Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice

2021 ◽  
Author(s):  
Shanzhi Wang ◽  
Kyeryoung Lee ◽  
Stephen Gray ◽  
Yongwei Zhang ◽  
Catherine Tang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Somatic Hypermutation ◽  
Nuclease Activity ◽  
Strand Break ◽  
Mutant Form ◽  
Metabolic Processes ◽  
Dna Mismatch ◽  
Antibody Diversification ◽  
Repair Pathways ◽  
V Region

ABSTRACTDNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1−/− and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1−/− mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1−/− mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1−/− mice was comparably defective, switch-switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1−/− mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1−/− mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.

scholarly journals DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1680
Author(s):  
Tassanee Lerksuthirat ◽  
Rakkreat Wikiniyadhanee ◽  
Sermsiri Chitphuk ◽  
Wasana Stitchantrakul ◽  
Somponnat Sampattavanich ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Fluorescent Proteins ◽  
Strand Break ◽  
Control Group ◽  
Focus Formation ◽  
Dna Damage And Repair ◽  
Biological Compounds ◽  
Repair Pathways ◽  
Garcinia Cowa

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

scholarly journals Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea

2021 ◽  
Vol 22 (24) ◽  
pp. 13296
Author(s):  
Mariarosaria De Falco ◽  
Mariarita De Felice
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Genome Stability ◽  
Developmental Disorders ◽  
Strand Break ◽  
Human Diseases ◽  
Dna Damages ◽  
Dna Repair Mechanisms ◽  
Repair Mechanisms ◽  
Repair Pathways

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.

Primary immunodeficiencies associated with DNA-repair disorders

2010 ◽  
Vol 12 ◽  
Author(s):  
Mary A. Slatter ◽  
Andrew R. Gennery
Keyword(s):  
Dna Repair ◽  
Adaptive Immunity ◽  
Somatic Hypermutation ◽  
Affinity Maturation ◽  
Antigen Receptors ◽  
Antibody Isotype ◽  
Vdj Recombination ◽  
Replication Errors ◽  
Clinical Consequences ◽  
Repair Pathways

DNA-repair pathways recognise and repair DNA damaged by exogenous and endogenous agents to maintain genomic integrity. Defects in these pathways lead to replication errors, loss or rearrangement of genomic material and eventually cell death or carcinogenesis. The creation of diverse lymphocyte receptors to identify potential pathogens requires breaking and randomly resorting gene segments encoding antigen receptors. Subsequent repair of the gene segments utilises ubiquitous DNA-repair proteins. Individuals with defective repair pathways are found to be immunodeficient and many are radiosensitive. The role of repair proteins in the development of adaptive immunity by VDJ recombination, antibody isotype class switching and affinity maturation by somatic hypermutation has become clearer over the past few years, partly because of identification of the genes involved in human disease. We describe the mechanisms involved in the development of adaptive immunity relating to DNA repair, and the clinical consequences and treatment of the primary immunodeficiency resulting from such defects.

scholarly journals Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

2015 ◽  
Vol 35 (21) ◽  
pp. 3657-3668 ◽  
Author(s):  
Han Lin ◽  
Kyungsoo Ha ◽  
Guojun Lu ◽  
Xiao Fang ◽  
Ranran Cheng ◽  
...  
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
Mitotic Exit ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Downstream Target ◽  
Damage Repair ◽  
Repair Pathways ◽  
Repair Defect

Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair.

scholarly journals Preserving genome integrity in human cells via DNA double-strand break repair

2020 ◽  
Vol 31 (9) ◽  
pp. 859-865 ◽  
Author(s):  
Ryan B. Jensen ◽  
Eli Rothenberg
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Human Cells ◽  
Cell Cycle Checkpoints ◽  
Human Diseases ◽  
Genome Integrity ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways

The efficient maintenance of genome integrity in the face of cellular stress is vital to protect against human diseases such as cancer. DNA replication, chromatin dynamics, cellular signaling, nuclear architecture, cell cycle checkpoints, and other cellular activities contribute to the delicate spatiotemporal control that cells utilize to regulate and maintain genome stability. This perspective will highlight DNA double-strand break (DSB) repair pathways in human cells, how DNA repair failures can lead to human disease, and how PARP inhibitors have emerged as a novel clinical therapy to treat homologous recombination-deficient tumors. We briefly discuss how failures in DNA repair produce a permissive genetic environment in which preneoplastic cells evolve to reach their full tumorigenic potential. Finally, we conclude that an in-depth understanding of DNA DSB repair pathways in human cells will lead to novel therapeutic strategies to treat cancer and potentially other human diseases.

scholarly journals Integration of Mutational Signature Analysis with 3D Chromatin Data Unveils Differential AID-Related Mutagenesis in Indolent Lymphomas

2021 ◽  
Vol 22 (23) ◽  
pp. 13015
Author(s):  
Julieta H. Sepulveda-Yanez ◽  
Diego Alvarez-Saravia ◽  
Jose Fernandez-Goycoolea ◽  
Jacqueline Aldridge ◽  
Cornelis A. M. van Bergen ◽  
...  
Keyword(s):  
Dna Repair ◽  
De Novo ◽  
Somatic Hypermutation ◽  
Excision Repair ◽  
Gene Mutations ◽  
Lymphocytic Leukemia ◽  
Sequencing Data ◽  
Repair Gene ◽  
Repair Pathways ◽  
Activation Induced Deaminase

Activation-induced deaminase (AID) is required for somatic hypermutation in immunoglobulin genes, but also induces off-target mutations. Follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL), the most frequent types of indolent B-cell tumors, are exposed to AID activity during lymphomagenesis. We designed a workflow integrating de novo mutational signatures extraction and fitting of COSMIC (Catalogue Of Somatic Mutations In Cancer) signatures, with tridimensional chromatin conformation data (Hi-C). We applied the workflow to exome sequencing data from lymphoma samples. In 33 FL and 30 CLL samples, 42% and 34% of the contextual mutations could be traced to a known AID motif. We demonstrate that both CLL and FL share mutational processes dominated by spontaneous deamination, failures in DNA repair, and AID activity. The processes had equiproportional distribution across active and nonactive chromatin compartments in CLL. In contrast, canonical AID activity and failures in DNA repair pathways in FL were significantly higher within the active chromatin compartment. Analysis of DNA repair genes revealed a higher prevalence of base excision repair gene mutations (p = 0.02) in FL than CLL. These data indicate that AID activity drives the genetic landscapes of FL and CLL. However, the final result of AID-induced mutagenesis differs between these lymphomas depending on chromatin compartmentalization and mutations in DNA repair pathways.

scholarly journals The HMCES DNA-protein cross-link functions as a constitutive DNA repair intermediate

2021 ◽  
Author(s):  
Daniel R Semlow ◽  
Victoria A MacKrell ◽  
Johannes Walter
Keyword(s):  
Dna Repair ◽  
Strand Break ◽  
S Phase ◽  
Cross Link ◽  
Link Functions ◽  
Cross Links ◽  
Ap Sites ◽  
Repair Pathways ◽  
Break Formation ◽  
Ap Site

The HMCES protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in ssDNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we show that an HMCES-DPC forms efficiently on the AP site generated during replication-coupled DNA interstrand cross-link (ICL) repair. We use this system to show that HMCES cross-links form on DNA after the replicative CMG helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses DSB formation, slows translesion synthesis (TLS) past the AP site, and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data show that HMCES-DPCs can form as constitutive intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

scholarly journals Evolution-based screening enables genome-wide prioritization and discovery of DNA repair genes

2019 ◽  
Vol 116 (39) ◽  
pp. 19593-19599 ◽  
Author(s):  
Gregory J. Brunette ◽  
Mohd A. Jamalruddin ◽  
Robert A. Baldock ◽  
Nathan L. Clark ◽  
Kara A. Bernstein
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Genome Stability ◽  
Strand Break ◽  
Evolutionary Analysis ◽  
Dna Double Strand Breaks ◽  
Atm Kinase ◽  
Functional Relationships ◽  
Genome Wide ◽  
Repair Pathways

DNA repair is critical for genome stability and is maintained through conserved pathways. Traditional genome-wide mammalian screens are both expensive and laborious. However, computational approaches circumvent these limitations and are a powerful tool to identify new DNA repair factors. By analyzing the evolutionary relationships between genes in the major DNA repair pathways, we uncovered functional relationships between individual genes and identified partners. Here we ranked 17,487 mammalian genes for coevolution with 6 distinct DNA repair pathways. Direct comparison to genetic screens for homologous recombination or Fanconi anemia factors indicates that our evolution-based screen is comparable, if not superior, to traditional screening approaches. Demonstrating the utility of our strategy, we identify a role for the DNA damage-induced apoptosis suppressor (DDIAS) gene in double-strand break repair based on its coevolution with homologous recombination. DDIAS knockdown results in DNA double-strand breaks, indicated by ATM kinase activation and 53BP1 foci induction. Additionally, DDIAS-depleted cells are deficient for homologous recombination. Our results reveal that evolutionary analysis is a powerful tool to uncover novel factors and functional relationships in DNA repair.

scholarly journals PDTM-41. INHIBITION OF DNA DOUBLE STRAND BREAK REPAIR PATHWAYS AS A PROMISING THERAPEUTIC TARGET IN DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi196-vi196
Author(s):  
Sharmistha Pal ◽  
Jie Bian ◽  
Brendan Price ◽  
Dipanjan Chowdhury ◽  
Daphne Haas-Kogan
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Diffuse Intrinsic Pontine Gliomas ◽  
Pathway Inhibition

Abstract New approaches to the treatment of diffuse intrinsic pontine gliomas (DIPGs) are desperately needed. DNA damage response is essential for cells to maintain genome integrity as DNA is damaged by both endogenous and exogenous stressors. Many cancer cells exhibit hyper-dependency on specific DNA repair pathways due to either defects in DNA repair mechanisms and/or high levels of endogenous stress leading to accumulation of DNA damage lesions. Identification of DIPG-specific DNA repair deficiencies and resultant dependencies may establish novel therapeutic strategies for DIPGs. METHODS To identify pathways critical for DIPG cell survival, genome wide CRISPR-Cas9 screen was performed on patient derived DIPG cell lines followed by gene set enrichment analyses. To monitor the effects of pathway inhibition on survival, apoptosis, DNA damage and repair, assays were performed to measure cell proliferation, cleaved-caspase3, gamma-H2AX and reporter based-DNA repair efficiency. RESULTS Our unbiased CRISPR approach to uncover vulnerabilities in DIPGs identified DNA double strand break (DSBs) repair pathways as essential for DIPG cell proliferation and survival. Further studies revealed high basal DSBs in DIPG cells compared to neural stem cells and primary astrocytes that suggest dependence of DIPG cell survival on specific DSB repair pathways. We confirmed the intrinsic reliance of DIPG cells on the specific DSB repair pathway of mutagenic end-joining, and defined a key role for DNA repair in suppressing endogenous DNA damage-induced apoptotic cell death. CONCLUSION DIPG cells have high endogenous DNA damage levels and escape catastrophic genomic instability and cell death by engaging DNA repair pathways, in particular the mutagenic end-joining DNA repair pathway. Inhibition of this specific DNA repair pathway represents a promising new avenue for the treatment of DIPGs.

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