FAM72A promotes mutagenic repair during antibody maturation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE183706 | GEO | 2021/09/11
REPOSITORIES: GEO
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Project description:FAM72A promotes mutagenic repair during antibody maturation
| PRJNA761718 | ENA
Project description:DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), but also unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, which was confirmed using mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA to promote genome stability in mammalian cells, specially in response to base damage.
2024-11-22 | GSE282260 | GEO
Project description:HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used to treat B-cell-derived malignancies. Such malignancies regularly carry mutational signatures that conform to off-target induction of uracil by the AID/APOBEC family of cytidine deaminases, or downstream processing of uracil. . HDACi suppress thymidylate synthase increasing the cellular dUTP/dTTP ratio and leading to increased pressure on uracil repair machinery due to misincorporated uracil lesions. To investigate potential effect upon other enzymes involved in genomic uracil induction and processing, Jurkat (T-cell lymphoma) and SUDHL5 (B-cell lymphoma) cells were treated with pan-HDACi SAHA prior to SILAC based MS/MS investigation. HDACi treatment mediated significant differential expression of xx and xx proteins in Jurkat and SUDHL5, respectively, and had a substantial impact upon enzymes involved in in pyrimidine metabolism. Surprisingly, uracil N-glycosylase, UNG, was strongly downregulated by HDACi treatment. Further analysis in HEK and HeLa cells revealed that HDACis induce specific loss of the nuclear isoform UNG2 independent of transcription and cell-cycle alterations. More than 80% of UNG2 is degraded proteasomally after 24 hours treatment with SAHA, MS275, Valproate or Na-butyrate, indicating a universal ability of HDACis to mediate loss of UNG2. Targeted MS/MS analysis in HEK cells against a panel of proteins involved in DNA repair, translesion synthesis and nucleotide metabolism, revealed that UNG2 was the most pronounced differentially expressed among these after HDACi treatment. 48 hour treatment lead to a 30-40% increase in uracil lesions in the nuclear genome of HeLa and HEK cells and MS275 treatment in murine CH12F3 cell line mediated robust UNG2-loss accompanied by reduced class switch recombination. Furthermore, our analysis identified the PCNA-associated factor PAF15 among the downregulated proteins. PAF15 is overexpressed in many cancers and suppress TLS by inducing double monoubiquitinylation of PCNA and recruitment of the replicative polymerases. In summary, our findings demonstrate that HDAC inhibition affects the levels of proteins involved in DNA base excision repair, translesion synthesis and pyrimidine metabolism. These findings are important for a wide range of clinical applications of HDACi, such as in rheumatology, HIV-, and cancer treatment.
2020-04-14 | PXD008293 | Pride
Project description:Data from Excision-seq experiments to map deoxyuridine and pyrimidine dimers in S. cerevisiae and E. coli For uracil mapping in pre-digestion Excision-seq, uracil was excised from genomic DNA from dut ung yeast and bacteria yielding double-stranded DNA fragments. Adapters were ligated to these fragments for Illumina library preparation. Using this method, the number of reads at a genomic location corresponds to the quantity of dU at that location. In post-digestion Excision-seq, uracil-containing library fragments are destroyed by UDG treatment, thus coverage is inversely proportional to uracil content. For pyrimidine dimer Excision-seq, yeast were irradiated with high doses of UVC light to generate a large number of DNA modifications. Using the excision repair enzyme UVDE from S. pombe, we excised pyrimidine dimers, cutting the phosphodieseter bond to release small double stranded fragments. These fragments were repaired with either CPD photolyase or 6-4 photolyase to generate independent libraries for each modification type. Illumina adapters were ligated to these fragments for library preparation. Using this method the number of reads at a genomic location corresponds to the quantity of the 3' pyrimidine of the dimer.
2014-05-15 | E-GEOD-51361 | biostudies-arrayexpress
Project description:Uracil in DNA can be generated as a result of cytosine deamination or dUMP misincorporation. However, its distribution in the human genome is poorly understood, due to the lack of a sensitive detection method. Here we present “dU-seq”, a selective labeling and pull-down technology, to profile uracil in the human genome. dU-seq specifically labels uracil-containing DNA via an in vitro base excision repair reaction and identifies thousands of uracil peaks in the human genome.
2018-05-24 | GSE99011 | GEO
Project description:Uracil DNA glycosylases (UDGs) excise uracil from DNA arising from dUMP misincorporation during replication or from cytosine deamination. Besides functioning in canonical uracil repair, UDGs cooperate with DNA base modifying enzymes to effect mutagenesis or DNA demethylation. Mammalian cells express four UDGs, the functional dissection of which represents a challenge. Here, we used Schizosaccharomyces pombe with only two UDGs, Ung1 and Thp1, as a simpler model to study functional interactions in uracil repair. We show that despite a predominance of Ung1 activity in cell extracts, both UDGs act redundantly against genomic uracil accumulation and mutations from cytosine deamination in cells. Notably, Thp1 but not Ung1-dependent repair is cytotoxic under genomic uracil stress induced by 5-fluorouracil exposure or AID expression. Also, Thp1- but not Ung1-mediated base excision is recombinogenic, accounting for more than 60% of spontaneous mitotic recombination events in a recombination assay. Hence, the qualitative outcome of uracil repair depends on the initiating UDG; while Ung1 shows expected features of a bona-fide DNA repair enzyme, Thp1-initiated repair appears slow and non-productive, implicating a function beyond canonical DNA repair. Given the epigenetic role of mammalian TDGs, we performed transcriptome analyses and identified a possible function of Thp1 in stabilizing gene expression.
2014-12-31 | GSE64585 | GEO
Project description:Using novel methods for mapping U/A base pairs in HIV DNA, we find that HIV proviruses within infected monocytes and macrophages contain high levels of U/A base pairs arising from the high levels of dUTP present during reverse transcription in these cells. Although U/A pairs retain the coding information of T/A pairs, they are the substrate for the nuclear uracil base excision repair (UBER) machinery that effectively degrades most of the uracilated viral DNA at the pre-integration stage of infection. Uracilated proviruses that successfully integrate either retain the uracils, undergo U/A T/A repair, or experience catastrophic mutagenesis induced by cytokine stimulated error-prone repair. Uracil is abundant in proviruses isolated from genomic DNA of short-lived blood monocytes, but not T cells, of HIV infected blood donors who show complete drug suppression plasma virus. This suggests that monocytes are recently infected by coming into contact with persistent virus producing cells in one or more tissue reservoirs. Data for HIV lockdown of Illumina libraries of MDM cells post viral infection with and without UDG treatment as compared to HT29-ugi cells treated with RTX.
2016-01-19 | E-GEOD-76091 | biostudies-arrayexpress
Project description:The nematode Caenorhabditis elegans has been used extensively to study responses to DNA damage. In contrast, little is known about DNA repair in this organism. C. elegans is unusual in that it encodes few DNA glycosylases and the uracil-DNA glycosylase (UDG) encoded by the ung-1 gene is the only known UDG. C. elegans could therefore become a valuable model organism for studies of the genetic interaction networks involving base excision repair (BER). As a first step towards characterization of BER in C. elegans, we show that the UNG-1 protein is an active uracil-DNA glycosylase. We demonstrate that an ung-1 mutant has reduced ability to repair uracil-containing DNA but that an alternative Ugi-inhibited activity is present in ung-1 nuclear extracts. Finally, we demonstrate that ung-1 mutants show altered levels of apoptotic cell corpses formed in response to DNA damaging agents. Increased apoptosis in the ung-1 mutant in response to ionizing radiation (IR) suggests that UNG-1 contributes to repair of IR-induced DNA base damage in vivo. Following treatment with paraquat however, the apoptotic corpse-formation was reduced. Gene expression profiling suggests that this phenotype is a consequence of compensatory transcriptomic shifts that modulate oxidative stress responses in the mutant and not an effect of reduced DNA damage signaling. C. elegans RNAi mutants deficient in ung-1 and the corresponding wild-type N2, were subjected to Affymetrix whole C. elegans genome microarrays. Triplicates were run for each sample group.
2010-06-08 | E-GEOD-21467 | biostudies-arrayexpress
Project description:Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3B, A3B) is a key molecular driver inducing mutations in multiple human cancer. A3B belongs to the APOBEC3 enzyme family, which consists seven closely related DNA deaminases that catalyse cytosine-to-uracil (C>U) editing of single-stranded DNA (ssDNA). Here, we used a lentiviral inducible system to overexpress A3B and hUGI in T-47D cells. This system will allow augmented genomic editing by A3B, while preserving the C>U editing sites from DNA repair by inhibiting base excision repair mechanism.
2025-01-06 | GSE193226 | GEO
Project description:Using novel methods for mapping U/A base pairs in HIV DNA, we find that HIV proviruses within infected monocytes and macrophages contain high levels of U/A base pairs arising from the high levels of dUTP present during reverse transcription in these cells. Although U/A pairs retain the coding information of T/A pairs, they are the substrate for the nuclear uracil base excision repair (UBER) machinery that effectively degrades most of the uracilated viral DNA at the pre-integration stage of infection. Uracilated proviruses that successfully integrate either retain the uracils, undergo U/A T/A repair, or experience catastrophic mutagenesis induced by cytokine stimulated error-prone repair. Uracil is abundant in proviruses isolated from genomic DNA of short-lived blood monocytes, but not T cells, of HIV infected blood donors who show complete drug suppression plasma virus. This suggests that monocytes are recently infected by coming into contact with persistent virus producing cells in one or more tissue reservoirs.
2016-01-19 | GSE76091 | GEO