scholarly journals iMUT-seq: high-resolution mapping of DSB mutational landscapes reveals new insights into the mutagenic mechanisms of DSB repair

2021 ◽  
Author(s):  
Aldo S Bader ◽  
Martin Bushell
Keyword(s):  
Cancer Biology ◽  
High Sensitivity ◽  
Primary Role ◽  
Dna Double Strand Breaks ◽  
Induced Mutations ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Resolution Mapping ◽  
Induced Mutagenesis ◽  
Nucleotide Resolution

DNA double-strand breaks (DSBs) are the most mutagenic form of DNA damage, and play a significant role in cancer biology, neurodegeneration and aging. However, studying DSB-induced mutagenesis is currently limited by the tools available for mapping these mutations. Here, we describe iMUT-seq, a technique that profiles DSB-induced mutations at high-sensitivity and single-nucleotide resolution around endogenous DSBs spread across the genome. By depleting 20 different DSB-repair factors we defined their mutational signatures in detail, revealing remarkable insights into the mechanisms of DSB-induced mutagenesis. We find that homologous-recombination (HR) is mutagenic in nature, displaying high levels of base substitutions and mononucleotide deletions due to DNA-polymerase errors, but simultaneously reduced translocation events, suggesting the primary role of HR is the specific suppression of genomic rearrangements. The results presented here offer new fundamental insights into DSB-induced mutagenesis and have significant implications for our understanding of cancer biology and the development of DDR-targeting chemotherapeutics.

scholarly journals Locally, Meiotic Double-Strand Breaks Targeted by Gal4BD-Spo11 Occur at Discrete Sites with a Sequence Preference

2009 ◽  
Vol 29 (13) ◽  
pp. 3500-3516 ◽  
Author(s):  
Hajime Murakami ◽  
Alain Nicolas
Keyword(s):  
Hot Spots ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Sequence Preference ◽  
Meiotic Dsbs ◽  
Resolution Mapping ◽  
Nucleotide Resolution ◽  
Recombination Hot Spots

ABSTRACT Meiotic recombination is initiated by DNA double-strand breaks (DSBs) that are catalyzed by the type II topoisomerase-like Spo11 protein. Locally, at recombination hot spots, Spo11 introduces DSBs at multiple positions within ∼75 to 250 bp, corresponding to accessible regions of the chromatin. The molecular basis of this multiplicity of cleavage positions, observed in a population of meiotic cells, remains elusive. To address this issue, we have examined the properties of the Gal4BD-Spo11 fusion protein, which targets meiotic DSBs to regions with Gal4 binding sites (UAS). By single-nucleotide resolution mapping of targeted DSBs, we found that DSB formation was restricted to discrete sites approximately 20 nucleotides from the UAS, defining a “DSB targeting window.” Thus, the multiplicity of cleavage positions at natural Spo11 hot spots likely represents binding of Spo11 to different distinct sites within the accessible DNA region in each different meiotic cell. Further, we showed that mutations in the Spo11 moiety affected the DSB distribution in the DSB targeting window and that mutations in the DNA at the Spo11 cleavage site affected DSB position. These results demonstrate that Spo11 itself has sequence preference and contributes to the choice of DSB positions.

scholarly journals Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

scholarly journals RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair

2021 ◽  
Author(s):  
Takaaki Yasuhara ◽  
Reona Kato ◽  
Motohiro Yamauchi ◽  
Yuki Uchihara ◽  
Lee Zou ◽  
...  
Keyword(s):  
Active Regions ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Critical Component ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Chromosome Translocations ◽  
Dna Double Strand Break

AbstractR-loops, consisting of ssDNA and DNA-RNA hybrids, are potentially vulnerable unless they are appropriately processed. Recent evidence suggests that R-loops can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. Yet, how the vulnerability of R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops and chromosome translocations and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end-joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

scholarly journals Emerging Technologies for Genome-Wide Profiling of DNA Breakage

2021 ◽  
Vol 11 ◽  
Author(s):  
Matthew J. Rybin ◽  
Melina Ramic ◽  
Natalie R. Ricciardi ◽  
Philipp Kapranov ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

scholarly journals ATM’s Role in the Repair of DNA Double-Strand Breaks

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1370
Author(s):  
Atsushi Shibata ◽  
Penny A. Jeggo
Keyword(s):  
Stress Responses ◽  
Ataxia Telangiectasia ◽  
Cell Cycle Checkpoint ◽  
Double Strand Breaks ◽  
Multiple Roles ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Cycle Checkpoint

Ataxia telangiectasia mutated (ATM) is a central kinase that activates an extensive network of responses to cellular stress via a signaling role. ATM is activated by DNA double strand breaks (DSBs) and by oxidative stress, subsequently phosphorylating a plethora of target proteins. In the last several decades, newly developed molecular biological techniques have uncovered multiple roles of ATM in response to DNA damage—e.g., DSB repair, cell cycle checkpoint arrest, apoptosis, and transcription arrest. Combinational dysfunction of these stress responses impairs the accuracy of repair, consequently leading to dramatic sensitivity to ionizing radiation (IR) in ataxia telangiectasia (A-T) cells. In this review, we summarize the roles of ATM that focus on DSB repair.

scholarly journals Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

2015 ◽  
Vol 112 (24) ◽  
pp. 7507-7512 ◽  
Author(s):  
Ozge Gursoy-Yuzugullu ◽  
Marina K. Ayrapetov ◽  
Brendan D. Price
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Histone H4 ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Nucleosome Dynamics ◽  
Chromatin Template ◽  
End Resection

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.

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

2019 ◽  
Vol 10 (1) ◽  
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.

scholarly journals CHD7 and 53BP1 regulate distinct pathways for the re-ligation of DNA double-strand breaks

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Magdalena B. Rother ◽  
Stefania Pellegrino ◽  
Rebecca Smith ◽  
Marco Gatti ◽  
Cornelia Meisenberg ◽  
...  
Keyword(s):  
Charge Syndrome ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Multiple Levels ◽  
Non Homologous End Joining ◽  
And Shifts ◽  
Chromatin Relaxation

AbstractChromatin structure is dynamically reorganized at multiple levels in response to DNA double-strand breaks (DSBs). Yet, how the different steps of chromatin reorganization are coordinated in space and time to differentially regulate DNA repair pathways is insufficiently understood. Here, we identify the Chromodomain Helicase DNA Binding Protein 7 (CHD7), which is frequently mutated in CHARGE syndrome, as an integral component of the non-homologous end-joining (NHEJ) DSB repair pathway. Upon recruitment via PARP1-triggered chromatin remodeling, CHD7 stimulates further chromatin relaxation around DNA break sites and brings in HDAC1/2 for localized chromatin de-acetylation. This counteracts the CHD7-induced chromatin expansion, thereby ensuring temporally and spatially controlled ‘chromatin breathing’ upon DNA damage, which we demonstrate fosters efficient and accurate DSB repair by controlling Ku and LIG4/XRCC4 activities. Loss of CHD7-HDAC1/2-dependent cNHEJ reinforces 53BP1 assembly at the damaged chromatin and shifts DSB repair to mutagenic NHEJ, revealing a backup function of 53BP1 when cNHEJ fails.

scholarly journals Hedgehog signaling enables repair of ribosomal DNA double-strand breaks

2020 ◽  
Vol 48 (18) ◽  
pp. 10342-10352
Author(s):  
Tshering D Lama-Sherpa ◽  
Victor T G Lin ◽  
Brandon J Metge ◽  
Shannon E Weeks ◽  
Dongquan Chen ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Ribosomal Dna ◽  
Hedgehog Signaling ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Rdna Sequences ◽  
Polymerase I

Abstract Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

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