DNA Repair Proteins Research Articles

Targeting HUWE1 in Multiple Myeloma Leads to Impaired Replicative Stress Response and Enhanced Sensitivity to Bortezomib

Introduction Despite continuous advances in therapies over the last few decades, Multiple Myeloma (MM) remains an incurable malignancy with almost all patients developing relapsed/refractory disease. This emphasizes the need for novel approaches that can improve the efficacy of current standard of care treatments. Previous work from our lab and others has identified dysregulation of the E3 ligase HUWE1 in MM and highlighted it's potential as a therapeutic target. Through its interaction with a diverse set of substrates, HUWE1 is implicated in many key cellular processes including stress responses and DNA replication and repair pathways and is being increasingly recognised as a major regulator of MYC activity. In this study, we uncover a novel role for HUWE1 in DNA replication and repair and assess the combination of HUWE1 and proteasome inhibition as a potential therapeutic strategy in MM. Methods MM cell lines were transfected with SMARTvector Inducible Human HUWE1 shRNA or a non-targeting control (NTC) shRNA (Dharmacon, USA). Replicative stress was induced by treatment with 2 mM Hydroxyurea (HU) and assessed using immunofluorescence staining and iPOND (isolation of proteins on nascent DNA) analysis. Viability was assessed using CellTiter® Glo and combination indices (CI) calculated using CompuSyn software. Commercially available HUWE1 inhibitor BI8622 (MedChemExpress) and novel HUWE1 inhibitors synthesised in house were used throughout the study. Results HUWE1 knockdown and/or inhibition in MM cell lines led to an accumulation of cells in S phase, consistent with previous studies indicating that HUWE1 is required for effective DNA replication. Using proteomic profiling and co-immunoprecipitation, we identified and validated Replication Protein A, 70 kDa (RPA70) as a novel substrate of HUWE1. RPA70 is a subunit of the heterotrimeric RPA complex which plays a critical role in the recruitment of DNA repair proteins. RPA70 is the first subunit to translocate to sites of DNA damage where it binds to DNA and recruits the other subunits (RPA32 and RPA14) forming the complex, thus allowing for initiation of DNA repair. We observed significantly less K63-linked ubiquitination of RPA70 (p=0.00213) in the absence of HUWE1, with an associated decrease in both phosphorylation (p=0.0064) and localisation of RPA subunit RPA32 to DNA following treatment with hydroxyurea (HU) to induce replicative stress. Subsequent iPOND analysis determined that this is associated with reduced recruitment of repair proteins, including RAD51, to DNA. Moreover, induced replicative stress in HUWE1 depleted/inhibited cells resulted in significantly higher levels of DNA damage at 6hrs (p=0.00421) and 24hrs (p=0.0219) post HU treatment. A recent study demonstrated that HUWE1 ubiquitination of MYC promotes the formation of MYC multimers that protect stalled replication forks to limit the formation of double strand breaks (DSB) (Nature 2022;612:148-155). In line with this we also observe decreased ubiquitination of MYC in the HUWE1 knockdown cell line compared to the NTC following HU treatment. Finally, we assessed the efficacy of combining HUWE1 inhibitors with bortezomib which is known to impair DSB repair. Dual inhibition of HUWE1 and proteasome activity resulted in synergistic effects (CI values < 1) and a corresponding increase in DNA damage compared to HUWE1 inhibitor or bortezomib alone as measured by levels of ƴH2AX. Conclusion We have identified RPA as a novel substrate for HUWE1 and demonstrate that targeting HUWE1 results in increased replication stress and a dampened DNA repair capacity, underpinned by reduced recruitment of repair proteins. This work outlines a clear role for HUWE1 in genome stability in MM cells and highlights that HUWE1 inhibitors represent a novel anti-myeloma strategy that acts in synergy with bortezomib to exacerbate DNA damage.

CellTool: An Open-Source Software Combining Bio-Image Analysis and Mathematical Modeling for the Study of DNA Repair Dynamics

Elucidating the dynamics of DNA repair proteins is essential to understanding the mechanisms that preserve genomic stability and prevent carcinogenesis. However, the measurement and modeling of protein dynamics at DNA lesions via currently available image analysis tools is cumbersome. Therefore, we developed CellTool—a stand-alone open-source software with a graphical user interface for the analysis of time-lapse microscopy images. It combines data management, image processing, mathematical modeling, and graphical presentation of data in a single package. Multiple image filters, segmentation, and particle tracking algorithms, combined with direct visualization of the obtained results, make CellTool an ideal application for the comprehensive analysis of DNA repair protein dynamics. This software enables the fitting of obtained kinetic data to predefined or custom mathematical models. Importantly, CellTool provides a platform for easy implementation of custom image analysis packages written in a variety of programing languages. Using CellTool, we demonstrate that the ALKB homolog 2 (ALKBH2) demethylase is excluded from DNA damage sites despite recruitment of its putative interaction partner proliferating cell nuclear antigen (PCNA). Further, CellTool facilitates the straightforward fluorescence recovery after photobleaching (FRAP) analysis of BRCA1 associated RING domain 1 (BARD1) exchange at complex DNA lesions. In summary, the software presented herein enables the time-efficient analysis of a wide range of time-lapse microscopy experiments through a user-friendly interface.

Colchicine prevents oxidative stress-induced endothelial cell senescence via blocking NF-κB and MAPKs: implications in vascular diseases

BackgroundSmoking, alcohol abuse, and hypertension are – among others, potential risk factors for cardiovascular diseases. These risk factors generate oxidative stress and cause oxidative stress-induced DNA damage, resulting in cellular senescence and senescence-associated secretory phenotype (SASP). The SASP factors in feed-forward response exacerbate inflammation and cause tissue remodeling, resulting in atherosclerotic plaque formation and rupture.ResultsColchicine inhibited ROS generation and mitigated oxidative stress-induced DNA damage. It dampened oxidative stress-induced endothelial cell senescence and improved the expression of DNA repair protein KU80 and aging marker Lamin B1. The drug attenuated the expression of senescence marker P21 at mRNA and protein levels. The pathway analysis showed that colchicine inhibited NF-κB and MAPKs pathways and subdued mTOR activation. Colchicine also attenuated mRNA expression of interleukin (IL)-1β, IL-6, IL-8, MCP-1, ICAM-1, and E-selectin. Furthermore, colchicine reduced the mRNA and protein expression of matrix metalloproteinase (MMP-2).ConclusionIn summary, colchicine blocked oxidative stress-induced senescence and SASP by inhibiting the activation of NF-κB and MAPKs pathways.Graphical

Decline of DNA damage response along with myogenic differentiation.

DNA integrity is incessantly confronted to agents inducing DNA lesions. All organisms are equipped with a network of DNA damage response mechanisms that will repair DNA lesions and restore proper cellular activities. Despite DNA repair mechanisms have been revealed in replicating cells, still little is known about how DNA lesions are repaired in postmitotic cells. Muscle fibers are highly specialized postmitotic cells organized in syncytia and they are vulnerable to age-related degeneration and atrophy after radiotherapy treatment. We have studied the DNA repair capacity of muscle fiber nuclei and compared it with the one measured in proliferative myoblasts here. We focused on the DNA repair mechanisms that correct ionizing radiation (IR)-induced lesions, namely the base excision repair, the nonhomologous end joining, and the homologous recombination (HR). We found that in the most differentiated myogenic cells, myotubes, these DNA repair mechanisms present weakened kinetics of recruitment of DNA repair proteins to IR-damaged DNA. For base excision repair and HR, this decline can be linked to reduced steady-state levels of key proteins involved in these processes.

Molecular Biomarkers for Predicting Cancer Patient Radiosensitivity and Radiotoxicity in Clinical Practice

Radiotherapy (RT) is a major part of cancer treatment. The reported variability in patient response to this modality can interfere with the continuation of best-possible care, promote side effects, and lead to long-term morbidity. Tools to predict a patient’s response to radiation could be highly useful in improving therapeutic outcomes while minimizing unnecessary and toxic exposure to radiation. This study investigates the potential of using molecular biomarkers as predictors of radiosensitivity in clinical practice. We review relative studies researching the positive correlation between various molecular biomarkers and patient radiosensitivity, including DNA damage response and repair proteins, inflammation and apoptosis markers, cell cycle regulators, and other biological markers. The clinical perspectives and applicability of these biomarkers in the prediction of radiosensitivity are also critically discussed. Conclusively, we underline the dynamics of molecular biomarkers to improve the efficacy and safety of radiotherapy in clinical practice and highlight the need for further research in this field. Identification of the most prominent markers is crucial for the personalization of therapies entailing ionizing radiation.

Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

H3.1K27me1 loss confers Arabidopsis resistance to Geminivirus by sequestering DNA repair proteins onto host genome

The H3 methyltransferases ATXR5 and ATXR6 deposit H3.1K27me1 to heterochromatin to prevent genomic instability and transposon re-activation. Here, we report that atxr5 atxr6 mutants display robust resistance to Geminivirus. The viral resistance is correlated with activation of DNA repair pathways, but not with transposon re-activation or heterochromatin amplification. We identify RAD51 and RPA1A as partners of virus-encoded Rep protein. The two DNA repair proteins show increased binding to heterochromatic regions and defense-related genes in atxr5 atxr6 vs wild-type plants. Consequently, the proteins have reduced binding to viral DNA in the mutant, thus hampering viral amplification. Additionally, RAD51 recruitment to the host genome arise via BRCA1, HOP2, and CYCB1;1, and this recruitment is essential for viral resistance in atxr5 atxr6. Thus, Geminiviruses adapt to healthy plants by hijacking DNA repair pathways, whereas the unstable genome, triggered by reduced H3.1K27me1, could retain DNA repairing proteins to suppress viral amplification in atxr5 atxr6.

Impact of vitamin B12 on rhamnose metabolism, stress defense and in-vitro virulence of Listeria monocytogenes

Listeria monocytogenes is a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. L. monocytogenes is capable of utilizing various nutrient sources including rhamnose, a naturally occurring deoxy sugar abundant in foods. L. monocytogenes can degrade rhamnose into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulated anaerobic growth of L. monocytogenes on rhamnose due to the activation of bacterial microcompartments for 1,2-propanediol utilization (pdu BMC) with concomitant production of propionate and propanol. Notably, anaerobic 1,2-propanediol metabolism has been linked to virulence of enteric pathogens including Salmonella spp. and L. monocytogenes. In this study we investigated the impact of B12 and BMC activation on i) aerobic and anerobic growth of L. monocytogenes on rhamnose and ii) the level of virulence. We observed B12-induced pdu BMC activation and growth stimulation only in anaerobically grown cells. Comparative Caco-2 virulence assays showed that these pdu BMC-induced cells have significantly higher translocation efficiency compared to non-induced cells (anaerobic growth without B12; aerobic growth with or without B12), while adhesion and invasion capacity is similar for all cells. Comparative proteome analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL, teichoic acid export ATP-binding protein TagH, DNA repair and protection proteins, RadA and DPS, and glutathione synthase GshAB, previously linked to activation of virulence response in L. monocytogenes, uniquely upregulated in anaerobically rhamnose grown pdu-induced cells. Our results shed light on possible effects of B12 on L. monocytogenes competitive fitness and virulence activation when utilizing rhamnose in anaerobic conditions encountered during transmission and the human intestine.

OGG1 promoted lung fibrosis by activating fibroblasts via interacting with Snail1

One of abundant DNA lesions induced by reactive oxygen species is 8-oxoguanine (8-oxoG), which compromises genetic instability. 8-oxoG is recognized by the DNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1) that not only participates in base excision repair but also involves in transcriptional regulation.OGG1 has an important role inIdiopathic Pulmonary Fibrosis (IPF) processing and targeting fibroblasts is a major strategy for the treatment of pulmonary fibrosis, but whether OGG1 activate fibroblast is not clear. In this study, we show that OGG1 expression level is increased at the fibroblast activation stage in mouse lungs induced by bleomycin (BLM) treatment. OGG1 promoted the expression level of fibroblast activation markers (CTGF, fibronectin, and collagen 1) in a pro-fibrotic gene transcriptional regulation pathway via interacting with Snail1, which dependent on 8-oxoG recognition. Global inhibition of OGG1 at the middle stage of lung fibrosis also relieved BLM-induced lung fibrosis in mice. Our results suggest that OGG1 is a target for inhibiting fibroblast activation and a potential therapeutic target for IPF.

MTOR Inhibitor Rapalink-1 Prevents Ethanol-Induced Senescence in Endothelial Cells.

The cardiovascular risk factors, including smoking, ethanol, and oxidative stress, can induce cellular senescence. The senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase (MMPs). These pro-inflammatory molecules and MMPs promote the infiltration and accumulation of inflammatory cells in the vascular tissue, exacerbating vascular tissue inflammation. MMPs damage vascular tissue by degenerating the extracellular matrix. Consequently, these cellular and molecular events promote the initiation and progression of cardiovascular diseases. We used Rapalink-1, an mTOR inhibitor, to block ethanol-induced senescence. Rapalink-1 inhibited oxidative-stress-induced DNA damage and senescence in endothelial cells exposed to ethanol. It attenuated the relative protein expression of senescence marker P21 and improved the relative protein expression of DNA repair protein KU70 and aging marker Lamin B1. It inhibited the activation of NF-κB, MAPKs (P38 and ERK), and mTOR pathway proteins (mTOR, 4EBP-1, and S6). Moreover, Rapalink-1 suppressed ethanol-induced mRNA expression of ICAM-1, E-selectin, MCP-1, IL-8, MMP-2, and TIMP-2. Rapalink-1 also reduced the relative protein expression of MMP-2. In summary, Rapalink-1 prevented senescence, inhibited pro-inflammatory pathway activation, and ameliorated pro-inflammatory molecule expression and MMP-2.

STEM-08. OVERCOMING GLIOBLASTOMA STEM CELL RESISTANCE TO RADIATION THROUGH TARGETING THIOREDOXIN REDUCTASE 1

Abstract Glioblastoma, the most aggressive primary tumor of the central nervous system, presents challenges due to its resistance to standard treatment, including radiation therapy (RT). Glioblastoma stem cells (GSCs), known for their chemo-radioresistance, employ adaptive mechanisms to counteract therapy-induced reactive oxygen species (ROS) levels. This includes modulating the activity of thioredoxin (Trx), a key player in the redox system. Furthermore, GSCs exhibit enhanced DNA repair capacity by upregulating DNA repair proteins, such as RAD51 to survive following ROS-induced DNA damage in response to radiation. Our TCGA analysis revealed elevated expression of thioredoxin reductase 1 (TrxR1) in patients diagnosed with GBM and a significant positive correlation between TrxR1 and RAD51. We hypothesized that GSCs might enhance their antioxidant capacity following RT to suppress RT-induced ROS elevation. Hence, combining RT with TrxR1 inhibitors might overcome GSCs radioresistance while reducing RAD51 expression. Auranofin (Au), a drug FDA-approved for rheumatoid arthritis inhibits TrxR1 activity with subsequent ROS increase. We assessed Au cytotoxicity on GSCs with and without ionizing radiation using Alamar Blue and neutrosphere formation assays and ROS assay for quantitative analysis of ROS levels. We evaluated the molecular mechanisms associated with Au and IR-induced cell death in GSCs using western blotting analysis. Our findings suggest that resistance of GSCs to IR was accompanied with increased expression of TrxR1 and RAD51. On the other hand, Au alone significantly decreased GSC viability and neurosphere formation and sensitized GSCs to IR within the nanomolar range (significantly lower than the corresponding IC50s). Au alone and combined with IR increased ROS, while expression of both RAD51 and TrxR1 was decreased. Our study provides insights into repurposing the TrxR1 inhibitor, Au and its combination with RT as a novel therapeutic strategy to overcome GSC radioresistance and ultimately improve the dismal outcome of patients diagnosed with GBM.

DDDR-05. TARGETED INHIBITION OF DNA REPAIR AND SURVIVAL SIGNALING IN DIFFUSE INTRINSIC PONTINE GLIOMAS

Abstract Therapeutic resistance remains a major obstacle to successful clinical management of Diffuse Intrinsic Pontine Glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood-brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. Based on these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrate that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Since radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of ATM to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.

TMET-13. DNA DAMAGE SIGNALING ACTIVATES GTP SYNTHESIS TO PROMOTE GLIOBLASTOMA TREATMENT RESISTANCE

Abstract Glioblastoma (GBM) is the most common type of invasive brain tumor in adults and is uniformly fatal due to inherent resistance to radiation therapy (RT) and chemotherapy. Our group and others have found that metabolites can regulate DNA repair and therapy resistance in brain tumors, but little is known about how DNA damage regulates metabolic pathway activity in cancer. Here we show that following treatment with RT, GBMs increase rates of de novo guanylate synthesis in vitro and in orthotopic patient-derived xenograft models via signaling through the DNA repair protein DNA-PK. To determine if disrupting this regulation can augment GBM treatment efficacy, we combined an FDA-approved inhibitor of purine synthesis (mycophenolate mofetil, MMF) with chemoradiation in a variety of mouse models of GBM. Critically, targeting GTP synthesis improved the efficacy of both RT alone and chemoradiation in multiple patient-derived and syngeneic intracranial models. Our translational studies have shown that mycophenolic acid, the active metabolite of MMF, penetrates the blood-brain barrier at concentrations sufficient to inhibit GTP synthesis in GBMs of human patients. The phase 1 arms of our study, which are ongoing, are testing the safety and efficacy of combining MMF with standard of care chemoradiation for patients with primary and recurrent GBM.

Linear Dichroism Measurements for the Study of Protein-DNA Interactions.

Linear dichroism (LD) is a differential polarized light absorption spectroscopy used for studying filamentous molecules such as DNA and protein filaments. In this study, we review the applications of LD for the analysis of DNA-protein interactions. LD signals can be measured in a solution by aligning the sample using flow-induced shear force or a strong electric field. The signal generated is related to the local orientation of chromophores, such as DNA bases, relative to the filament axis. LD can thus assess the tilt and roll of DNA bases and distinguish intercalating from groove-binding ligands. The intensity of the LD signal depends upon the degree of macroscopic orientation. Therefore, DNA shortening and bending can be detected by a decrease in LD signal intensity. As examples of LD applications, we present a kinetic study of DNA digestion by restriction enzymes and structural analyses of homologous recombination intermediates, i.e., RecA and Rad51 recombinase complexes with single-stranded DNA. LD shows that the DNA bases in these complexes are preferentially oriented perpendicular to the filament axis only in the presence of activators, suggesting the importance of organized base orientation for the reaction. LD measurements detect DNA bending by the CRP transcription activator protein, as well as by the UvrB DNA repair protein. LD can thus provide information about the structures of protein-DNA complexes under various conditions and in real time.

TRANSLATIONAL RESEARCH-03 MULTIPLE MECHANISMS MODULATE TEMOZOLOMIDE RESISTANCE AND ARE POTENTIAL TARGETS FOR GLIOBLASTOMA THERAPY.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Temozolomide (TMZ) is the only drug currently been used for treatment of newly diagnosed GBM patients but the benefit from this agent has been hampered by the primary and secondary resistance. Thus, there is a strong need for uncovering the mechanisms of TMZ resistance. The DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) is the main driver of primary TMZ resistance and lacking MGMT expression due to promoter hypermethylation has been associated with a significant survival prolongation. However, the MGMT-independent resistance mechanisms remain poorly understood. To investigate TMZ resistance mechanisms, we developed in vivo TMZ resistant models using GBM patient derived xenografts (PDXs) by treating animals bearing flank PDXs with clinically relevant 3-cyles of TMZ. The recurrent tumors were all resistant to TMZ through multiple mechanisms, including MGMT re-expression, elevated homologous recombination (HR) and/or non-homologous end joining (NHEJ) repair activities, and loss of mismatch repair (MMR). However, other tumors developed resistance due to unknown mechanisms. To identify novel modulators of TMZ sensitivity, we performed genome wide screening using shRNA library, and we identified over 600 druggable targets. Validation experiments identified RBBP4 gene as one of the candidates involved in TMZ sensitivity. Silencing RBBP4 in GBM cells sensitized both MGMT-methylated and -unmethylated GBM cells to TMZ. Mechanistically, RBBP4 interacts with p300 to form a chromatin remodeling complex that control expression of DNA damage repair genes, and silencing RBBP4 or p300 significantly blocked the recovery of TMZ induced DNA double strand breaks causing cell death. We also found the nuclear pore exporter XPO1 is the modulator of TMZ sensitivity, and the XPO1 inhibitor Selinexor is currently being evaluated for GBM treatment in a clinical trial. Collectively, our work has uncovered multiple mechanisms of TMZ resistance that are potential targets for GBM therapy.

PKCδ Regulates Chromatin Remodeling and DNA Repair through SIRT6.

PKCδ controls sensitivity to irradiation by regulating DNA repair.

Rad53 regulates the lifetime of Rdh54 at homologous recombination intermediates.

Rdh54 is a conserved DNA translocase that participates in homologous recombination (HR), DNA checkpoint adaptation, and chromosome segregation. Saccharomyces cerevisiae Rdh54 is a known target of the Mec1/Rad53 signaling axis, which globally protects genome integrity during DNA metabolism. While phosphorylation of DNA repair proteins by Mec1/Rad53 is critical for HR progression little is known about how specific post translational modifications alter HR reactions. Phosphorylation of Rdh54 is linked to protection of genomic integrity but the consequences of modification remain poorly understood. Here, we demonstrate that phosphorylation of the Rdh54 C-terminus by the effector kinase Rad53 regulates Rdh54 clustering activity as revealed by single molecule imaging. This stems from phosphorylation dependent and independent interactions between Rdh54 and Rad53. Genetic assays reveal that loss of phosphorylation leads to phenotypic changes resulting in loss-of-heterozygosity (LOH) outcomes. Our data highlight Rad53 as a key regulator of HR intermediates through activation and attenuation of Rdh54 motor function.

Caseinolytic mitochondrial matrix peptidase X is essential for homologous chromosome synapsis and recombination during meiosis of male mouse germ cells.

Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins. The mitochondrial protease complex (ClpXP), which consists of caseinolytic mitochondrial matrix peptidase X (ClpX) and caseinolytic protease P (ClpP) and mediates the degradation of misfolded, damaged, and oxidized proteins, is essential for maintaining mitochondrial homeostasis. ClpXP has been implicated in meiosis regulation, but its precise role is currently unknown. In this study, we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X ( ClpxcKO ) mouse model to investigate the function of ClpX in meiosis. We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis. Specifically, it caused asynapsis of homologous chromosomes and impaired meiotic recombination, resulting in meiotic arrest in the zygotene-to-pachytene transition phase. The loss of ClpX compromised the double-strand break (DSB) repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1 (RAD51) to DSB sites. This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in ClpxcKO spermatocytes, as discerned by electron microscopy. Furthermore, ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in ClpxcKO spermatocytes. Taken together, we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing, synapsis, and recombination in spermatocytes during meiotic prophase I.

Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin.

Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.

Analysis of the Relationship between PARP1 and BRCA1 Suggests PARP1 Gene Has a Role in Breast Cancer

Poly [ADP-ribose] polymerase 1 (PARP1), one of the genes in the PARP family, is mainly involved in the detection and repair of DNA damage in cells, and its upregulation has been associated with tumorigenesis. PARP1 is essential to all cells in the body as it ensures that DNA is replicated correctly. The PARP1 protein addresses repair of single-stranded breaks (SSBs) in DNA by initially binding near the point of break in the DNA. While it is bound to the region of the SSB, PARP1 transfers ADP ribosyl moiety from NAD+ to acceptor proteins. This leads to the recruitment of DNA repair proteins to the region of DNA breaks. The PARP1 protein also aids the double-stranded break (DSB) repair of DNA by recruiting the homologous recombination (HR) pathway proteins. Due to this role of PARP1 in DNA repair, it has been associated with cancer growth. Using databases, including UniProt, Xena Browser, and CBioPortal, this article explores the relationship between PARP1 mutations and BRCA1-mutated breast cancer. BRCA1 is a tumor suppressor gene, which is involved in DNA repair through the HR pathway. Therefore, in BRCA1-mutated cells, the lack of tumor suppressor factors leads to cancer growth. However, in a cell that has both BRCA1 mutations and PARP1 inhibition, DNA damage cannot be repaired, leading to apoptosis. Hence, PARP1 inhibitors have become essential in BRCA1-mutated breast cancers. There are several PARP1 inhibitors that are currently being used to treat breast cancer, which work using several mechanisms of action, including PARP1 trapping, which prevents the repair, transcription, and replication of DNA. This article also focuses on the clinical trials of three specific drugs, Olaparib, Iniparib, and Veliparib, which are used to treat breast cancer. However, due to the lack of understanding surrounding PARP1 inhibition mechanisms and potential drug combinations, more research needs to be done to understand potential biomarker targets and PARP1 inhibitor resistance.