RPA accumulation during class switch recombination represents 5'-3' DNA end resection during the S-G2/M phase of the cell cycle
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE42451 | GEO | 2013/01/02
SECONDARY ACCESSION(S): PRJNA182307
REPOSITORIES: GEO
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Project description:AID promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in G1. RPA is a ssDNA-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR) such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in S-G2/M is extensive, ATM-independent, and associated with Rad51 accumulation. RPA in S-G2/M increases in NHEJ-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during CSR represents salvage of un-repaired breaks by homology-based pathways during the S-G2/M phases of the cell cycle. Chip-Seq of RPA from mouse activated B cells (n = 40), mouse thymocytes (n = 6), and MEFs (n = 1). Different genotypes and/or inhibitors were used.
2013-01-02 | E-GEOD-42451 | biostudies-arrayexpress
Project description:Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by HR, suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. Strand-specific transcriptome analysis of biological replicates of WT cells (JKM139 strain) at T0, or 60 and 240 minutes after HO induction, and of xrn1∆, rrp6∆ and trf4∆ cells at T0.
2014-11-20 | E-GEOD-63444 | biostudies-arrayexpress
Project description:Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) and proceeds via binding of RPA, RAD51 and DMC1 to single-stranded DNA (ssDNA) substrates created after the formation of DSBs. Here, we report high-resolution in vivo maps of RPA and RAD51 binding in meiosis, mapping their binding locations and lifespans in a B6 and a genetically modified B6xCAST F1 mouse. We ascribe signals separately to the individual homologous chromosomes in the hybrid mouse, thereby separating the signal of binding to the chromosome where DSBs occurred and the chromosome that was used as template for repair. Together with super-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: whereas DMC1 binds near the break-site, RAD51 binds away from it. We characterize the D-loop, a critical intermediate bound by RPA, in vivo. These data show that DMC1, not RAD51, performs strand exchange in mammalian meiosis. We find that the localisation of D-loop intermediates is similar in crossover and non-crossover pathways, with a longer lifespan for crossover-destined intermediates. These findings answer long-standing questions about the molecular intermediates of recombination.
2020-06-30 | GSE143582 | GEO
Project description:Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by HR, suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.
2014-11-20 | GSE63444 | GEO
Project description:DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether it does so directly is not known. Here we identify Rapl-interacting factor 1 (Rif1) as an Ataxia-Telangiectasia Mutated (ATM) phosphorylation-dependent interactor of 53BP1, and show that absence of Rif1 results in 5M-bM-^@M-^Y-3M-bM-^@M-^Y DNA end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G1 and S phases of the cell cycle, interferes with class switch recombination (CSR) in B lymphocytes, and leads to accumulation of chromosome DSBs. Study of Rif1 DNA-end protection activity against resection via analysis of single-stranded DNA binding protein RPA and Rad51 accumulation at sites of AID-induced DNA damage by ChIP-seq. All samples shown in Fig. 4 are included (controls and test samples, 7 samples in total).
2013-03-01 | E-GEOD-42298 | biostudies-arrayexpress
Project description:During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strand can displace the parental strand to generate single-strand DNA (ssDNA) flaps. Many programmed DSBs and thus many ssDNA flaps occur during meiosis. However, how these flaps are removed remains enigmatic. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) in Saccharomyces cerevisiae causes an abundant accumulation of RPA-ssDNA flaps and an expansion of RPA from DSBs to broader regions. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, inducing Dna2 expression at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreased and increased RPA accumulation in dna2-md, respectively. Together, our findings show that meiotic DSB repair requires Dna2 to remove RPA-ssDNA flaps generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.
2023-08-30 | GSE217628 | GEO
Project description:DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins DNA double strand breaks (DSBs) in B lymphocytes are thought to arise stochastically during replication (S phase) or as a result of targeted DNA damage by activation induced cytidine deaminase (AID) in G1. Here we identify a novel class of recurrent, early replicating and AID independent DNA lesions, termed early replication fragile sites (ERFS), by genome-wide localization of DNA repair proteins RPA, SMC5, gamma-H2AX, and BRCA1 in B cells subjected to replication stress. Protein-DNA association for four DNA damage response proteins (RPA, SMC5, g-H2AX, BRCA1), BrdU incorporation, and gene transcription in B lymphocytes with and without hydroxyurea treatment were examined.
2013-02-04 | E-GEOD-43504 | biostudies-arrayexpress
Project description:Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks (DSBs). This process requires choreographed binding of RPA, DMC1 and RAD51 to single-stranded DNA (ssDNA) substrates and in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the homologous chromosome used as template for repair, which reveals further mechanistic detail and enables the structure of the recombination intermediates to be inferred. By applying CRISPR/Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this powerful analysis technique to explore the molecular detail of recombination when a key component is knocked-out. As a proof-of-concept, we have generated biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in these knockout hybrid mice. Dmc1 mutants undergo meiotic arrest and comparison of these maps with those from wild-type mice is informative about the structure and timing of recombination intermediates in both genotypes. We confirm a complete abrogation of strand exchange in Dmc1 mutants, and observe a redistribution of RAD51 binding across both the distal and proximal ends of the resected DNA. We observe unexpected RPA and DMC1 binding in the wild-type, which suggests multiple rounds of strand invasion with template-switching in mouse. The methodology used involves direct phenotyping of hybrid “founder” mice following CRISPR mutagenesis and provides a high-throughput approach for the analysis of gene function during meiotic recombination, at low animal cost.
2023-10-16 | GSE239997 | 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.
2015-07-16 | GSE66424 | GEO
Project description:After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). Using a candidate approach, AID has been shown to initiate mutations at numerous non-Ig genes in B cells in Peyer’s patches. However, it is not known if during B cell activation, AID also induces DNA breaks at genes other than IgH genes, which would lead to genomic instability and malignancy. Using a non-biased genome-wide approach, we have identified hundreds of reproducible AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. The AID-dependent DSBs are not restricted to transcribed regions, and they frequently occur within repeated sequence elements, including CA and non-CA repeats, and SINEs. Comparison of Nbs1 binding sites in wild-type vs. aid-/- splenic B cells induced to undergo CSR; aid-dependent Nbs1 sites correlated with gene transcription (RNA Pol2).
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