons is a hallmark of Parkinsons disease, the aetiology of which is associated with increased levels of oxidative stress. We used C. elegans to screen for genes that protect dopaminergic neurons against oxidative stress and isolated glit-1 (gliotactin (Drosophila neuroligin-like) homologue). Loss of the C. elegans neuroligin-like glit-1 causes increased dopaminergic neurodegeneration after treatment with 6-hydroxydopamine (6-OHDA), an oxidative- stress inducing drug that is specifically taken up into dopaminergic neurons. Furthermore, glit-1 mutants exhibit increased sensitivity to oxidative stress induced by H2O2 and paraquat. We provide evidence that GLIT-1 acts in the same genetic pathway as the previously identified tetraspanin TSP-17. After exposure to 6-OHDA and paraquat, glit-1 and tsp-17 mutants show almost identical, non-additive hypersensitivity phenotypes and exhibit highly increased induction of oxidative stress reporters. TSP-17 and GLIT-1 are both expressed in dopaminergic neurons. In addition, the neuroligin-like GLIT-1 is expressed in pharynx, intestine and several unidentified cells in the head. GLIT-1 is homologous, but not orthologous to neuroligins, transmembrane proteins required for the function of synapses. The Drosophila GLIT-1 homologue Gliotactin in contrast is required for epithelial junction formation. We report that GLIT-1 likely acts in multiple tissues to protect against 6-OHDA, and that the epithelial barrier of C. elegans glit-1 mutants does not appear to be compromised. We further describe that hyperactivation of the SKN-1 oxidative stress response pathway alleviates 6-OHDA-induced neurodegeneration. In addition, we find that mutations in the canonical apoptosis pathway and the calcium chaperone crt-1 cause increased 6-OHDA-induced dopaminergic neuron loss. In summary, we report that the neuroligin-like GLIT-1, the canonical apoptosis pathway and the calreticulin CRT-1 are required to prevent 6-OHDA-induced dopaminergic neurodegeneration.\n\nAuthor summaryNeurons use dopamine as a chemical messenger to mediate diverse behaviours. The gradual loss of dopaminergic neurons in specific brain areas is a hallmark of Parkinsons disease. The increased occurrence of highly reactive oxygen radicals, also called oxidative stress, is assumed to contribute to the demise of dopaminergic neurons. In this study we searched for genes that protect dopaminergic neurons against oxidative stress. We used the nematode C. elegans, a well- characterised model organism whose dopamine signalling system is very similar to that of humans. When C. elegans is exposed to 6-hydroxydopamine, an oxidative stress-inducing compound, dopaminergic neurons gradually die. Our major findings include: (i) absence of the neuroligin-like gene glit-1 causes highly increased cell death of dopaminergic neurons after 6-OHDA exposure; (ii) GLIT-1 acts in a similar manner as the previously identified tetraspanin TSP-17; (iii) GLIT-1 and TSP-17 also protect C. elegans from other types of oxidative stress; and (iv) the programmed cell death pathway and a calcium chaperone protect dopaminergic neurons as well. Collectively, this study shows that apoptosis proteins, the calcium chaperone CRT-1 and the neuroligin-like GLIT-1 protect against neurodegeneration after oxidative stress exposure.

genetics↗

bioRxiv2017

Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers

1.Throughout their lifetime cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. One of these, DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. While it is possible to associate mutational signatures extracted from human cancers with possible mutational processes the exact causation is often unknown. Here we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase {varepsilon} subunit pole-4. Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures one of which closely resembles the C. elegans MMR spectrum. A characteristic difference between human and worm MMR deficiency is the lack of elevated levels of NCG>NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms. The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes.

genomics↗