PRP19 Transforms into a Sensor of RPA-ssDNA after DNA Damage and Drives ATR Activation via a Ubiquitin-Mediated Circuitry

Maréchal, Alexandre; Li, Ju Mei; Ji, Xiao Ye; Wu, Chenwei; Yazinski, Stephanie A.; Nguyen, Hai Dang; Liu, Shizhou; Jimenez, Amanda E.; Jin, Jianping; Zou, Lee

doi:10.1016/j.molcel.2013.11.002

Cited by 214 publications

(261 citation statements)

References 68 publications

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“…We recently showed that the full activation of ATR pathway is dependent on a ubiquitylation circuitry mediated by RPA-ssDNA and the ubiquitin ligase PRP19 (Marechal et al 2014). In this study, we found that UBC9 is required for efficient ATR activation, suggesting that the ATR pathway is also intertwined with the SUMOylation circuitry.…”

Section: Discussionmentioning

confidence: 59%

“…In addition to the phosphorylation events mediated by the ATM and ATR pathways, several other types of post-translational modifications (PTMs), such as ubiquitylation, SUMOylation, methylation, acetylation, and poly-ADP ribosylation, are also implicated in the DDR (Bekker-Jensen and Mailand 2010; Huen et al 2010;Lukas et al 2011;Jackson and Durocher 2013). We recently found that the efficient activation of ATR relies on a ubiquitylation circuitry mediated by RPA-ssDNA (RPA-coated ssDNA) and PRP19 (Marechal et al 2014). This new finding raises a question as to whether other PTMs also participate in DNA damage signaling through the ATR pathway.…”

mentioning

confidence: 99%

“…ATR, through its functional partner, ATRIP, is recruited to RPA-ssDNA at sites of DNA damage and stressed replication forks (Costanzo et al 2003;Ball et al 2005;Namiki and Zou 2006). ATRIP not only binds RPA-ssDNA directly but also recognizes the ubiquitin chains (Marechal et al 2014). Three regulators of ATR, the RAD17 complex, the RAD9-RAD1-HUS1 (9-1-1) complex, and RHINO, are recruited to junctions of ssDNA and dsDNA (Zou et al 2002Ellison and Stillman 2003;Cotta-Ramusino et al 2011).…”

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confidence: 99%

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SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

Ouyang

Mori

et al. 2014

Genes Dev.

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The ATR (ATM [ataxia telangiectasia-mutated]-and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.[Keywords: ATR; ATRIP; checkpoint; SUMOylation] Supplemental material is available for this article.Received January 18, 2014; revised version accepted June 2, 2014.The maintenance of genomic stability requires not only DNA repair machineries but also signal transduction pathways that regulate and coordinate the DNA damage response (DDR) (Ciccia and Elledge 2010). In human cells, DNA damage signaling is primarily initiated by the ataxia telangiectasia-mutated (ATM) and the ATMand Rad3-related (ATR) kinases. Whereas ATM is activated by DNA double-stranded breaks (DSBs), ATR is elicited by a much broader spectrum of DNA damage and replication stress (Cimprich and Cortez 2008;Marechal and Zou 2013;Shiloh and Ziv 2013). Once activated, ATM and ATR phosphorylate and activate their effector kinases, Chk2 and Chk1, respectively. Together, the ATMChk2 and ATR-Chk1 kinase cascades phosphorylate a number of substrates involved in DNA repair, DNA replication, and cell cycle transitions, coordinating these processes to suppress genomic instability. In addition to the phosphorylation events mediated by the ATM and ATR pathways, several other types of post-translational modifications (PTMs), such as ubiquitylation, SUMOylation, methylation, acetylation, and poly-ADP ribosylation, are also implicated in the DDR (Bekker-Jensen and Mailand 2010;Huen et al. 2010;Lukas et al. 2011;Jackson and Durocher 2013). We recently found that the efficient activation of ATR relies on a ubiquitylation circuitry mediated by RPA-ssDNA (RPA-coated ssDNA) and PRP19 (Marechal et al. 2014). This new finding raises a question as to whether other PTMs also participate in DNA damage signaling through the ATR path...

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Section: Discussionmentioning

confidence: 59%

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confidence: 99%

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confidence: 99%

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SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

Ouyang

Mori

et al. 2014

Genes Dev.

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“…For example, RNAs complementary to ssDNAs may interfere with processes downstream of end resection in HR unless they are removed by DDX1. In agreement with its role in maintaining ssDNA, DDX1 has been shown to interact with ssDNA coated with RPA (63).…”

Section: Discussionmentioning

confidence: 61%

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

Germain

Poon

et al. 2016

Molecular and Cellular Biology

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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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“…We then treated PTEN +/+ and PTEN −/− HCT116 cells with MG132, a proteasome inhibitor that inhibits ubiquitin-mediated proteolysis, and observed that RPA1 expression was largely rescued in PTEN −/− cells (Supplementary information, Figure S4A). Post-translational modification is recognized as a regulatory mechanism of RPA1 function, and ubiquitination is related to protein stability [26,27]. We therefore tested if RPA1 ubiquitination is affected in cells lacking PTEN.…”

Section: Pten Promotes Rpa1 Protein Stability Through Regulation Of Rmentioning

confidence: 99%

PTEN regulates RPA1 and protects DNA replication forks

Wang

et al. 2015

Cell Res

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Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication.

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PRP19 Transforms into a Sensor of RPA-ssDNA after DNA Damage and Drives ATR Activation via a Ubiquitin-Mediated Circuitry

Cited by 214 publications

References 68 publications

SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

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

PTEN regulates RPA1 and protects DNA replication forks

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