REV7 is required for processing AID initiated DNA lesions in activated B cells.
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ABSTRACT:
SUBMITTER: Yang D
PROVIDER: S-EPMC7272641 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Activation-induced deaminase (AID) initiates U:G mismatches, causing point mutations or DNA double-stranded breaks at Ig loci. How AID-initiated lesions are prevented from inducing genome-wide damage remains elusive. A differential DNA repair mechanism might protect certain non-Ig loci such as c-myc from AID attack. However, determinants regulating such protective mechanisms are largely unknown. To test whether target DNA sequences modulate protective mechanisms via altering the processing manner of AID-initiated lesions, we established a knock-in model by inserting an S?2b region, a bona fide AID target, into the first intron of c-myc. Unexpectedly, we found that the inserted S region did not mutate or enhance c-myc genomic instability, due to error-free repair of AID-initiated lesions, in Ag-stimulated germinal center B cells. In contrast, in vitro cytokine-activated B cells display a much higher level of c-myc genomic instability in an AID- and S region-dependent manner. Furthermore, we observe a comparable frequency of AID deamination events between the c-myc intronic sequence and inserted S region in different B cell populations, demonstrating a similar frequency of AID targeting. Thus, our study reveals a clear difference between germinal center and cytokine-activated B cells in their ability to develop genomic instability, attributable to a differential processing of AID-initiated lesions in distinct B cell populations. We propose that locus-specific regulatory mechanisms (e.g., transcription) appear to not only override the effects of S region sequence on AID targeting frequency but also influence the repair manner of AID-initiated lesions.
| S-EPMC4239163 | biostudies-literature
Project description:Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core S? and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity.
| S-EPMC4760233 | biostudies-literature
Project description:During B-cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent C? constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200 kilobases downstream in the same transcriptional orientation as V(D)J and C? exons. Long repetitive switch (S) regions precede C? and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing C? with a downstream CH (ref. 2). Activation-induced cytidine deaminase (AID) initiates CSR by promoting deamination lesions within S? and a downstream acceptor S region; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an S? DSB to the downstream end of an acceptor S-region DSB. However, the relative frequency of deletional to inversional CSR junctions has not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved is unknown. To address this question, we adapt high-throughput genome-wide translocation sequencing into a highly sensitive DSB end-joining assay and apply it to endogenous AID-initiated S-region DSBs in mouse B cells. We show that CSR is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis Igh organizational features in combination with frequent S-region DSBs initiated by AID. We further implicate ATM-dependent DSB-response factors in enforcing this mechanism and provide an explanation of why CSR is so reliant on the 53BP1 DSB-response factor.
| S-EPMC4592165 | biostudies-literature
Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor. We performed high-throughput genome-wide translocation sequencing (HTGTS) with different B cell genotypes that induces either I-SceI or AID-initiated DSBs as bait to study their joining pattern to AID-initiated S region breaks Please note that the 'ΔSγ1_2xI/ΔSµ_2xI-3' raw data files were analyzed twice with different reference assemblies to address specific points, and associated with both 'ΔSγ1_2xI_ΔSµ_2xI-3'.txt and Sγ1_2xI_ΔSµ_2xI-3'_trans-Sm.txt processed data files. As for the reference genome mm9_129_IgHC, it is a custom built generated based on mm9 (Bl6 line based mouse genome) and the partial genome sequence of another mouse line 129sv. It is not currently available in any public database however can be easily obtained either from the authors or self-built using information provided in the associated manuscript.
2015-08-26 | E-GEOD-71005 | biostudies-arrayexpress
Project description:REV7 is a small multifunctional protein that participates in multiple DNA repair pathways, most notably translesion DNA synthesis and double-strand break (DSB) repair. While the role of REV7 in translesion synthesis has been known for several decades, its function in DSB repair is a recent discovery. Investigations into the DSB repair function of REV7 have led to the discovery of a new DNA repair complex known as Shieldin. Recent studies have also highlighted the importance of REV7's HORMA domain, an ancient structural motif, in REV7 function and have identified the HORMA regulators, TRIP13 and p31, as novel DNA repair factors. In this review, we discuss these recent findings and their implications for repair pathway choice, at both DSBs and replication forks. We suggest that REV7, in particular the activation state of its HORMA domain, can act as a critical determinant of mutagenic versus error-free repair in multiple contexts.
| S-EPMC8608404 | biostudies-literature
Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor.
2015-08-26 | GSE71005 | GEO
Project description:Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7(C70R)) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase ? (Pol?), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Pol?. Rev7(C70R/C70R) cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive ?H2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7(C70R) mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Pol? preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.
| S-EPMC3916577 | biostudies-literature
Project description:Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.
2015-07-16 | GSE66424 | GEO
Project description:Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.
| S-EPMC4532491 | biostudies-literature
Project description:To exploit vulnerabilities of tumors, it is urgent to identify associated defects in genome maintenance. One unsolved problem is the mechanism of regulation of DNA double-strand break repair by REV7 in complex with 53BP1 and RIF1, and its influence on repair pathway choice between homologous recombination and non-homologous end-joining. We searched for REV7-associated factors in human cells and found FAM35A, a previously unstudied protein with an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded DNA-binding protein RPA, as novel interactor of REV7/RIF1/53BP1. FAM35A re-localized in damaged cell nuclei, and its knockdown caused sensitivity to DNA-damaging agents. In a BRCA1-mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. We found FAM35A absent in one widely used BRCA1-mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases, revealing promise for FAM35A as a therapeutically relevant cancer marker.
| S-EPMC6003645 | biostudies-literature