scholarly journals Repair Kinetics of Genomic Interstrand DNA Cross-Links: Evidence for DNA Double-Strand Break-Dependent Activation of the Fanconi Anemia/BRCA Pathway

2004 ◽  
Vol 24 (1) ◽  
pp. 123-134 ◽  
Author(s):  
Andreas Rothfuss ◽  
Markus Grompe
Keyword(s):  
Fanconi Anemia ◽  
Strand Break ◽  
S Phase ◽  
Dna Double Strand Breaks ◽  
Subsequent Formation ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Cross Links ◽  
Kinetics Of

ABSTRACT The detailed mechanisms of DNA interstrand cross-link (ICL) repair and the involvement of the Fanconi anemia (FA)/BRCA pathway in this process are not known. Present models suggest that recognition and repair of ICL in human cells occur primarily during the S phase. Here we provide evidence for a refined model in which ICLs are recognized and are rapidly incised by ERCC1/XPF independent of DNA replication. However, the incised ICLs are then processed further and DNA double-strand breaks (DSB) form exclusively in the S phase. FA cells are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB, suggesting a role of the FA/BRCA pathway downstream in ICL repair. In fact, activation of FANCD2 occurs slowly after ICL treatment and correlates with the appearance of DSB in the S phase. In contrast, activation is rapid after ionizing radiation, indicating that the FA/BRCA pathway is specifically activated upon DSB formation. Furthermore, the formation of FANCD2 foci is restricted to a subpopulation of cells, which can be labeled by bromodeoxyuridine incorporation. We therefore conclude that the FA/BRCA pathway, while being dispensable for the early events in ICL repair, is activated in S-phase cells after DSB have formed.

scholarly journals Jack of all trades? The versatility of RNA in DNA double-strand break repair

2020 ◽  
Vol 64 (5) ◽  
pp. 721-735 ◽  
Author(s):  
Ruth F. Ketley ◽  
Monika Gullerova
Keyword(s):  
Dna Damage ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Non Coding Rna ◽  
Dna Double Strand Break ◽  
Rna:Dna Hybrid ◽  
Cellular Stressors ◽  
Long Non Coding Rna

Abstract The mechanisms by which RNA acts in the DNA damage response (DDR), specifically in the repair of DNA double-strand breaks (DSBs), are emerging as multifaceted and complex. Different RNA species, including but not limited to; microRNA (miRNA), long non-coding RNA (lncRNA), RNA:DNA hybrid structures, the recently identified damage-induced lncRNA (dilncRNA), damage-responsive transcripts (DARTs), and DNA damage-dependent small RNAs (DDRNAs), have been shown to play integral roles in the DSB response. The diverse properties of these RNAs, such as sequence, structure, and binding partners, enable them to fulfil a variety of functions in different cellular contexts. Additionally, RNA can be modified post-transcriptionally, a process which is regulated in response to cellular stressors such as DNA damage. Many of these mechanisms are not yet understood and the literature contradictory, reflecting the complexity and expansive nature of the roles of RNA in the DDR. However, it is clear that RNA is pivotal in ensuring the maintenance of genome integrity. In this review, we will discuss and summarise recent evidence which highlights the roles of these various RNAs in preserving genomic integrity, with a particular focus on the emerging role of RNA in the DSB repair response.

scholarly journals Physiological Roles of DNA Double-Strand Breaks

2017 ◽  
Vol 2017 ◽  
pp. 1-20 ◽  
Author(s):  
Farhaan A. Khan ◽  
Syed O. Ali
Keyword(s):  
Double Helix ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Spatiotemporal Regulation ◽  
Dna Double Strand Break

Genomic integrity is constantly threatened by sources of DNA damage, internal and external alike. Among the most cytotoxic lesions is the DNA double-strand break (DSB) which arises from the cleavage of both strands of the double helix. Cells boast a considerable set of defences to both prevent and repair these breaks and drugs which derail these processes represent an important category of anticancer therapeutics. And yet, bizarrely, cells deploy this very machinery for the intentional and calculated disruption of genomic integrity, harnessing potentially destructive DSBs in delicate genetic transactions. Under tight spatiotemporal regulation, DSBs serve as a tool for genetic modification, widely used across cellular biology to generate diverse functionalities, ranging from the fundamental upkeep of DNA replication, transcription, and the chromatin landscape to the diversification of immunity and the germline. Growing evidence points to a role of aberrant DSB physiology in human disease and an understanding of these processes may both inform the design of new therapeutic strategies and reduce off-target effects of existing drugs. Here, we review the wide-ranging roles of physiological DSBs and the emerging network of their multilateral regulation to consider how the cell is able to harness DNA breaks as a critical biochemical tool.

scholarly journals i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Anna Biernacka ◽  
Yingjie Zhu ◽  
Magdalena Skrzypczak ◽  
Romain Forey ◽  
Benjamin Pardo ◽  
...  
Keyword(s):  
Cell Fate ◽  
Genome Stability ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Universal Method ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Agarose Beads ◽  
Genome Wide ◽  
Dna Double Strand Break

AbstractMaintenance of genome stability is a key issue for cell fate that could be compromised by chromosome deletions and translocations caused by DNA double-strand breaks (DSBs). Thus development of precise and sensitive tools for DSBs labeling is of great importance for understanding mechanisms of DSB formation, their sensing and repair. Until now there has been no high resolution and specific DSB detection technique that would be applicable to any cells regardless of their size. Here, we present i-BLESS, a universal method for direct genome-wide DNA double-strand break labeling in cells immobilized in agarose beads. i-BLESS has three key advantages: it is the only unbiased method applicable to yeast, achieves a sensitivity of one break at a given position in 100,000 cells, and eliminates background noise while still allowing for fixation of samples. The method allows detection of ultra-rare breaks such as those forming spontaneously at G-quadruplexes.

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 Regulation of DNA Double-Strand Break Repair by Non-Coding RNAs

2018 ◽  
Author(s):  
Roopa Thapar
Keyword(s):  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Protein Dna Interactions ◽  
Damage Response ◽  
Non Coding Rnas ◽  
Non Homologous End Joining

DNA double-strand breaks (DSBs) are deleterious lesions that are generated in response to ionizing radiation or replication fork collapse that can lead to genomic instability and cancer.  Eukaryotes have evolved two major pathways, namely homologous recombination (HR) and non-homologous end joining (NHEJ) to repair DSBs.  Whereas the roles of protein-DNA interactions in HR and NHEJ have been fairly well defined, the functions of small and long non-coding RNAs and RNA-DNA hybrids in the DNA damage response is just beginning to be elucidated.  This review summarizes recent discoveries on the identification of non-coding RNAs and RNA-mediated regulation of DSB repair

scholarly journals Staphylococcal DNA repair is required for infection

2020 ◽  
Author(s):  
Kam Pou Ha ◽  
Rebecca S. Clarke ◽  
Gyu-Lee Kim ◽  
Jane L. Brittan ◽  
Jessica E. Rowley ◽  
...  
Keyword(s):  
Human Blood ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Reporter System ◽  
Gram Positive ◽  
Fluorescent Reporter ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Transposon Mutants

AbstractThe repair of DNA damage is essential for bacterial viability and contributes to adaptation via increased rates of mutation and recombination. However, the mechanisms by which DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double strand breaks via the oxidative burst, which are repaired by RexAB, leading to induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted survival of these pathogens in human blood, suggesting that DNA double strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection.

scholarly journals Parp3 promotes long-range end joining in murine cells

2018 ◽  
Vol 115 (40) ◽  
pp. 10076-10081 ◽  
Author(s):  
Jacob V. Layer ◽  
J. Patrick Cleary ◽  
Alexander J. Brown ◽  
Kristen E. Stevenson ◽  
Sara N. Morrow ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Embryonic Stem ◽  
Cell Types ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class–switch recombination in primary B cells, and inversions in tail fibroblasts that generateEml4–Alkfusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing ofEml4–Alkjunctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

scholarly journals Structural snapshots of human DNA polymerase μ engaged on a DNA double-strand break

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Andrea M. Kaminski ◽  
John M. Pryor ◽  
Dale A. Ramsden ◽  
Thomas A. Kunkel ◽  
Lars C. Pedersen ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Template Strand ◽  
Single Strand Breaks ◽  
Dna Double Strand Break ◽  
Break Site

Abstract Genomic integrity is threatened by cytotoxic DNA double-strand breaks (DSBs), which must be resolved efficiently to prevent sequence loss, chromosomal rearrangements/translocations, or cell death. Polymerase μ (Polμ) participates in DSB repair via the nonhomologous end-joining (NHEJ) pathway, by filling small sequence gaps in broken ends to create substrates ultimately ligatable by DNA Ligase IV. Here we present structures of human Polμ engaging a DSB substrate. Synapsis is mediated solely by Polμ, facilitated by single-nucleotide homology at the break site, wherein both ends of the discontinuous template strand are stabilized by a hydrogen bonding network. The active site in the quaternary Pol μ complex is poised for catalysis and nucleotide incoporation proceeds in crystallo. These structures demonstrate that Polμ may address complementary DSB substrates during NHEJ in a manner indistinguishable from single-strand breaks.

scholarly journals The ubiquitin landscape at DNA double-strand breaks

2009 ◽  
Vol 187 (3) ◽  
pp. 319-326 ◽  
Author(s):  
Troy E. Messick ◽  
Roger A. Greenberg
Keyword(s):  
Dna Damage ◽  
Cancer Susceptibility ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Checkpoint Signaling ◽  
Strand Breaks ◽  
Parallel Processes ◽  
Dna Double Strand Break ◽  
Cellular Dna

The intimate relationship between DNA double-strand break (DSB) repair and cancer susceptibility has sparked profound interest in how transactions on DNA and chromatin surrounding DNA damage influence genome integrity. Recent evidence implicates a substantial commitment of the cellular DNA damage response machinery to the synthesis, recognition, and hydrolysis of ubiquitin chains at DNA damage sites. In this review, we propose that, in order to accommodate parallel processes involved in DSB repair and checkpoint signaling, DSB-associated ubiquitin structures must be nonuniform, using different linkages for distinct functional outputs. We highlight recent advances in the study of nondegradative ubiquitin signaling at DSBs, and discuss how recognition of different ubiquitin structures may influence DNA damage responses.

scholarly journals A synergistic interaction between HDAC- and PARP inhibitors in childhood tumors with chromothripsis

2021 ◽  
Author(s):  
Umar Khalid ◽  
Milena Simovic ◽  
Murat Iskar ◽  
John KL Wong ◽  
Rithu Kumar ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Parp Inhibitor ◽  
Strand Break ◽  
Parp Inhibitors ◽  
Synergistic Interaction ◽  
Parp Inhibition ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Double Strand Break

ABSTRACTChromothripsis is a form of genomic instability characterized by the occurrence of tens to hundreds of clustered DNA double-strand breaks in a one-off catastrophic event. Rearrangements associated with chromothripsis are detectable in numerous tumor entities and linked with poor prognosis in some of these, such as Sonic Hedgehog medulloblastoma, neuroblastoma and osteosarcoma. Hence, there is a need for therapeutic strategies eliminating tumor cells with chromothripsis. Defects in DNA double-strand break repair, and in particular homologous recombination repair, have been linked with chromothripsis. Targeting DNA repair deficiencies by synthetic lethality approaches, we performed a synergy screen using drug libraries (n = 375 compounds, 15 models) combined with either a PARP inhibitor or cisplatin. This revealed a synergistic interaction between the HDAC inhibitor romidepsin and PARP inhibition. Functional assays, transcriptome analyses, and in vivo validation in patient-derived xenograft mouse models confirmed the efficacy of the combinatorial treatment.

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