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Molecular Biology of the Cell Apr 2022Homology-directed repair of DNA double-strand breaks (DSBs) represents a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely...
Homology-directed repair of DNA double-strand breaks (DSBs) represents a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). How homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair often involves multiple site-specific and Rad51-dependent colocalization events between the DSB and donor sequence. Upon loss of the RecQ helicase Rqh1 (BLM in humans) we observe rapid repair possibly involving a single strand invasion event, suggesting that multiple strand invasion cycles antagonized by Rqh1 could reflect ongoing SDSA. However, failure to colocalize with the donor sequence and execute repair is also more likely in cells, possibly reflecting erroneous strand invasion. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.
Topics: DNA Breaks, Double-Stranded; DNA Helicases; DNA Repair; DNA Replication; Humans; Recombinational DNA Repair; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 35080989
DOI: 10.1091/mbc.E20-07-0433 -
Pediatric Rheumatology Online Journal Jul 2021Respiratory conditions are the leading cause of hospitalization and death in children with Trisomy 21 (T21). Diffuse alveolar hemorrhage (DAH) occurs at higher frequency... (Review)
Review
BACKGROUND
Respiratory conditions are the leading cause of hospitalization and death in children with Trisomy 21 (T21). Diffuse alveolar hemorrhage (DAH) occurs at higher frequency in children with T21; yet, it is not widely studied nor is there a standardized approach to diagnosis or management. The objective of this study was to identify children with T21 and DAH in order to understand contributing factors and identify opportunities to improve outcomes. We identified 5 children with T21 at a single institution with histology-proven DAH over 10 years and discuss their presentation, evaluation, management, and outcomes. We also reviewed the cases in the literature.
CASE PRESENTATION
Patient 1 died at age seven due to secondary hemophagocytic lymphohistiocytosis. DAH was seen on autopsy. Patient 2 was a three-year-old with systemic-onset juvenile idiopathic arthritis diagnosed with DAH after presenting for hypoxia. Patient 3 was diagnosed with DAH at age nine after presenting with recurrent suspected pneumonia and aspiration. Patient 4 was diagnosed with DAH at age eight after presenting with pallor and fatigue. She had additional ICU admissions for DAH with infections. Patient 5 developed hemoptysis at age three and had recurrent DAH for 10 years. Four patients responded positively to immune-modulation such as intravenous immunoglobulin, glucocorticoids, and rituximab. Of the 19 patients identified in the literature, only one was from the United States. The majority had anemia, respiratory distress, autoantibodies, and recurrences. Very few patients had hemoptysis. Idiopathic pulmonary hemosiderosis was the most common diagnosis. Almost all received glucocorticoids with or without additional immunosuppression. The majority of our patients and those in the literature had positive auto-antibodies such as anti-neutrophil cytoplasmic antibodies and anti-nuclear antigen antibodies. Diagnostic clues included respiratory distress, hypoxia, anemia, recurrent pneumonia, and/or ground glass opacities on imaging. We identified four contributors to DAH: structural lung abnormalities, pulmonary arterial hypertension, infection/aspiration, and autoimmune disease/immune dysregulation.
CONCLUSION
These cases demonstrate the need for an increased index of suspicion for DAH in children with T21, particularly given the low frequency of hemoptysis at presentation, enrich the understanding of risk factors, and highlight the favorable response to immunosuppressive therapies in this vulnerable population.
Topics: Child; Child, Preschool; Down Syndrome; Female; Hemorrhage; Humans; Lung Diseases; Male; Pulmonary Alveoli
PubMed: 34273981
DOI: 10.1186/s12969-021-00592-4 -
Nature Communications Jan 2021The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce...
The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.
Topics: Amino Acid Motifs; Bloom Syndrome; CRISPR-Cas Systems; DNA Damage; DNA Replication; DNA Topoisomerases, Type I; DNA, Single-Stranded; DNA-Binding Proteins; Gene Knockdown Techniques; HeLa Cells; Humans; Mitosis; Mutation; Protein Binding; Protein Domains; RecQ Helicases; Recombinant Proteins; Recombinational DNA Repair; Replication Protein A
PubMed: 33500419
DOI: 10.1038/s41467-020-20818-5 -
International Journal of Oncology Jan 2023Prostate cancer (PCa) is one of the most fundamental causes of cancer‑related mortality and morbidity among males. However, the underlying mechanisms have not yet been...
Prostate cancer (PCa) is one of the most fundamental causes of cancer‑related mortality and morbidity among males. However, the underlying mechanisms have not yet been fully clarified. The present study aimed to investigate the effects of plasmacytoma variant translocation 1 (PVT1) on the malignant behaviors of PCa cells and to explore the possible molecular mechanisms involved. The expression levels of PVT1 and microRNA (miRNA/miR)‑27b‑3p in PCa tissues and cell lines were measured using reverse‑transcritpion‑quantitative polymerase chain reaction. Methyltransferase 3 (METTL3)‑mediated PVT1 N‑methyladenosine (mA) modifications were detected using RNA immunoprecipitation (RIP) and RNA pull‑down assays. Bioinformatics analysis was used to predict the interactions of miR‑27b‑3p with PVT1 and bloom syndrome protein (BLM), and these interactions were validated using RIP, dual‑luciferase reporter and biotin pull‑down assays. The functional importance of miR‑27b‑3p, PVT1 and BLM within PCa cells was assessed through the utilization of Cell Counting Kit‑8, Transwell, wound healing and colony formation assays, and the use of a mouse xenograft model. The results revealed the high expression level of PVT1 in PCa tissues and cells, and epigenetic analyses revealed the upregulation of PVT1 expression following METTL3‑mediated mA modification. PVT1 overexpression induced PCa cells to become more proliferative, migratory and invasive, whereas PVT1 knockdown led to the opposite phenotype. Furthermore, miR‑27b‑3p was found to target both PVT1 and BLM, and PVT1 functioned to sequester miR‑27b‑3p within cells, thereby indirectly promoting the BLM expression level. BLM overexpression reversed the adverse effects of PVT1 knockdown on the migratory, proliferative and invasive capabilities of PCa cells and . The overexpression of PVT1 contributed to the aggressive phenotype of PCa cells by regulating the miR‑27b‑3p/BLM axis. On the whole, the findings of the present study may provide novel potential targets for the treatment of PCa.
Topics: Humans; Mice; Animals; Male; RNA, Long Noncoding; Prostatic Neoplasms; MicroRNAs; Methyltransferases
PubMed: 36484368
DOI: 10.3892/ijo.2022.5464 -
Molecules (Basel, Switzerland) Dec 2022Using standard DNA-damaging medicines with DNA repair inhibitors is a promising anticancer tool to achieve better therapeutic responses and reduce therapy-related side...
Using standard DNA-damaging medicines with DNA repair inhibitors is a promising anticancer tool to achieve better therapeutic responses and reduce therapy-related side effects. Cell viability assay, neutral comet assay, western blotting (WB), and cell cycle and apoptosis analysis were used to determine the synergistic effect and mechanism of ML216, a Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, in PCa cells. Based on the online database research, our findings revealed that BLM was substantially expressed in PCa, which is associated with a bad prognosis for PCa patients. The combination of ML216 and CDDP improved the antiproliferative properties of three PCa cell lines. As indicated by the increased production of γH2AX and caspase-3 cleavage, ML216 significantly reduced the DNA damage-induced high expression of BLM, making PC3 more susceptible to apoptosis and DNA damage caused by CDDP. Furthermore, the combination of ML216 and CDDP increased p-Chk1 and p-Chk2 expression. The DNA damage may have triggered the ATR-Chk1 and ATM-Chk2 pathways simultaneously. Our results demonstrated that ML216 and CDDP combination therapy exhibited synergistic effects, and combination chemotherapy could be a novel anticancer tactic.
Topics: Humans; Cisplatin; Antineoplastic Agents; RecQ Helicases; Apoptosis; DNA Damage; DNA; Cell Line, Tumor
PubMed: 36557923
DOI: 10.3390/molecules27248790 -
Genes & Development Oct 2020TRF1 facilitates the replication of telomeric DNA in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Lagging-strand...
TRF1 facilitates the replication of telomeric DNA in part by recruiting the BLM helicase, which can resolve G-quadruplexes on the lagging-strand template. Lagging-strand telomeres lacking TRF1 or BLM form fragile telomeres-structures that resemble common fragile sites (CFSs)-but how they are formed is not known. We report that analogous to CFSs, fragile telomeres in BLM-deficient cells involved double-strand break (DSB) formation, in this case by the SLX4/SLX1 nuclease. The DSBs were repaired by POLD3/POLD4-dependent break-induced replication (BIR), resulting in fragile telomeres containing conservatively replicated DNA. BIR also promoted fragile telomere formation in cells with FokI-induced telomeric DSBs and in alternative lengthening of telomeres (ALT) cells, which have spontaneous telomeric damage. BIR of telomeric DSBs competed with PARP1-, LIG3-, and XPF-dependent alternative nonhomologous end joining (alt-NHEJ), which did not generate fragile telomeres. Collectively, these findings indicate that fragile telomeres can arise from BIR-mediated repair of telomeric DSBs.
Topics: Animals; Cell Line; Cells, Cultured; Chromosome Fragile Sites; DNA Breaks, Double-Stranded; DNA Repair; DNA Replication; Endodeoxyribonucleases; Fibroblasts; Humans; Mice; RecQ Helicases; Recombinases; Telomere
PubMed: 32883681
DOI: 10.1101/gad.328575.119 -
American Journal of Respiratory and... Jan 2022Studies have suggested some patients with asthma are at risk of severe coronavirus disease (COVID-19), but they have had limited data on asthma phenotype and have not... (Observational Study)
Observational Study
Studies have suggested some patients with asthma are at risk of severe coronavirus disease (COVID-19), but they have had limited data on asthma phenotype and have not considered if risks are specific to COVID-19. To determine the effect of asthma phenotype on three levels of COVID-19 outcomes. Compare hospitalization rates with influenza and pneumonia. Electronic medical records were used to identify patients with asthma and match them to the general population. Patient-level data were linked to Public Health England severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test data, hospital, and mortality data. Asthma was phenotyped by medication, exacerbation history, and type 2 inflammation. The risk of each outcome, adjusted for major risk factors, was measured using Cox regression. A total of 434,348 patients with asthma and 748,327 matched patients were included. All patients with asthma had a significantly increased risk of a General Practice diagnosis of COVID-19. Asthma with regular inhaled corticosteroid (ICS) use (hazard ratio [HR], 1.27; 95% confidence interval [CI], 1.01-1.61), intermittent ICS plus add-on asthma medication use (HR, 2.00; 95% CI, 1.43-2.79), regular ICS plus add-on use (HR, 1.63; 95% CI, 1.37-1.94), or with frequent exacerbations (HR, 1.82; 95% CI, 1.34-2.47) was significantly associated with hospitalization. These phenotypes were significantly associated with influenza and pneumonia hospitalizations. Only patients with regular ICS plus add-on asthma therapy (HR, 1.70; 95% CI, 1.27-2.26) or frequent exacerbations (HR, 1.66; 95% CI, 1.03-2.68) had a significantly higher risk of ICU admission or death. Atopy and blood eosinophil count were not associated with severe COVID-19 outcomes. More severe asthma was associated with more severe COVID-19 outcomes, but type 2 inflammation was not. The risk of COVID-19 hospitalization appeared to be similar to the risk with influenza or pneumonia.
Topics: Administration, Inhalation; Adrenal Cortex Hormones; Adult; Asthma; COVID-19; Critical Care; Death; England; Female; Hospitalization; Humans; Influenza, Human; Longitudinal Studies; Male; Middle Aged; Patient Acuity; Phenotype; Pneumonia; Proportional Hazards Models; SARS-CoV-2
PubMed: 34669568
DOI: 10.1164/rccm.202107-1704OC -
Journal of the American College of... Mar 2023
Topics: Humans; Cardiovascular Abnormalities; Loeys-Dietz Syndrome; Subclavian Artery; Marfan Syndrome
PubMed: 36889878
DOI: 10.1016/j.jacc.2023.01.020 -
BioRxiv : the Preprint Server For... Apr 2024Bloom Syndrome helicase (Blm) is a RecQ family helicase involved in DNA repair, cell-cycle progression, and development. Pathogenic variants in human cause the...
Bloom Syndrome helicase (Blm) is a RecQ family helicase involved in DNA repair, cell-cycle progression, and development. Pathogenic variants in human cause the autosomal recessive disorder Bloom Syndrome, characterized by predisposition to numerous types of cancer. Prior studies of mutants lacking helicase activity or protein have shown sensitivity to DNA damaging agents, defects in repairing DNA double-strand breaks (DSBs), female sterility, and improper segregation of chromosomes in meiosis. Blm orthologs have a well conserved and highly structured RecQ helicase domain, but more than half of the protein, particularly in the N-terminus, is predicted to be unstructured. Because this region is poorly conserved across multicellular organisms, we compared closely related species to identify regions of conservation, potentially indicating important functions. We deleted two of these -conserved regions in using CRISPR/Cas9 gene editing and assessed the effects on different Blm functions. Each deletion had distinct effects on different Blm activities. Deletion of either conserved region 1 (CR1) or conserved region 2 (CR2) compromised DSB repair through synthesis-dependent strand annealing and resulted in increased mitotic crossovers. In contrast, CR2 is critical for embryonic development but CR1 is not as important. CR1 deletion allows for proficient meiotic chromosome segregation but does lead to defects in meiotic crossover designation and patterning. Finally, deletion of CR2 does not lead to significant meiotic defects, indicating that while each region has overlapping functions, there are discreet roles facilitated by each. These results provide novel insights into functions of the N-terminal disordered region of Blm.
PubMed: 38659896
DOI: 10.1101/2024.04.12.589165 -
Molecular Therapy : the Journal of the... Feb 2020The recycling activity of cytidine deaminase (CDA) within the pyrimidine salvage pathway is essential to DNA and RNA synthesis. As such, CDA deficiency can lead to... (Review)
Review
The recycling activity of cytidine deaminase (CDA) within the pyrimidine salvage pathway is essential to DNA and RNA synthesis. As such, CDA deficiency can lead to replicative stress, notably in Bloom syndrome. Alternatively, CDA also can deaminate cytidine and deoxycytidine analog-based therapies, such as gemcitabine. Thus, CDA overexpression is often associated with lower systemic, chemotherapy-related, adverse effects but also with resistance to treatment. Considering the increasing interest of CDA in cancer chemoresistance, the aims of this review are to describe CDA structure, regulation of expression, and activity, and to report the therapeutic strategies based on CDA expression that recently emerged for tumor treatment.
Topics: Animals; Cytidine Deaminase; Disease Susceptibility; Gene Expression Regulation; Genetic Loci; Genetic Predisposition to Disease; Genetic Structures; Genetic Therapy; Humans; Structure-Activity Relationship
PubMed: 31870623
DOI: 10.1016/j.ymthe.2019.11.026