scholarly journals PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Gergely Rona ◽  
Domenico Roberti ◽  
Yandong Yin ◽  
Julia K Pagan ◽  
Harrison Homer ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Ubiquitin Ligase ◽  
Transcriptional Repression ◽  
Genotoxic Stress ◽  
Strand Break ◽  
Homologous Recombination Repair ◽  
Dependent Manner ◽  
Ubiquitin Ligase Complex ◽  
Recombination Repair

The mammalian FBXL10-RNF68-RNF2 ubiquitin ligase complex (FRRUC) mono-ubiquitylates H2A at Lys119 to repress transcription in unstressed cells. We found that the FRRUC is rapidly and transiently recruited to sites of DNA damage in a PARP1- and TIMELESS-dependent manner to promote mono-ubiquitylation of H2A at Lys119, a local decrease of H2A levels, and an increase of H2A.Z incorporation. Both the FRRUC and H2A.Z promote transcriptional repression, double strand break signaling, and homologous recombination repair (HRR). All these events require both the presence and activity of the FRRUC. Moreover, the FRRUC and its activity are required for the proper recruitment of BMI1-RNF2 and MEL18-RNF2, two other ubiquitin ligases that mono-ubiquitylate Lys119 in H2A upon genotoxic stress. Notably, whereas H2A.Z is not required for H2A mono-ubiquitylation, impairment of the latter results in the inhibition of H2A.Z incorporation. We propose that the recruitment of the FRRUC represents an early and critical regulatory step in HRR.

scholarly journals HDAC Inhibition Induces microRNA-182 Which Targets Rad51 Protein and Impairs Homologous Recombination Repair to Sensitize Cells to the Double Strand Break Inducing Nucleoside Analog, Sapacitabine in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3639-3639 ◽  
Author(s):  
Tzung-Huei Lai ◽  
Alma Zecevic ◽  
Brett Ewald ◽  
Liu Chaomei ◽  
Lara Rizzotto ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Ectopic Expression ◽  
Strand Break ◽  
Nucleoside Analog ◽  
Homologous Recombination Repair ◽  
Hdac Inhibition ◽  
Recombination Repair

Abstract Acute myelogenous leukemia (AML) is characterized by multiple genetic and epigenetic abnormalities including a profound dysregulation of microRNA expression. Effective clinical treatment of AML has largely depended on a class of antimetabolites - the nucleoside analogs. Of these, sapacitabine is a nucleoside analog prodrug that is in development for the therapy of AML. It is converted to its active metabolite 2-C-cyano-2-deoxy-1-β(-D-arabino-pentafuranosyl) cytosine (CNDAC), which interferes with DNA synthesis by initially causing a single stranded DNA break that is converted into a double strand break in the subsequent replicative cycle. Such double strand breaks are primarily repaired by the homologous recombination repair (HR) pathway. Consequently, efficient HR may offer a potential resistance mechanism to therapy with sapacitabine. Rad51 is a protein plays a critical role in HR, and high levels of Rad51 are linked to resistance to DNA damaging therapies. Histone deacetylases (HDACs) are chromatin modulating agents that decrease levels of acetylation of histones, repress gene expression. HDAC inhibitors (HDACis) function by modifying chromatin to epigenetically reverse gene silencing of coding and non-coding genes such as the microRNAs (miRs). miRs are endogenous noncoding RNAs 19-25 nucleotides in length that bind to complimentary sequences in target RNA to either destabilize it or prevent its transcription. In this study, we determined that determined primary AML blasts and cell lines express low levels of microRNA-182. Recruitment of HDAC1 and its co-repressors were linked to the epigenetic silencing of miR-182 in AML. Conversely, HDAC inhibition led to accumulation of activating chromatin modifications followed by the upregulation of miR-182 in AML blasts and cell lines. The HDACi-induced increases in miR-182 were linked to decreases in the levels of Rad51, an inhibition in the ability of cells to conduct homologous recombination repair as measured by the Homologous recombination directed repair (HDR) assay, persistent levels of DNA damage as measured by the levels of Ɣ-H2AX and sensitization to sapacitabine. We then mechanistically defined the relation between miR-182 and Rad51. Ectopic expression of miR-182 in AML cell lines identified that Rad51 was a target of miR-182. An assay with luciferase constructs bearing full length or mutated Rad51 3'UTR indentified that Rad51 was a direct target of miR-182. We also determined that ectopic expression of miR-182 attenuated the ability of AML cells to conduct homologus repair as measured by the Homologous recombination directed repair (HDR) assay which resulted in sensitizing AML cells to the cytotoxic action of CNDAC as measured by colony forming assays. In conclusion, our data show that HDAC inhibitors target Rad51 via miR-182 to compromise HR repair to result in higher levels of residual DNA damage and sensitize AML cells to double strand damaging agents such as CNDAC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

2018 ◽  
Author(s):  
Xiangduo Kong ◽  
Gladys Mae Saquilabon Cruz ◽  
Sally Loyal Trinh ◽  
Xu-Dong Zhu ◽  
Michael W. Berns ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Sister Chromatid ◽  
Strand Break ◽  
Homologous Recombination Repair ◽  
Telomeric Dna ◽  
Dna Double Strand Break ◽  
Mre11 Complex ◽  
Recombination Repair ◽  
Step Mechanism

AbstractTRF2 binds to telomeric repeats and is critical for telomere integrity. Evidence suggests that it also localizes to non-telomeric DNA damage sites. However, this recruitment appears to be precarious and functionally controversial. We find that TRF2 recruitment to damage sites occurs by a two-step mechanism: the initial rapid recruitment (phase I) and stable and prolonged association with damage sites (phase II). Phase I is poly(ADP-ribose) polymerase (PARP)-dependent and requires the N-terminal basic domain. The phase II recruitment requires the C-terminal MYB/SANT domain and the iDDR region in the hinge domain, which is mediated by the MRE11 complex and is stimulated by hTERT. PARP-dependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates two phases. TRF2 binds to the I-PpoI-induced DNA double-strand break sites, which is enhanced by the presence of complex damage and is dependent on PARP and the MRE11 complex. TRF2 depletion affects non-sister chromatid homologous recombination (HR) repair, but not HR between sister chromatids or non-homologous endjoining pathways. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.Summary StatementTRF2 is recruited to DNA double-strand break damage sites by a two-step mechanism and functions in non-sister chromatid homologous recombination repair

scholarly journals Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

2018 ◽  
Vol 131 (23) ◽  
pp. jcs219311 ◽  
Author(s):  
Xiangduo Kong ◽  
Gladys Mae Saquilabon Cruz ◽  
Sally Loyal Trinh ◽  
Xu-Dong Zhu ◽  
Michael W. Berns ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Sister Chromatid ◽  
Homologous Recombination Repair ◽  
Recombination Repair

scholarly journals Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 889 ◽  
Author(s):  
Klaudia Szymonowicz ◽  
Adam Krysztofiak ◽  
Jansje van der Linden ◽  
Ajvar Kern ◽  
Simon Deycmar ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Proton Irradiation ◽  
Negative Impact ◽  
Particle Therapy ◽  
Homologous Recombination Repair ◽  
End Joining ◽  
X Ray ◽  
Recombination Repair ◽  
Non Homologous End Joining

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.

141 INDICATIONS FOR DNA DOUBLE-STRAND BREAK REPAIR IN EARLY BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 188
Author(s):  
A. Brero ◽  
D. Koehler ◽  
T. Cremer ◽  
E. Wolf ◽  
V. Zakhartchenko
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Dna Lesions ◽  
Homologous Recombination Repair ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Male And Female ◽  
Γh2ax Foci ◽  
Recombination Repair

DNA double-strand breaks (DSBs) are considered the most severe type of DNA lesions, because such lesions, if unrepaired, lead to a loss of genome integrity. Soon after induction of DSBs, chromatin surrounding the damage is modified by phosphorylation of the histone variant H2AX, generating so-called γH2AX, which is a hallmark of DSBs (Takahashi et al. 2005 Cancer Lett. 229, 171–179). γH2AX appears to be a signal for the recruitment of proteins constituting the DNA repair machinery. Depending on the type of damage and the cell cycle stage of the affected cell, DSBs are repaired either by nonhomologous end joining or by homologous recombination using the sister chromatid DNA as template (Hoeijmakers 2001 Nature 411, 366–374). We used immunofluorescence to analyze chromatin composition during bovine development and found γH2AX foci in both male and female pronuclei of IVF embryos. The number and size of foci varied considerably between embryos and between the male and female pronuclei. To test whether the observed γH2AX foci represented sites of active DNA repair, we co-stained IVF zygotes for γH2AX and 3 different proteins involved in homologous recombination repair of DSBs: NBS1 (phosphorylated at amino acid serine 343), 53BP1, and Rad51. We found co-localization of γH2AX foci with phosphorylated NBS1 as well as with Rad51 but did not observe the presence of 53BP1 at γH2AX foci in IVF zygotes. Our finding shows the presence of DSBs in IVF zygotes and suggests the capability of homologous recombination repair. The lack of 53BP1, a component of homologous recombination repair, which usually co-localizes with γH2AX foci at exogenously induced DSBs (Schultz et al. 2000 J. Cell. Biol. 151, 1381–1390) poses the possibility that the mechanism present in early embryos differs substantially from that involved in DNA repair of DSBs in somatic cells.

scholarly journals Hsp90 induces increased genomic instability toward DNA-damaging agents by tuning down RAD53 transcription

2016 ◽  
Vol 27 (15) ◽  
pp. 2463-2478 ◽  
Author(s):  
Nidhi Khurana ◽  
Shyamasree Laskar ◽  
Mrinal K. Bhattacharyya ◽  
Sunanda Bhattacharyya
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Transcriptional Repression ◽  
Strand Break ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Checkpoint Activation ◽  
Dna Damage Signaling ◽  
Dna Damaging Agents ◽  
G1 Cells

It is well documented that elevated body temperature causes tumors to regress upon radiotherapy. However, how hyperthermia induces DNA damage sensitivity is not clear. We show that a transient heat shock and particularly the concomitant induction of Hsp90 lead to increased genomic instability under DNA-damaging conditions. Using Saccharomyces cerevisiae as a model eukaryote, we demonstrate that elevated levels of Hsp90 attenuate efficient DNA damage signaling and dictate preferential use of the potentially mutagenic double-strand break repair pathway. We show that under normal physiological conditions, Hsp90 negatively regulates RAD53 transcription to suppress DNA damage checkpoint activation. However, under DNA damaging conditions, RAD53 is derepressed, and the increased level of Rad53p triggers an efficient DNA damage response. A higher abundance of Hsp90 causes increased transcriptional repression on RAD53 in a dose-dependent manner, which could not be fully derepressed even in the presence of DNA damage. Accordingly, cells behave like a rad53 loss-of-function mutant and show reduced NHEJ efficiency, with a drastic failure to up-regulate RAD51 expression and manifestly faster accumulation of CLN1 and CLN2 in DNA-damaged G1, cells leading to premature release from checkpoint arrest. We further demonstrate that Rad53 overexpression is able to rescue all of the aforementioned deleterious effects caused by Hsp90 overproduction.

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