scholarly journals Surveillance of Different Recombination Defects in Mouse Spermatocytes Yields Distinct Responses despite Elimination at an Identical Developmental Stage

2005 ◽  
Vol 25 (16) ◽  
pp. 7203-7215 ◽  
Author(s):  
Marco Barchi ◽  
Shantha Mahadevaiah ◽  
Monica Di Giacomo ◽  
Frédéric Baudat ◽  
Dirk G. de Rooij ◽  
...  
Keyword(s):  
Cellular Response ◽  
Stage Iv ◽  
Development Stage ◽  
Male Meiosis ◽  
Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Epistasis Analysis ◽  
Associated Proteins ◽  
Normal Males

ABSTRACT Fundamentally different recombination defects cause apoptosis of mouse spermatocytes at the same stage in development, stage IV of the seminiferous epithelium cycle, equivalent to mid-pachynema in normal males. To understand the cellular response(s) that triggers apoptosis, we examined markers of spermatocyte development in mice with different recombination defects. In Spo11 − / − mutants, which lack the double-strand breaks (DSBs) that initiate recombination, spermatocytes express markers of early to mid-pachynema, forming chromatin domains that contain sex body-associated proteins but that rarely encompass the sex chromosomes. Dmc1 − / − spermatocytes, impaired in DSB repair, appear to arrest at or about late zygonema. Epistasis analysis reveals that this earlier arrest is a response to unrepaired DSBs, and cytological analysis implicates the BRCT-containing checkpoint protein TOPBP1. Atm − / − spermatocytes show similarities to Dmc1 − / − spermatocytes, suggesting that ATM promotes meiotic DSB repair. Msh5 − / − mutants display a set of characteristics distinct from these other mutants. Thus, despite equivalent stages of spermatocyte elimination, different recombination-defective mutants manifest distinct responses, providing insight into surveillance mechanisms in male meiosis.

scholarly journals DEAD Box 1 Facilitates Removal of RNA and Homologous Recombination at DNA Double-Strand Breaks

2016 ◽  
Vol 36 (22) ◽  
pp. 2794-2810 ◽  
Author(s):  
Lei Li ◽  
Devon R. Germain ◽  
Ho-Yin Poon ◽  
Matthew R. Hildebrandt ◽  
Elizabeth A. Monckton ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cellular Response ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Repair Process ◽  
Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dead Box ◽  
End Resection

Although RNA and RNA-binding proteins have been linked to double-strand breaks (DSBs), little is known regarding their roles in the cellular response to DSBs and, if any, in the repair process. Here, we provide direct evidence for the presence of RNA-DNA hybrids at DSBs and suggest that binding of RNA to DNA at DSBs may impact repair efficiency. Our data indicate that the RNA-unwinding protein DEAD box 1 (DDX1) is required for efficient DSB repair and cell survival after ionizing radiation (IR), with depletion of DDX1 resulting in reduced DSB repair by homologous recombination (HR). While DDX1 is not essential for end resection, a key step in homology-directed DSB repair, DDX1 is required for maintenance of the single-stranded DNA once generated by end resection. We show that transcription deregulation has a significant effect on DSB repair by HR in DDX1-depleted cells and that RNA-DNA duplexes are elevated at DSBs in DDX1-depleted cells. Based on our combined data, we propose a role for DDX1 in resolving RNA-DNA structures that accumulate at DSBs located at sites of active transcription. Our findings point to a previously uncharacterized requirement for clearing RNA at DSBs for efficient repair by HR.

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 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 The ATM protein: The importance of being active

2012 ◽  
Vol 198 (3) ◽  
pp. 273-275 ◽  
Author(s):  
Yosef Shiloh ◽  
Yael Ziv
Keyword(s):  
Cellular Response ◽  
Cancer Therapeutics ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Ataxia Telangiectasia Mutated ◽  
Strand Breaks ◽  
Cell Biol ◽  
Response Network ◽  
Damage Response ◽  
Atm Protein

The ataxia telangiectasia mutated (ATM) protein kinase regulates the cellular response to deoxyribonucleic acid (DNA) double-strand breaks by phosphorylating numerous players in the extensive DNA damage response network. Two papers in this issue (Daniel et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb201204035; Yamamoto et al. 2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb201204098) strikingly show that, in mice, the presence of a catalytically inactive version of ATM is embryonically lethal. This is surprising because mice completely lacking ATM have a much more moderate phenotype. The findings impact on basic cancer research and cancer therapeutics.

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.

Double-strand-break-induced homologous recombination in mammalian cells

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
Author(s):  
R. D. Johnson ◽  
M. Jasin
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

scholarly journals The role of ubiquitin-dependent segregase p97 (VCP or Cdc48) in chromatin dynamics after DNA double strand breaks

2017 ◽  
Vol 372 (1731) ◽  
pp. 20160282 ◽  
Author(s):  
Ignacio Torrecilla ◽  
Judith Oehler ◽  
Kristijan Ramadan
Keyword(s):  
Dna Repair ◽  
Current Knowledge ◽  
Dna Lesions ◽  
Chromatin Remodelling ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Chromatin Dynamics ◽  
Strand Breaks ◽  
Signalling Molecules

DNA double strand breaks (DSBs) are the most cytotoxic DNA lesions and, if not repaired, lead to chromosomal rearrangement, genomic instability and cell death. Cells have evolved a complex network of DNA repair and signalling molecules which promptly detect and repair DSBs, commonly known as the DNA damage response (DDR). The DDR is orchestrated by various post-translational modifications such as phosphorylation, methylation, ubiquitination or SUMOylation. As DSBs are located in complex chromatin structures, the repair of DSBs is engineered at two levels: (i) at sites of broken DNA and (ii) at chromatin structures that surround DNA lesions. Thus, DNA repair and chromatin remodelling machineries must work together to efficiently repair DSBs. Here, we summarize the current knowledge of the ubiquitin-dependent molecular unfoldase/segregase p97 (VCP in vertebrates and Cdc48 in worms and lower eukaryotes) in DSB repair. We identify p97 as an essential factor that regulates DSB repair. p97-dependent extraction of ubiquitinated substrates mediates spatio-temporal protein turnover at and around the sites of DSBs, thus orchestrating chromatin remodelling and DSB repair. As p97 is a druggable target, p97 inhibition in the context of DDR has great potential for cancer therapy, as shown for other DDR components such as PARP, ATR and CHK1. This article is part of the themed issue ‘Chromatin modifiers and remodellers in DNA repair and signalling’.

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