Biology subjects

Black, J. A.

Publications and source records attributed to Black, J. A..

6 recordsLinked to original sources

bioRxiv2025

Loss of PIMREG impairs double-strand break signaling and repair

Glioblastoma (GBM) is the most common primary brain tumor in adults. It is an aggressive type of tumor with an average overall survival of 12-15 months post-diagnosis. Standard treatment involves surgical resection, followed by radiotherapy and chemotherapy with temozolomide (TMZ). However, resistance to treatment is frequently observed. PIMREG is a marker of proliferation highly expressed across different tumor types, with particularly elevated levels in GBM. Our previous study revealed that PIMREG silencing sensitizes GBM cells to TMZ treatment and affects ATM activation, a key apical kinase involved in DNA damage signaling. Given the emerging significance of PIMREG in the DNA damage response, we sought to explore PIMREGs role in DNA damage repair and signaling in GBM cells exposed to genotoxic agents. Here we assessed the functionally enriched pathways associated with high PIMREG expression in GBM patient samples, analyzed the interactome of PIMREG upon treatment of GBM cells with TMZ and evaluated the impact of PIMREG loss on DNA damage signaling, DNA break formation and the repair of double-strand breaks. Our findings reveal that PIMREG functions as a direct substrate for ATM/ATR kinases, interacts with key DNA repair proteins upon TMZ treatment and actively facilitates the cellular response to chemotherapy- and radiotherapy-induced DNA damage. Taken together, PIMREG may function in the DNA damage response, particularly in the signaling and repair of double strand breaks via homologous recombination in GBM cells. In all, our data suggested high PIMREG expression may affect resistance of GBM cells to treatment.

cancer biology↗

bioRxiv2025

ATR, a DNA damage kinase, modulates DNA replication timing in Leishmania major.

All cells possess mechanisms to maintain and replicate their genomes, whose integrity and transmission are constantly challenged by DNA damage and replication impediments. In eukaryotes, the protein kinase Ataxia-Telangiectasia and Rad3-related (ATR), a member of the phosphatidylinositol 3-kinase-like family acts as a master regulator of the eukaryotic response to DNA injuries, ensuring DNA replication completion and genome stability. Here we aimed to investigate the functional relevance of the ATR homolog in the DNA metabolism of Leishmania major, a protozoan parasite with a remarkably plastic genome. CRISPR/cas9 genome editing was used to generate a Myc-tagged ATR cell line (mycATR), and a Myc-tagged C-terminal knockout of ATR (mycATR{Delta}C-/-). We show that the nuclear localisation of ATR depends upon its C-terminus. Moreover, its deletion results in single-stranded DNA accumulation, impaired cell cycle control, increased levels of DNA damage, and delayed DNA replication restart after replication stress. In addition, we show that ATR plays a key role in maintaining L. majors unusual DNA replication program, where larger chromosomes duplicate later than smaller chromosomes. Our data reveals loss of the ATR C-terminus promotes the accumulation of replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that these alterations to the DNA replication program promote chromosome instability. In summary, our work shows that ATR acts to moderate DNA replication timing thus limiting the plasticity of the Leishmania genome.

molecular biology↗

bioRxiv2024

Genome-wide mapping of RPA1 and RAD9 reveals the management of polycistronic transcription, replication initiation, and responses to replication stress in Leishmania

When exposed single-stranded DNA accumulates at stalled or collapsed replication forks, the replication stress response is triggered to prevent genome instability. Leishmania are parasitic eukaryotes where gene expression is universally polycistronic and whose plastic genomes facilitate rapid adaptations in response to stress, with evidence implicating intrinsic replication stress as a source. Little is known about the Leishmania replication stress response. In this study, we reveal the global dynamics of the replication stress response in L. major promastigotes by performing ChIP-seq on three key replication stress response proteins, {gamma}H2A, RPA1 and RAD9, in the absence and presence of replication stress. We show that common hotspots of replication stress correlate with DNA replication initiation and transcription termination in Leishmania. When DNA replication is stalled, replication stress response factors accumulate at early S-phase origins, with a signal pattern reminiscent of bidirectional replication fork progression. Under conditions of chronic replication stress, increased accumulation of replication stress response factors emerges at wider sites of transcription initiation, suggesting Leishmania may possess compensatory strategies to limit the effects of replication stress and ensure DNA replication can complete under these conditions. In contrast, chronic replication stress enhances RSR factor accumulation at transcription termination sites, highlighting these regions as key replication stress hotspots in Leishmania. Lastly, variations in RPA dynamics in ATR-deficient cells uncover crucial roles of this protein kinase in managing polycistronic transcription and DNA replication, particularly under replication stress, in Leishmania. SummaryStrict controls operate to precisely copy an organisms DNA. However, cells need ways to rapidly adapt and respond to stimuli. In some cases, these beneficial adaptations come from problems during replication. Leishmania parasites cause serious neglected infections in humans and animals across the worlds tropics and sub-tropics. Remarkably, recent evidence suggests that Leishmania DNA experiences enhanced stress during replication that can drive its ability to rapidly adapt in response to stress. How Leishmania respond to DNA replication stress is still poorly understood. Here, using a genome-wide approach to map the locations of key proteins that manage DNA replication stress and maintain genome integrity, we show hotspots of DNA replication stress coincide with start sites of DNA replication and regions of transcription termination.

molecular biology↗

bioRxiv2024

AUK3 is required for faithful nuclear segregation in the bloodstream form of Trypanosoma brucei

Eukaryotic chromosomes segregate faithfully prior to nuclear division to ensure genome stability. If segregation becomes defective, the chromosome copy number of the cell may alter leading to aneuploidy and/or polyploidy, both common hallmarks of cancers. In eukaryotes, aurora kinases regulate chromosome segregation during mitosis and meiosis, but their functions in the divergent, single-celled eukaryotic pathogen Trypanosoma brucei are less understood. Here, we focused on one of three aurora kinases in these parasites, TbAUK3, a homologue of the human aurora kinase AURKC, whose functions are primarily restricted to meiosis. We show that RNAi targeted depletion of TbAUK3 correlates with nuclear segregation defects, reduced proliferation, and decreased DNA synthesis, suggestive of a role for TbAUK3 during mitotic, not meiotic, chromosome segregation. Moreover, we uncover a putative role for TbAUK3 during the parasites response to DNA damage since we show that depletion of TbAUK3 enhances DNA instability and sensitivity to genotoxic agents. HIGHLIGHTSO_LIThe C-terminus of TbAUK3 is disordered C_LIO_LITbAUK3 depletion coincides with nuclear segregation defects C_LIO_LIDepletion of TbAUK3 enhances DNA instability C_LI

cell biology↗

bioRxiv2021

Widespread roles of Trypanosoma brucei ATR in nuclear genome function and transmission are linked to R-loops

Inheritance of aberrant chromosomes can compromise genome integrity and affect cellular fitness. In eukaryotes, surveillance pathways and cell cycle checkpoints monitor for aberrant DNA transmission and the ATR kinase, a regulator of the DNA damage response, plays a pivotal role. Prior work revealed that ATR acts during antigenic variation in Trypanosoma brucei mammal-infective life cycle forms and that its loss is lethal, but how widely ATR operates in genome maintenance is largely unknown. Here, we show that after prolonged ATR depletion by RNAi T. brucei continues to synthesise DNA and enters new rounds of cell division, despite increased genome damage. Furthermore, we detect defective chromosome segregation, micronuclei formation and disruption of the nuclear architecture. RNA-seq revealed that loss of ATR affects the expression of nearly half the genes in the genome, including both RNA Polymerase I and II transcription. Using ChIP-seq of yH2A and DRIP-seq, we reveal overlapping signals for genome damage and R-loops after ATR depletion in all intergenic regions. In addition, we report reduced R-loop levels and accumulation of yH2A signal within centromeres. Together, our data indicates widespread roles of ATR in T. brucei, including differing roles in R-loop homeostasis during multigene transcription and in chromosome segregation.

cell biology↗

bioRxiv2018

The ATR kinase of Trypanosoma brucei links DNA damage signalling and monoallelic control of surface antigen gene expression during antigenic variation

To evade mammalian immunity, Trypanosoma brucei switches the variant surface glycoprotein (VSG) expressed on its surface. Key to this reaction are controls exerted to ensure only one of many subtelomeric multigene VSG expression sites are transcribed at a time. DNA repair activities have to date been implicated only in catalysis of VSG switching by recombination, not transcriptional control. However, how VSG switching is signalled to guide the appropriate reaction, or to integrate switching into parasite growth, is unknown. Here we show that loss of ATR, a DNA damage signalling protein kinase, is lethal and causes increased nuclear genome lesions. ATR depletion also causes expression of mixed VSGs on the cell surface, increased transcription of genes from silent expression sites, and altered localisation of RNA Polymerase I and VEX1, factors involved in VSG transcription. The work therefore reveals that VSG expression control is mediated by a nuclear DNA damage signalling factor.

molecular biology↗