Actinomycin D Induces Histone γ-H2AX Foci and Complex Formation of γ-H2AX with Ku70 and Nuclear DNA Helicase II

Mischo, Hannah E.; Hemmerich, Peter; Grosse, Frank; Zhang, Suisheng

doi:10.1074/jbc.m411444200

Cited by 89 publications

(76 citation statements)

References 62 publications

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“…We have previously shown that pretreatment of HeLa cells with the transcription inhibitor actinomycin D prior to IR exposure abolishes formation of discrete DDX1 foci at DSBs (9). A caveat to this experiment is that actinomycin D has the potential to induce ␥-H2AX foci as well as to inhibit transcription (48). To ensure that the effect observed upon actinomycin D treatment is the consequence of transcription inhibition, we tested a second transcription inhibitor, cordycepin, an adenosine analogue that terminates RNA chain elongation (49).…”

Section: Ddx1 Contributes To Dsb Repair and Cell Survival Post-irmentioning

confidence: 85%

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

Germain

Poon

et al. 2016

Molecular and Cellular Biology

133

138

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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 RNAunwinding 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. DEAD box proteins are a family of putative RNA helicases that function by altering RNA secondary structure. This protein family has been implicated in all aspects of RNA metabolism. The DEAD box 1 gene (DDX1) is a widely expressed gene that is misexpressed in a number of cancers, including retinoblastoma, neuroblastoma, and breast cancer (1, 2). Knockout of DDX1 leads to early embryonic lethality in mice and severely reduced fertility in flies (3, 4). DDX1 is involved in the transport of RNAs from the nucleus to the cytoplasm and regulates cytoplasmic localization of the splicing-regulatory protein KSRP (5). In neurons, DDX1 resides in RNA-transporting granules, cytoplasmic organelles that regulate the localization and expression of target mRNAs (6, 7). DDX1 has also been identified as a core subunit of the human tRNA ligase complex which is essential for tRNA splicing (8).In addition to its roles in RNA metabolism, DDX1 has been implicated in the cellular response to DNA double-strand breaks (DSBs). Upon treatment of cells with ionizing radiation (IR), DDX1 rapidly accumulates at a subset of DNA DSBs (ϳ30%), where it forms IR-induced foci that colocalize with ␥-H2AX, a marker for DSBs (9). DDX1 coimmunoprecipitates with the MRN (MRE11-RAD50-NBS1) complex, the early sensor of DNA DSBs, and ATM (ataxia telangiectasia mutated) protein, the key transducer of the signaling cascade in response to DSBs (10, 11). DSBs induce DDX1 phosphorylation in an ATM-dependent manner. Notably, IR-induced DDX1 foci are lost when cells are treated with RNase H, an enzyme that specifically digests RNA from RNA-DNA hybrids (9). These results suggest that RNA-DNA double-stranded structures are required for the presence and/or retention of DDX1 at DSBs. In line with this observation, biochemical analysis has shown that DDX1 can unwind both...

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Section: Ddx1 Contributes To Dsb Repair and Cell Survival Post-irmentioning

confidence: 85%

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

Germain

Poon

et al. 2016

Molecular and Cellular Biology

133

138

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“…We found that downregulation of endogenous TTAGGGbinding protein TRF1 increases the frequency of spontaneous chromosome aberrations in Chinese hamster cells (94) but the mechanism remains unknown. It has been shown that an inhibition of transcription by actinomycin D (ActD) induces formation of g-H2AX foci (95) and TRF1 may be required for transcription at ITS. Stalled transcription at TTAGGG arrays in the absence of TRF1 may lead to double-strand breaks and instability.…”

Section: Global Sine Clustering and Line Depletion In Gc-rich Gene-rimentioning

confidence: 99%

Regulation of mammalian gene expression by retroelements and non‐coding tandem repeats

Tomilin

2008

BioEssays

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Genomes of higher eukaryotes contain abundant non‐coding repeated sequences whose overall biological impact is unclear. They comprise two categories. The first consists of retrotransposon‐derived elements. These are three major families of retroelements (LINEs, SINEs and LTRs). SINEs are clustered in gene‐rich regions and are found in promoters of genes while LINEs are concentrated in gene‐poor regions and are depleted from promoters. The second class consists of non‐coding tandem repeats (satellite DNAs and TTAGGG arrays), which are associated with mammalian centromeres, heterochromatin and telomeres. Terminal TTAGGG arrays are involved in telomere capping and satellite DNAs are located in heterochromatin, which is implicated in transcription silencing by gene repositioning (relocalization). It is unknown whether interstitial TTAGGG sequences, which are present in many vertebrates, have a function. Here, evidence will be presented that retroelements and TTAGGG arrays are involved in regulation of gene expression. Retroelements can provide binding sites for transcription factors and protect promoter CpG islands from repressive chromatin modifications, and may be also involved in nuclear compartmentalization of transcriptionally active and inactive domains. Interstitial telomere‐like sequences can form dynamically maintained three‐dimensional nuclear networks of transcriptionally inactive domains, which may be involved in transcription silencing like classic heterochromatin. BioEssays 30:338–348, 2008. © 2008 Wiley Periodicals, Inc.

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“…H2AX, a variant form of histone H2A, is rapidly phosphorylated at serine 139 in response to DNA double-strand breaks (DSB) originating from exogenous DNA damage (Rogakou et al, 1998(Rogakou et al, , 1999 replication fork collision (Ward and Chen, 2001;Furuta et al, 2003), shortened telomeres (Takai et al, 2003;d'Adda di Fagagna et al, 2003), apoptosis (Rogakou et al, 2000) and transcription inhibition (Mischo et al, 2005). Phosphorylated histone H2AX, designated as g-H2AX, forms distinct nuclear foci at or in the vicinity of DSB sites (Rogakou et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Histone H2AX is a critical factor for cellular protection against DNA alkylating agents

Meador

Zhao

et al. 2008

Oncogene

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Histone H2A variant H2AX is a dose-dependent suppressor of oncogenic chromosome translocations. H2AX participates in DNA double-strand break repair, but its role in other DNA repair pathways is not known. In this study, role of H2AX in cellular response to alkylation DNA damage was investigated. Cellular sensitivity to two monofunctional alkylating agents (methyl methane sulfonate and N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG)) was dependent on H2AX dosage, and H2AX null cells were more sensitive than heterozygous cells. In contrast to wild-type cells, H2AX-deficient cells displayed extensive apoptotic death due to a lack of cell-cycle arrest at G 2 /M phase. Lack of G 2 /M checkpoint in H2AX null cells correlated well with increased mitotic irregularities involving anaphase bridges and gross chromosomal instability. Observation of elevated poly(ADP) ribose polymerase 1 (PARP-1) cleavage suggests that MNNG-induced apoptosis occurs by PARP-1-dependent manner in H2AX-deficient cells. Consistent with this, increased activities of PARP and poly(ADP) ribose (PAR) polymer synthesis were detected in both H2AX heterozygous and null cells. Further, we demonstrate that the increased PAR synthesis and apoptotic death induced by MNNG in H2AX-deficient cells are due to impaired activation of mitogen-activated protein kinase pathway. Collectively, our novel study demonstrates that H2AX, similar to PARP-1, confers cellular protection against alkylation-induced DNA damage. Therefore, targeting either PARP-1 or histone H2AX may provide an effective way of maximizing the chemotherapeutic value of alkylating agents for cancer treatment.

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Actinomycin D Induces Histone γ-H2AX Foci and Complex Formation of γ-H2AX with Ku70 and Nuclear DNA Helicase II

Cited by 89 publications

References 62 publications

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

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

Regulation of mammalian gene expression by retroelements and non‐coding tandem repeats

Histone H2AX is a critical factor for cellular protection against DNA alkylating agents

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