Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form γ-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers.

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RIF1 acts in DNA repair through phosphopeptide recognition of 53BP1

10.1101/2021.04.26.441429 ◽

2021 ◽

Author(s):

Dheva Setiaputra ◽

Cristina Escribano-Diaz ◽

Julia K. Reinert ◽

Pooja Sadana ◽

Dali Zong ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Binding Sites ◽

Binding Protein ◽

Strand Break ◽

Chromatin Binding ◽

Alternative Mode ◽

Downstream Effectors ◽

Dna Double Strand Break ◽

Radiation Induced

SummaryThe chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1 and shieldin to DNA double-strand break sites. While how PTIP recognizes 53BP1 is known, the molecular details of RIF1 recruitment to DNA damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing radiation-induced foci, but surprisingly only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.

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A SWI/SNF subunit regulates chromosomal dissociation of structural maintenance complex 5 during DNA repair in plant cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1900308116 ◽

2019 ◽

Vol 116 (30) ◽

pp. 15288-15296 ◽

Cited By ~ 1

Author(s):

Jieming Jiang ◽

Ning Mao ◽

Huan Hu ◽

Jiahang Tang ◽

Danlu Han ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Genome Stability ◽

Damage Accumulation ◽

Protein Complexes ◽

Plant Cells ◽

Strand Break ◽

Interacting Protein ◽

Dna Double Strand Break ◽

A Subunit

DNA damage decreases genome stability and alters genetic information in all organisms. Conserved protein complexes have been evolved for DNA repair in eukaryotes, such as the structural maintenance complex 5/6 (SMC5/6), a chromosomal ATPase involved in DNA double-strand break (DSB) repair. Several factors have been identified for recruitment of SMC5/6 to DSBs, but this complex is also associated with chromosomes under normal conditions; how SMC5/6 dissociates from its original location and moves to DSB sites is completely unknown. In this study, we determined that SWI3B, a subunit of the SWI/SNF complex, is an SMC5-interacting protein in Arabidopsis thialiana. Knockdown of SWI3B or SMC5 results in increased DNA damage accumulation. During DNA damage, SWI3B expression is induced, but the SWI3B protein is not localized at DSBs. Notably, either knockdown or overexpression of SWI3B disrupts the DSB recruitment of SMC5 in response to DNA damage. Overexpression of a cotranscriptional activator ADA2b rescues the DSB localization of SMC5 dramatically in the SWI3B-overexpressing cells but only weakly in the SWI3B knockdown cells. Biochemical data confirmed that ADA2b attenuates the interaction between SWI3B and SMC5 and that SWI3B promotes the dissociation of SMC5 from chromosomes. In addition, overexpression of SMC5 reduces DNA damage accumulation in the SWI3B knockdown plants. Collectively, these results indicate that the presence of an appropriate level of SWI3B enhances dissociation of SMC5 from chromosomes for its further recruitment at DSBs during DNA damage in plant cells.

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Human SIRT6 Promotes DNA End Resection Through CtIP Deacetylation

Science ◽

10.1126/science.1192049 ◽

2010 ◽

Vol 329 (5997) ◽

pp. 1348-1353 ◽

Cited By ~ 261

Author(s):

Abderrahmane Kaidi ◽

Brian T. Weinert ◽

Chunaram Choudhary ◽

Stephen P. Jackson

Keyword(s):

Homologous Recombination ◽

Genome Stability ◽

Protein A ◽

Strand Break ◽

Dsb Repair ◽

Interacting Protein ◽

Dna Double Strand Break ◽

Dna End Resection ◽

Lysine Deacetylases ◽

End Resection

SIRT6 belongs to the sirtuin family of protein lysine deacetylases, which regulate aging and genome stability. We found that human SIRT6 has a role in promoting DNA end resection, a crucial step in DNA double-strand break (DSB) repair by homologous recombination. SIRT6 depletion impaired the accumulation of replication protein A and single-stranded DNA at DNA damage sites, reduced rates of homologous recombination, and sensitized cells to DSB-inducing agents. We identified the DSB resection protein CtIP [C-terminal binding protein (CtBP) interacting protein] as a SIRT6 interaction partner and showed that SIRT6-dependent CtIP deacetylation promotes resection. A nonacetylatable CtIP mutant alleviated the effect of SIRT6 depletion on resection, thus identifying CtIP as a key substrate by which SIRT6 facilitates DSB processing and homologous recombination. These findings further clarify how SIRT6 promotes genome stability.

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The complex matter of DNA double-strand break detection

Biochemical Society Transactions ◽

10.1042/bst0310040 ◽

2003 ◽

Vol 31 (1) ◽

pp. 40-44 ◽

Cited By ~ 26

Author(s):

J.M. Bradbury ◽

S.P. Jackson

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Strand Break ◽

Damage Sensing ◽

Dna Double Strand Break ◽

Mre11 Complex ◽

Damage Response ◽

Radiation Induced ◽

Dna Damage Signalling ◽

Complex Matter

To maintain genomic stability, despite constant exposure to agents that damage DNA, eukaryotic cells have developed elaborate and highly conserved pathways of DNA damage sensing, signalling and repair. In this review, we concentrate mainly on what we know about DNA damage sensing with particular reference to Lcd1p, a yeast protein that functions early in DNA damage signalling, and MDC1 (mediator of DNA damage checkpoint 1), a recently identified human protein that may be involved in recruiting the MRE11 complex to radiation-induced nuclear foci. We describe a model for the DNA damage response in which factors are recruited sequentially to sites of DNA damage to form complexes that can amplify the original signal and propagate it to the multitude of response pathways necessary for genome stability.

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Perfecting DNA double-strand break repair on transcribed chromatin

Essays in Biochemistry ◽

10.1042/ebc20190094 ◽

2020 ◽

Vol 64 (5) ◽

pp. 705-719 ◽

Cited By ~ 5

Author(s):

Xin Yi Tan ◽

Michael S.Y. Huen

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Chromosomal Aberrations ◽

Chromatin Structure ◽

Genome Stability ◽

Transcriptional Control ◽

Strand Break ◽

Double Strand Break ◽

Dna Double Strand Break ◽

Molecular Bases

Abstract Timely repair of DNA double-strand break (DSB) entails coordination with the local higher order chromatin structure and its transaction activities, including transcription. Recent studies are uncovering how DSBs trigger transient suppression of nearby transcription to permit faithful DNA repair, failing of which leads to elevated chromosomal aberrations and cell hypersensitivity to DNA damage. Here, we summarize the molecular bases for transcriptional control during DSB metabolism, and discuss how the exquisite coordination between the two DNA-templated processes may underlie maintenance of genome stability and cell homeostasis.

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Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

The Journal of Cell Biology ◽

10.1083/jcb.201401146 ◽

2014 ◽

Vol 206 (7) ◽

pp. 877-894 ◽

Cited By ~ 64

Author(s):

Olivia Barton ◽

Steffen C. Naumann ◽

Ronja Diemer-Biehs ◽

Julia Künzel ◽

Monika Steinlage ◽

...

Keyword(s):

Large Scale ◽

Strand Break ◽

Dna Double Strand Breaks ◽

End Joining ◽

Interacting Protein ◽

Genomic Deletions ◽

Cellular Processes ◽

Dna Double Strand Break ◽

Radiation Induced ◽

Alternative Nhej

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

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Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage

Biochemical Journal ◽

10.1042/bj20050379 ◽

2005 ◽

Vol 391 (3) ◽

pp. 473-480 ◽

Cited By ~ 43

Author(s):

Xiaoming Wu ◽

Zhengguan Yang ◽

Yiyong Liu ◽

Yue Zou

Keyword(s):

Dna Damage ◽

Protein A ◽

Replication Protein A ◽

Strand Break ◽

Double Strand Break ◽

Replication Protein ◽

Double Strand Break Repair ◽

Dsb Repair ◽

Checkpoint Activation ◽

In Cells

RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.

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Poly(ADP-ribosyl)ation temporally confines SUMO-dependent ataxin-3 recruitment to control DNA double-strand break repair

Journal of Cell Science ◽

10.1242/jcs.247809 ◽

2021 ◽

pp. jcs.247809

Author(s):

Annika Pfeiffer ◽

Laura K. Herzog ◽

Martijn S. Luijsterburg ◽

Rashmi G. Shah ◽

Magdalena B. Rother ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Time Window ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Dna Repair Proteins ◽

Short Time Window ◽

Short Time

DNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent recruitment of the deubiquitylating enzyme ataxin-3 to DSBs, unlike recruitment of the ubiquitin ligase RNF4, additionally depends on PARP1-mediated poly(ADP-ribosyl)ation (PARylation). The co-dependence of ataxin-3 recruitment on PARylation and SUMOylation temporally confines its presence at DSBs to a short time window directly following detection of the DNA damage. We propose that this mechanism ensures that ataxin-3 prevents the premature removal of DNA repair proteins only during the early phase of the DSB response and does not interfere with the subsequent timely displacement of DNA repair proteins by RNF4. Thus, our data show that PARylation differentially regulates SUMO-dependent recruitment of ataxin-3 and RNF4 to DSBs, explaining how both proteins can play a stimulatory role at DSBs despite their opposing activities.

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ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair

Nature Communications ◽

10.1038/s41467-021-25790-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sonia Jimeno ◽

Rosario Prados-Carvajal ◽

María Jesús Fernández-Ávila ◽

Sonia Silva ◽

Domenico Alessandro Silvestris ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Rna Editing ◽

Genomic Stability ◽

Strand Break ◽

Dna Lesions ◽

Dna Breaks ◽

Dna Double Strand Break ◽

Editing Enzyme

AbstractThe maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

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Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy

Scientific Reports ◽

10.1038/srep45385 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Fei Xu ◽

Yixuan Fang ◽

Lili Yan ◽

Lan Xu ◽

Suping Zhang ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Strand Break ◽

Beclin 1 ◽

Dsb Repair ◽

Dna Topoisomerase ◽

Damage Repair ◽

Dna Double Strand Break ◽

Radiation Induced

Abstract Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Here, we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice. Surprisingly, deletion of beclin1 in in vitro human cells did not block an autophagy response, but attenuated the expression of several DNA double-strand break (DSB) repair proteins and formation of repair complexes, and reduced an ability to repair DNA in the cells exposed to ionizing radiation (IR). Overexpressing Beclin 1 improved the repair of IR-induced DSB, but did not restore an autophagy response in cells lacking autophagy gene Atg7, suggesting that Beclin 1 may regulate DSB repair independent of autophagy in the cells exposed to IR. Indeed, we found that Beclin 1 could directly interact with DNA topoisomerase IIβ and was recruited to the DSB sites by the interaction. These findings reveal a novel function of Beclin 1 in regulation of DNA damage repair independent of its role in autophagy particularly when the cells are under radiation insult.

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