NBS1 is regulated by two kind of mechanisms: ATM-dependent complex formation with MRE11 and RAD50, and cell cycle–dependent degradation of protein

Zhou, Hui; Kawamura, Kasumi; Yanagihara, Hirokazu; Kobayashi, Junya; Zhang-Akiyama, Qiu-Mei

doi:10.1093/jrr/rrx014

Cited by 15 publications

(15 citation statements)

References 24 publications

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“…After DSB, ATM is recruited to the site of DNA damage and activated via phosphorylation, converting H2AX into γH2AX (Nickoloff, ). ATM itself can recruit additional HR pathway DNA damage repair proteins, such as MRE, NBS1, and RAD51, to form the MRN complex, which promotes the HR pathway repair process (Lavin et al ., ; Zhou et al ., ). In the present study, we demonstrated that ATM was upregulated after DSB, using both immunofluorescence and WB approaches.…”

Section: Discussionmentioning

confidence: 97%

Preliminary study of the homologous recombination repair pathway in mouse spermatogonial stem cells

et al. 2018

Andrology

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The present study was designed to detect DNA repair response through the homologous recombination pathway in mouse spermatogonial stem cells. Mouse spermatogonial stem cells (mSSCs) were obtained from the adult DBA/2 mouse testes by MACS sorting. mSSCs and mice animals were divided into four groups (30 min, 2, 24 h, control) and treated with ionizing irradiation while the control group received pseudo-irradiation. Proteins involved in the homologous recombination pathway (γH2AX, ATM, RAD51, CtIP, and RPA2) were assessed in mSSCs both in vitro and in vivo. Moreover, the non-homologous end-joining or homologous recombination (NHEJ/HR) reporter plasmids were transfected into mSSCs to assess NHEJ/HR pathway activity after DNA double-strand break (DSB). γH2AX, a classical DNA DSB marker, was absent in mSSCs both in vivo and in vitro after DSB repair, but was highly expressed in other tissue stem cells. In addition, ATM and phosphorylated ATM (p-ATM) were involved in DNA damage response (DDR) in mSSCs. p-ATM foci were overexpressed immediately after irradiation (30 min and 2 h), but gradually decreased over the repair time. The HR pathway-related proteins, CtIP and RPA2 were negatively regulated after treatment in Western blot (WB). NHEJ/HR reporter plasmid transfection indicated that the HR pathway played a minor role in mSSCs during DDR, consistent with the WB findings. This study demonstrates that mSSCs may have a unique response to DNA damage since crucial proteins involved in HR pathway were negatively regulated after DSB. In addition, the expression level of p-ATM, but not γH2AX, was increased after DSB, suggesting that DNA damage repair in mSSCs might be a γH2AX-independent response. Furthermore, the HR pathway may play a minor role during DDR in mSSCs.

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Section: Discussionmentioning

confidence: 97%

Preliminary study of the homologous recombination repair pathway in mouse spermatogonial stem cells

et al. 2018

Andrology

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“…Using human proteins instead, we found that the phosphorylation of CtIP was largely but not completely dispensable for the activation of MR in the absence of NBS1. Furthermore, it has been reported that the physical interaction of NBS1 with MR may be differentially regulated in G1 vs. S-G2, upon DNA damage or at various subcellular locations, such as at telomeres (Zhu et al, 2000;Zhou et al, 2017;Limbo et al, 2018). It is well established that CDK-and ATM-dependent phosphorylation of CtIP is necessary for the activation of extended resection and hence homologous recombination in S-G2 (Huertas et al, 2008;Huertas & Jackson, 2009;Wang et al, 2013).…”

Section: A B Cmentioning

confidence: 99%

“…While MRE11 and RAD50 form a constitutively interacting dimer of dimers complex, there are conflicting reports about the stoichiometry of NBS1 within the MRN complex (Trujillo et al, 1998;Paull & Gellert, 1999;Schiller et al, 2012). Furthermore, it has been reported that the physical interaction of NBS1 with MR may be differentially regulated in G1 vs. S-G2, upon DNA damage or at various subcellular locations, such as at telomeres (Zhu et al, 2000;Zhou et al, 2017;Limbo et al, 2018). Specifically, the MR complex was shown to localize to DSBs in the absence of NBS1 (Limbo et al, 2018), and NBS1 co-localized with MR to telomeres in S but not during interphase (Zhu et al, 2000).…”

Section: A B Cmentioning

confidence: 99%

NBS1 promotes the endonuclease activity of the MRE11‐RAD50 complex by sensing CtIP phosphorylation

et al. 2019

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DNA end resection initiates DNA double‐strand break repair by homologous recombination. MRE11‐RAD50‐NBS1 and phosphorylated CtIP perform the first resection step via MRE11‐catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11‐RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11‐RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation‐dependent mode through NBS1 and a phosphorylation‐independent mode without NBS1. In support, we show that limited DNA end resection occurs in vivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11‐RAD50 nuclease to S‐G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.

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“…HeLa, U2OS, and hTERT-immortalized human fibroblasts (48BR) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics [13].…”

Section: Methodsmentioning

confidence: 99%

Nucleolar protein nucleolin functions in replication stress–induced DNA damage responses

Kawamura

Meng

et al. 2019

Journal of Radiation Research

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The nucleolus contains multiple copies of ribosomal (r)DNA, which indicate sites of frequent replication stress and suggest the existence of a mechanism to prevent replication stress–related rDNA instability and the possibility that such a mechanism contributes to the whole genomic stability against replication stress. We have previously reported that nucleolin, a major nucleolar protein, is involved in ionizing radiation–induced DNA damage responses (DDRs) such as ataxia telangiectasia mutated (ATM)-dependent cell cycle checkpoints and homologous recombination (HR) repair. Here, we investigated the role of nucleolin in DDR due to replication stress. The results indicate that following replication stress, nucleolin interacted with the histone γH2AX, proliferating cell nuclear antigen (PCNA), and replication protein A (RPA)32, suggesting that it may be recruited to DNA damage sites on the replication fork. Furthermore, the knockdown of nucleolin by siRNA reduced the activation of ATM and RAD3-related (ATR) kinase and the formation of RAD51 and RPA32 foci after replication stress due to UV or camptothecin exposure, whereas nucleolin overexpression augmented ATR-dependent phosphorylation and RAD51 and RPA accumulation on chromatin. Moreover, these overexpressing cells seemed to increase repair activity and resistance to replication stress. Our results indicate that nucleolin plays an important role in replication stress–induced DDRs such as ATR activation and HR repair. Given that nucleolin overexpression is often observed in many types of cancer cells, our findings suggest that nucleolin is involved in the regulation of resistance to replication stress that may otherwise lead to tumorigenesis and it could be a possible target for chemotherapy and radiotherapy.

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NBS1 is regulated by two kind of mechanisms: ATM-dependent complex formation with MRE11 and RAD50, and cell cycle–dependent degradation of protein

Cited by 15 publications

References 24 publications

Preliminary study of the homologous recombination repair pathway in mouse spermatogonial stem cells

Preliminary study of the homologous recombination repair pathway in mouse spermatogonial stem cells

NBS1 promotes the endonuclease activity of the MRE11‐RAD50 complex by sensing CtIP phosphorylation

Nucleolar protein nucleolin functions in replication stress–induced DNA damage responses

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