Removal of Spo11 from meiotic DNA breaksin vitrobut notin vivoby Tyrosyl DNA Phosphodiesterase 2

ABSTRACTMeiotic recombination events are initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein, Spo11. Similar to type-II topoisomerases, Spo11 becomes covalently linked to the 5′ ends generated on each side of the DSB. Whilst Spo11-oligos—the product of nucleolytic removal by Mre11—have been detected in a number of biological systems, the lifetime of the covalent Spo11-DSB precursor has not been systematically determined and may be subject to alternative processing reactions. Here we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2, on Spo11-DSBs isolated fromS. cerevisiaecells. We demonstrate that TDP2 can remove Spo11 from natural ssDNA-oligos, and dsDNA ends even when in the presence of excess competitor genomic DNA. Interestingly, TDP2-processed Spo11-DSBs are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essentialin vivoto facilitate resection-dependent HR at Spo11-DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptidesin vitro, TDP2 was unable to remove Spo11in vivo—unlike during the repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher order protein complexes resident at sites of meiotic recombination.

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The internal region of CtIP negatively regulates DNA end resection

Nucleic Acids Research ◽

10.1093/nar/gkaa273 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5485-5498 ◽

Cited By ~ 2

Author(s):

Sean Michael Howard ◽

Ilaria Ceppi ◽

Roopesh Anand ◽

Roger Geiger ◽

Petr Cejka

Keyword(s):

Homologous Recombination ◽

Regulatory Function ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Internal Region ◽

Dna End Resection ◽

End Resection

Abstract DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11–RAD50–NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM–DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350–600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN–CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550–600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

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A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

Nature Communications ◽

10.1038/s41467-019-12802-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 21

Author(s):

William H. Gittens ◽

Dominic J. Johnson ◽

Rachal M. Allison ◽

Tim J. Cooper ◽

Holly Thomas ◽

...

Keyword(s):

Dna Cleavage ◽

Genome Stability ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

Strand Breaks ◽

Single Strand Breaks ◽

Human Genomes ◽

Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

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A global view of meiotic double-strand break end resection

Science ◽

10.1126/science.aak9704 ◽

2017 ◽

Vol 355 (6320) ◽

pp. 40-45 ◽

Cited By ~ 79

Author(s):

Eleni P. Mimitou ◽

Shintaro Yamada ◽

Scott Keeney

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Naked Dna ◽

Global View ◽

Genome Wide ◽

End Resection

DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution inSaccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.

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Erratum for Kiziltepe et al., JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells

Blood ◽

10.1182/blood-2007-08-106435 ◽

2007 ◽

Vol 110 (13) ◽

pp. 4187-4187

Keyword(s):

Nitric Oxide ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Myeloma Cells ◽

Strand Breaks ◽

Damage Response ◽

Human Multiple Myeloma

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ATR is required to complete meiotic recombination in mice

10.1101/133744 ◽

2017 ◽

Cited By ~ 3

Author(s):

Sarai Pacheco ◽

Andros Maldonado-Linares ◽

Marina Marcet-Ortega ◽

Cristina Rojas ◽

Ana Martínez-Marchal ◽

...

Keyword(s):

Meiotic Recombination ◽

Meiotic Prophase ◽

Protein A ◽

Homology Search ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Molecular Features ◽

Chromosome Synapsis ◽

Meiotic Dsbs

ABSTRACTPrecise execution of recombination during meiosis is essential for forming chromosomally balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Binding of replication protein A (RPA) at resected DSBs fosters association of RAD51 and DMC1, the primary effectors of homology search. It is well appreciated that cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these responses remain poorly understood, particularly in mammals. Here we provide evidence that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

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A Role for DEAD Box 1 at DNA Double-Strand Breaks

Molecular and Cellular Biology ◽

10.1128/mcb.01053-08 ◽

2008 ◽

Vol 28 (20) ◽

pp. 6413-6425 ◽

Cited By ~ 57

Author(s):

Lei Li ◽

Elizabeth A. Monckton ◽

Roseline Godbout

Keyword(s):

Ionizing Radiation ◽

Neuroblastoma Cell ◽

Rnase H ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Rna Helicases ◽

Strand Breaks ◽

Dead Box

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

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Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Blood ◽

10.1182/blood-2008-07-165167 ◽

2009 ◽

Vol 113 (13) ◽

pp. 2965-2975 ◽

Cited By ~ 35

Author(s):

William Giblin ◽

Monalisa Chatterji ◽

Gerwin Westfield ◽

Tehmina Masud ◽

Brian Theisen ◽

...

Keyword(s):

Immune System ◽

Dna Cleavage ◽

Severe Combined Immunodeficiency ◽

Double Strand Breaks ◽

Mutant Form ◽

Dna Rearrangements ◽

Strand Breaks ◽

Immune System Dysfunction

Abstract The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating double-strand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence.

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Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin

The Journal of Cell Biology ◽

10.1083/jcb.200709008 ◽

2008 ◽

Vol 181 (2) ◽

pp. 227-240 ◽

Cited By ~ 146

Author(s):

Christoph Spycher ◽

Edward S. Miller ◽

Kelly Townsend ◽

Lucijana Pavic ◽

Nicholas A. Morrice ◽

...

Keyword(s):

Nijmegen Breakage Syndrome ◽

Dependent Manner ◽

Sequence Motifs ◽

Dna Double Strand Breaks ◽

Focus Formation ◽

Strand Breaks ◽

Mrn Complex ◽

Interfering Rna

The MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin.

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A versatile system to introduce clusters of genomic double-strand breaks in large cell populations

10.1101/2020.04.27.064105 ◽

2020 ◽

Author(s):

Thorsten Kolb ◽

Umar Khalid ◽

Milena Simović ◽

Manasi Ratnaparkhe ◽

John Wong ◽

...

Keyword(s):

Primary Cultures ◽

Large Cell ◽

Time Lapse ◽

Genome Rearrangements ◽

Dna Lesions ◽

Double Strand Breaks ◽

Brdu Incorporation ◽

Dna Double Strand Breaks ◽

Strand Breaks

ABSTRACTIn vitro assays for clustered DNA lesions will facilitate the analysis of the mechanisms underlying complex genome rearrangements such as chromothripsis, including the recruitment of repair factors to sites of DNA double-strand breaks. We present a novel method generating localized DNA double-strand breaks using UV-irradiation with photomasks. The size of the damage foci and the spacing between lesions are fully adjustable, making the assay suitable for different cell types and targeted areas. We validated this set-up with genomically stable epithelial cells, normal fibroblasts, pluripotent stem cells and patient-derived primary cultures. Our method does not require a specialized device such as a laser, making it accessible to a broad range of users. Sensitization by BrdU incorporation is not required, which enables analyzing the DNA damage response in post-mitotic cells. Irradiated cells can be cultivated further, followed by time-lapse imaging or used for downstream biochemical analyses, thanks to the high-throughput of the system. Importantly, we showed genome rearrangements in the irradiated cells, providing a proof of principle for the induction of structural variants by localized DNA lesions.

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