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Cited by 46DNA Repair MechanismsEffects of Radiation ExposureNuclear Structure and Function

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle

Open access
Ray, Alo, Blevins, Chessica, Wani, Gulzar, Wani, Altaf A.·2016·PLOS ONE
DOI 10.1371/journal.pone.0159344PMC PMC4956099
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Abstract

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

AI snapshot

Written from the paper’s full text.

TL;DR

This study dissects how UVR-induced ATR and ATM recruitment and H2AX phosphorylation depend on nucleotide excision repair (NER) proteins in different cell-cycle phases. It reports that ATR/ATM activation at UV lesions requires DDB2, XPC, and XPA in G1, but not in S phase, where stalled replication forks and processed ssDNA/DSBs likely drive recruitment independently of NER assembly.

Key findings

  • ATR and ATM are recruited to UVR damage sites as early as 10 min in asynchronous and G1-arrested OSU-2 cells, co-localizing with XPC, with H2AX phosphorylation also evident at 10 min.
  • In XP-A cells, ATR and ATM recruitment/activation and downstream Chk1/Chk2 phosphorylation were substantially reduced: ATR foci were 19% vs 44% in OSU-2, γH2AX 18% vs 46%; pATM foci 18% vs 41%, γH2AX 15% vs 41%.
  • In G1-arrested cells, ATR/ATM recruitment and H2AX phosphorylation were moderately reduced in XP-E but almost completely abrogated in XP-C and XP-A cells; e.g., XP-E showed 18% ATR and 16% γH2AX foci vs 34% and 31% in OSU-2, and 20% pATM and 18% γH2AX vs 33% and 32% in OSU-2.
  • In S phase EdU-positive cells, γH2AX foci were comparable or slightly higher in repair-deficient cells: XP-E 58%, XP-C 65%, XP-A 80%, vs 59% in OSU-2; pATM foci were 57%, 61%, and 57%, respectively, vs 45% in OSU-2, indicating NER-independent ATM/H2AX responses in S phase.

Methods

  • Used normal human fibroblasts OSU-2 and patient-derived XP-E (GM01389, DDB2-mutated), XP-C (GM02096, XPC-mutated), and XP-A (GM05509, XPA-mutated) cells.
  • Cells were UV-irradiated at 254 nm, typically 20 J/m² for Western blotting or 100 J/m² through a 5-µm micropore filter for immunofluorescence foci; G1 arrest was by 48-h serum starvation (~85% G1 by FACS), and S phase cells were identified by EdU labeling.
  • Main analyses were Western blotting for phosphorylated ATR, ATM, Chk1, Chk2, and H2AX, plus immunofluorescence quantitation of ATR, pATM, γH2AX, XPC, and CPD foci; quantitation used three independent experiments with at least 100 random samples per experiment and two-sided t-tests, with p<0.05 considered significant.

Limitations

  • The authors state they cannot rule out that during S phase DDB2, XPC, and XPA also regulate ATR/ATM recruitment to some UVR lesions in non-replicating regions not encountered by stalled forks, but detecting such signals would be difficult due to abundant signal from stalled replication sites.
  • The authors also state it is difficult to define the types of lesions where ATR and ATM are recruited within the cell cycle.
  • Evident from the text: the time-course data are shown only for initial 10- and 20-min points, with no further increase beyond 30 min stated as data not shown; G1 arrest was ~85%, not complete; and S phase assignment relied on EdU labeling in asynchronous populations.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0159344

Cites

72 of 72 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 79 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    H2AX phosphorylation in discrete foci is evident as early as 10 min in G1 arrested OSU-2 cells.

    “Accordingly, H2AX phosphorylation in discrete foci was also evident as early as 10 min in these cells (Fig 3D).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Levels of ATR and ATM phosphorylation are significantly reduced in XP-A cells compared to OSU-2 cells.

    “As shown in Fig 2A, the levels of ATR and ATM phosphorylation were significantly reduced in XP-A cells as compared to OSU-2 cells, indicating that XPA is required for ATR and ATM recruitment.”
    Quote in the paperSubstantial claim
  • Findingown data

    H2AX phosphorylation is evident at ten minutes post-UVR exposure in OSU-2 cells.

    “Consequently, H2AX phosphorylation is also evident at the same ten minute post-UVR exposure time in OSU-2 cells (Fig 1C).”
    Quote in the paperNot a substantial claim
  • Findingown data

    About 15-20% of XP-E, XP-C, and XP-A cells showed H2AX phosphorylation after 20 min post-repair.

    “We observed that about 15–20% cells showed H2AX phosphorylation in XP-E, XP-C, and XP-A cells.”
    Quote in the paperNot a substantial claim
  • Findingown data

    ATR and ATM activation and accumulation to UVR-induced damage depends on DDB2, XPC, and XPA.

    “We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA”
    Quote in the paperSubstantial claim
  • May combine two claims. 25
  • Classified as probably not a substantial claim. 30

  • May combine two claims.

    Findingown data

    ATR and ATM as well as target substrate phosphorylation are drastically impaired in all repair-deficient G1 arrested cells.

    “In contrast, ATR and ATM as well as the target substrate phosphorylation were drastically impaired in all repair-deficient G1 arrested cells (Fig 4A, lanes 3–8).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both ATR and ATM are promptly recruited to UVR damage sites in G1 arrested OSU-2 cells as early as 10 min, and foci intensified at 20 min.

    “We found that both ATR and ATM are promptly recruited to UVR damage sites in G1 arrested OSU-2 cells as early as 10 min, and the foci intensified further at 20 min time indicating an increasing congregation of these proteins at the UVR damage sites with elapsing time (Fig 3B & 3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ATR and ATM recruitment and H2AX phosphorylation are not dependent on NER proteins in S phase.

    “but not in S phase.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In asynchronous OSU-2 cells, both ATR and ATM are recruited as early as ten minutes and co-localize with XPC foci.

    “We found that in asynchronous OSU-2 cells, both ATR and ATM are recruited as early as ten minutes and co-localize with XPC foci (Fig 1A & 1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ATR foci were present in 19% and γH2AX foci in 18% of XP-A cells compared to 44% and 46% in OSU-2 cells.

    “When ATR and γH2AX co-localization was performed, ATR foci were present in 19% and γH2AX foci were present in 18% of XP-A cells as compared to 44% and 46% in OSU-2 cells, respectively (Fig 2C, top panel).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ATR and ATM are activated by phosphorylation in G1 arrested OSU-2 cells, and Chk1 and Chk2 are phosphorylated.

    “Using these G1 arrested cells, we demonstrated that ATR and ATM were activated by phosphorylation in OSU-2 cells, as evident by the levels of phosphorylated ATR and ATM (Fig 4A, lane 1 and 2). As a result, ATR and ATM substrates Chk1 and Chk2 respectively, were also phosphorylated in OSU-2 cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The frequency of γH2AX foci was essentially comparable in all cell types, with no significant reduction of foci formation in repair-deficient or repair-proficient cells.

    “More importantly, the frequency of γH2AX foci was essentially comparable in all cell types as no significant reduction of foci formation could be observed in repair-deficient or repair-proficient cells (Fig 7A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    ATR, ATM, and H2AX phosphorylation is moderately affected in XP-E cells, but completely abrogated in XP-C and XP-A cells.

    “Our quantitative analysis revealed that ATR, ATM, and H2AX phosphorylation is moderately affected in XP-E cells, but completely abrogated in XP-C and XP-A cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In repair-deficient cells, the frequency of CPD foci remained unaffected but the corresponding damage response phosphorylation events were undetectable.

    “In repair-deficient cells, the frequency of CPD foci remained unaffected but the corresponding damage response phosphorylation events were undetectable.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The mammalian nucleotide excision repair (NER) pathway removes a wide range of chemically and conformationally diverse DNA adducts, including UVR-induced bulky DNA adducts such as cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP).

    “The mammalian nucleotide excision repair (NER) pathway removes a wide range of chemically and conformationally diverse DNA adducts, including ultraviolet radiation (UVR)-induced bulky DNA adducts, e.g., cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6-4PP) [3].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] WL De Laat, 1999, Molecular mechanism of nucleotide excision repair, Genes Dev source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    6-4PP lesions are directly recognized by XPC independent of DDB.

    “whereas 6-4PP lesions are directly recognized by XPC independent of DDB [5–8].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [5] M Wakasugi, 2002, DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair, J Biol Chem source

      Not checked in the free run.

    • cites [6] ME Fitch, 2003, In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product, J Biol Chem source

      Not checked in the free run.

    • cites [7] K Sugasawa, 2001, A multistep damage recognition mechanism for global genomic nucleotide excision repair, Genes Dev source

      Not checked in the free run.

    • cites [8] MS Luijsterburg, 2007, Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC, J Cell Sci source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In global genomic NER (GG-NER), damage is recognized by the DDB complex involving DDB1 and DDB2, and by XPC-RAD23B complexes.

    “In GG-NER, damage is recognized by the DDB (damaged DNA binding protein), involving DDB1 and DDB2, and XPC (Xeroderma pigmentosum complementation group C)-RAD23B complexes [5;6].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [5] M Wakasugi, 2002, DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair, J Biol Chem source

      Not checked in the free run.

    • cites [6] ME Fitch, 2003, In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product, J Biol Chem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The DDB complex initially recognizes CPD lesions and helps recruit XPC.

    “The DDB complex initially recognizes the CPD lesions and helps in recruiting XPC”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [5] M Wakasugi, 2002, DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair, J Biol Chem source

      Not checked in the free run.

    • cites [6] ME Fitch, 2003, In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product, J Biol Chem source

      Not checked in the free run.

    • cites [7] K Sugasawa, 2001, A multistep damage recognition mechanism for global genomic nucleotide excision repair, Genes Dev source

      Not checked in the free run.

    • cites [8] MS Luijsterburg, 2007, Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC, J Cell Sci source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The MRN complex recognizes resulting DSBs and facilitates ATM recruitment and activation.

    “The Mre11/Rad50/Nbs1 (MRN) complex recognizes the resulting DSBs and facilitates ATM recruitment and activation [20–23].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [20] JH Lee, 2007, Activation and regulation of ATM kinase activity in response to DNA double-strand breaks, Oncogene source

      Not checked in the free run.

    • cites [21] JH Lee, 2005, ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex, Science source

      Not checked in the free run.

    • cites [22] BE Nelms, 1998, In situ visualization of DNA double-strand break repair in human fibroblasts, Science source

      Not checked in the free run.

    • cites [23] T Uziel, 2003, Requirement of the MRN complex for ATM activation by DNA damage, EMBO J source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    ATR interacts with XPA upon UV irradiation.

    “Furthermore, ATR was shown to interact with XPA upon UV irradiation [51]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [51] SM Shell, 2009, Checkpoint kinase ATR promotes nucleotide excision repair of UV-induced DNA damage via physical interaction with xeroderma pigmentosum group A, J Biol Chem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    XPA stabilizes the bubble and helps position XPF and XPG endonucleases for 5′ and 3′ incisions to excise a 24-32 bp oligonucleotide containing the damaged lesion.

    “XPA stabilizes the bubble and helps in positioning XPF and XPG endonucleases for respective 5′ and 3′ incisions to excise out a 24–32 bp oligonucleotide containing damaged lesion.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [3] WL De Laat, 1999, Molecular mechanism of nucleotide excision repair, Genes Dev source

      Not checked in the free run.

    • cites [11] A Sancar, 1996, DNA excision repair, Annu Rev Biochem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    During S phase, replication forks encounter CPD and 6-4PP lesions that provoke stalling of replication forks at single-strand breaks.

    “In addition, during S phase, replication forks encounter the CPD and 6-4PP lesions that provoke stalling of the replication forks at the single-strand breaks (SSBs).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [17] D Cortez, 2005, Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling, Genes Dev source

      Not checked in the free run.

    • cites [18] L Zou, 2003, Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes, Science source

      Not checked in the free run.

    • cites [19] L Zou, 2001, Sensing and signaling DNA damage: roles of Rad17 and Rad9 complexes in the cellular response to DNA damage, Harvey Lect

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    UVR-induced DNA damage and replication stress also activates ATM, which in turn phosphorylates H2AX.

    “Recently, we and others have shown that UVR-induced DNA damage and replication stress also activates ATM [39–42], which in turn phosphorylates H2AX [40;41].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [39] CL Limoli, 2002, UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, gamma -H2AX formation, and Mre11 relocalization, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [40] H Yajima, 2009, DNA double-strand break formation upon UV-induced replication stress activates ATM and DNA-PKcs kinases, J Mol Biol source

      Not checked in the free run.

    • cites [41] A Ray, 2013, NER initiation factors, DDB2 and XPC, regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites, DNA Repair (Amst) source

      Not checked in the free run.

    • cites [42] M Wakasugi, 2014, Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells, J Biol Chem source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisereasoning

    XPA is an integral component of DNA damage processing by both GG-NER and TC-NER.

    “Therefore, XPA is an integral component of DNA damage processing by both GG-NER and TC-NER.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Short single-stranded DNA gaps caused by UV damage result in activation of ATR-dependent repair and checkpoint pathways.

    “Several studies implicated that short ssDNA (single-stranded DNA) gaps caused by UV damage results in activation of ATR-dependent repair and checkpoint pathways [14–16].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [14] KA Cimprich, 2008, ATR: an essential regulator of genome integrity, Nat Rev Mol Cell Biol source

      Not checked in the free run.

    • cites [15] F Lazzaro, 2009, Checkpoint mechanisms at the intersection between DNA damage and repair, DNA Repair (Amst) source

      Not checked in the free run.

    • cites [16] D Novarina, 2011, Mind the gap: keeping UV lesions in check, DNA Repair (Amst) source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    DDB2 and XPC influence ATR and ATM recruitment to the damage site and promote their activation by phosphorylation.

    “Moreover, we showed that DDB2 and XPC influence ATR and ATM recruitment to the damage site and promote their activation by phosphorylation [41].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] A Ray, 2013, NER initiation factors, DDB2 and XPC, regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites, DNA Repair (Amst) source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ATR and ATM phosphorylate histone H2AX at Ser-139 (γH2AX) in response to DNA double-strand breaks, and γH2AX spreads along DNA up to 200-400 kb and helps recruit proteins involved in DNA damage repair and checkpoint activation.

    “ATR and ATM also phosphorylate histone H2AX at Ser-139 (γH2AX) in response to DNA double-strand breaks (DSBs), which spreads along the DNA up to 200–400 kb and helps in the recruitment of proteins involved in DNA damage repair and checkpoint activation [29].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] V Savic, 2009, Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin, Mol Cell source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    H2AX is phosphorylated both in G1 and S phases by ATR in response to UV irradiation.

    “H2AX is phosphorylated both in G1 and S phases by ATR in response to UV irradiation [26;32–38].”
    Quote in the paperNot a substantial claim0 of 8 sources checked
    • cites [26] D Branzei, 2008, Regulation of DNA repair throughout the cell cycle, Nat Rev Mol Cell Biol source

      Not checked in the free run.

    • cites [32] S Hanasoge, 2007, H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase, car

      Not checked in the free run.

    • cites [33] TM Marti, 2006, H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [34] RS Tibbetts, 2000, Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress, Genes Dev source

      Not checked in the free run.

    • cites [35] D Branzei, 2010, Maintaining genome stability at the replication fork, Nat Rev Mol Cell Biol source

      Not checked in the free run.

    • cites [36] KA Cimprich, 2008, ATR: an essential regulator of genome integrity, Nat Rev Mol Cell Biol source

      Not checked in the free run.

    • cites [37] Y Auclair, 2008, ATR kinase is required for global genomic nucleotide excision repair exclusively during S phase in human cells, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [38] TP Heffernan, 2002, An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following UVC-induced DNA damage, Mol Cell Biol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ATR phosphorylates XPA.

    “and phosphorylates XPA [52].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] X Wu, 2006, Phosphorylation of nucleotide excision repair factor xeroderma pigmentosum group A by ataxia telangiectasia mutated and Rad3-related-dependent checkpoint pathway promotes cell survival in response to UV irradiation, Cancer Res source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    ATR is predominantly responsible for the DNA damage response upon UVR exposure.

    “Previous studies support that ATR is predominantly responsible for DDR upon UVR exposure”
    Quote in the paperNot a substantial claim0 of 8 sources checked
    • cites [26] D Branzei, 2008, Regulation of DNA repair throughout the cell cycle, Nat Rev Mol Cell Biol source

      Not checked in the free run.

    • cites [32] S Hanasoge, 2007, H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase, car

      Not checked in the free run.

    • cites [33] TM Marti, 2006, H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [34] RS Tibbetts, 2000, Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress, Genes Dev source

      Not checked in the free run.

    • cites [35] D Branzei, 2010, Maintaining genome stability at the replication fork, Nat Rev Mol Cell Biol source

      Not checked in the free run.

    • cites [36] KA Cimprich, 2008, ATR: an essential regulator of genome integrity, Nat Rev Mol Cell Biol source

      Not checked in the free run.

    • cites [37] Y Auclair, 2008, ATR kinase is required for global genomic nucleotide excision repair exclusively during S phase in human cells, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [38] TP Heffernan, 2002, An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following UVC-induced DNA damage, Mol Cell Biol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Stalled replication forks are processed to long ssDNA, where RPA binds and initiates recruitment of DDR proteins including ATRIP-ATR, TopBP1, MRE11, Rad50, Rad17, and the 9-1-1 complex.

    “These breaks are processed to long ssDNA, where RPA binds and initiates the recruitment of a complex array of DNA damage response (DDR) proteins, including ATRIP-ATR complex, TopBP1, MRE11, Rad50, Rad17, and 9-1-1 complex [17–19].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [17] D Cortez, 2005, Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling, Genes Dev source

      Not checked in the free run.

    • cites [18] L Zou, 2003, Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes, Science source

      Not checked in the free run.

    • cites [19] L Zou, 2001, Sensing and signaling DNA damage: roles of Rad17 and Rad9 complexes in the cellular response to DNA damage, Harvey Lect

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Upon recruitment, ATR phosphorylates H2AX near stalled replication forks.

    “Upon recruitment, ATR phosphorylates H2AX near the stalled replication forks.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisenone given

    Collapsing of replication forks generates DSBs.

    “Collapsing of the replication forks generates DSBs.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    In G1, checkpoint activation is due to ssDNA and not from DSBs.

    “Previous studies showed that in G1, checkpoint activation is due to the ssDNA and not from DSBs [33;46;49]”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [33] TM Marti, 2006, H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [46] S Sertic, 2011, Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [49] M Giannattasio, 2004, Physical and functional interactions between nucleotide excision repair and DNA damage checkpoint, EMBO J source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    DDB2 and XPC recruitment can be immuno-detected at damage sites by ~5 minutes post-irradiation.

    “DDB2 and XPC recruitment can be immuno-detected at the damage sites by ~5 minutes post-irradiation [57;60].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [57] R Nishi, 2009, UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells, DNA Repair (Amst) source

      Not checked in the free run.

    • cites [60] J Moser, 2005, The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions, DNA Repair (Amst) source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    ATR and ATM physically and functionally interact with XPC.

    “Our previous study demonstrated that ATR and ATM physically and functionally interact with XPC.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] A Ray, 2013, NER initiation factors, DDB2 and XPC, regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites, DNA Repair (Amst) source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    ATR and ATM localization as well as H2AX phosphorylation are regulated by NER proteins during G1, but not in S phase of cell cycle.

    “Our results of ATR and ATM recruitment to the UVR damage sites showing that ATR and ATM localization as well as H2AX phosphorylation are regulated by NER proteins during G1, but not in S phase of cell cycle are quite intriguing.”
    • Also stated in: § Discussion · boosted
      “Collectively, our data unambiguously show that ATM and ATR recruitment and H2AX phosphorylation during G1 phase is dependent on NER proteins, but independent of NER proteins in S phase.”
    • Also stated in: § Discussion · boosted
      “our approach using single-cell analysis enabled us to show that defects in DDB2, XPC, and XPA do interfere with UV-induced ATR and ATM recruitment in G1, but not in S phase of cell cycle.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    hEXO1 association to NER processed ssDNA is restricted to non-replicating cells and is needed for checkpoint activation in G1.

    “Additionally, Sertic et. al. also found that hEXO1 association to NER processed ssDNA is restricted to non-replicating cells, and is needed for checkpoint activation in G1 [46].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [46] S Sertic, 2011, Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation, Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    XP-C cells despite accumulation of ssDNA do not exhibit an apparent defect in S phase progression whereas Polη-deficiency resulted in S phase arrest.

    “Nevertheless, our data is supported by recent results of Quinet et. al., which show that XP-C cells despite the accumulation of ssDNA do not exhibit an apparent defect in S phase progression whereas Polη-deficiency resulted in S phase arrest [66].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [66] A Quinet, 2014, Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome, DNA Repair (Amst) source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    UV-induced Chk1 phosphorylation is restricted in G1 and G2/M phases in XP-A mutated cells, but not in S phase cells.

    “This data is supported by Marini et. al. showing that UV-induced Chk1 phosphorylation is restricted in G1 and G2/M phases in XP-A mutated cells, but not in S phase cells [50].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] F Marini, 2006, DNA nucleotide excision repair-dependent signaling to checkpoint activation, Proc Natl Acad Sci U S A source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    DSB recruits MRN complex, which facilitates ATM recruitment, H2AX phosphorylation, and DNA repair by HR pathway.

    “DSB recruits MRN complex, which subsequently facilitates ATM recruitment, H2AX phosphorylation, and DNA repair by HR pathway [20;21;40].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [20] JH Lee, 2007, Activation and regulation of ATM kinase activity in response to DNA double-strand breaks, Oncogene source

      Not checked in the free run.

    • cites [21] JH Lee, 2005, ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex, Science source

      Not checked in the free run.

    • cites [40] H Yajima, 2009, DNA double-strand break formation upon UV-induced replication stress activates ATM and DNA-PKcs kinases, J Mol Biol source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    H2AX phosphorylation upon UV radiation is triggered by both NER-dependent and independent pathways.

    “Previous studies in this area showed that H2AX phosphorylation upon UV radiation is triggered by both NER-dependent and independent pathways [50;61–63].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [50] F Marini, 2006, DNA nucleotide excision repair-dependent signaling to checkpoint activation, Proc Natl Acad Sci U S A source

      Not checked in the free run.

    • cites [61] M Matsumoto, 2007, Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells, J Cell Sci source

      Not checked in the free run.

    • cites [62] MG Vrouwe, 2011, UV-induced photolesions elicit ATR-kinase-dependent signaling in non-cycling cells through nucleotide excision repair-dependent and -independent pathways, J Cell Sci source

      Not checked in the free run.

    • cites [63] LA Lindsey-Boltz, 2014, Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system, J Biol Chem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    DDB2 and XPC have inherent recognition capacity for UVR-induced DNA damage and instigate prompt formation of pre-incision complex at damage sites.

    “DDB2 and XPC have the inherent recognition capacity for UVR-induced DNA damage, and instigate the prompt formation of pre-incision complex at the damage sites [5;57–59].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [5] M Wakasugi, 2002, DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair, J Biol Chem source

      Not checked in the free run.

    • cites [57] R Nishi, 2009, UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells, DNA Repair (Amst) source

      Not checked in the free run.

    • cites [58] K Sugasawa, 2009, Two-step recognition of DNA damage for mammalian nucleotide excision repair: Directional binding of the XPC complex and DNA strand scanning, Mol Cell source

      Not checked in the free run.

    • cites [59] D Hoogstraten, 2008, Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC, J Cell Sci source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    NER-dependent DSBs in quiescent G0 cells activate ATM kinase and trigger H2AX phosphorylation upon UVR exposure.

    “Indeed, recent studies by Wakasugi et. al. showed that NER-dependent DSBs in quiescent G0 cells activate ATM kinase and trigger H2AX phosphorylation upon UVR exposure [64].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [64] M Wakasugi, 2014, Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells, J Biol Chem source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ATRIP localization and Chk1 phosphorylation is not affected in XP-C, XP-F, and XP-G cells, but is affected in XP-A cells.

    “However, Bomgarden et. al. showed that even though ATRIP localization and Chk1 phosphorylation is not affected in XP-C, XP-F, and XP-G cells, it is affected in XP-A cells [65].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [65] RD Bomgarden, 2006, Opposing effects of the UV lesion repair protein XPA and UV bypass polymerase eta on ATR checkpoint signaling, EMBO J source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Upon UV irradiation and replication fork stalling, replication repair factors such as BRCA1, RAD51 and FANCD2 form nuclear foci.

    “Accordingly, upon UV irradiation and replication fork stalling, replication repair factors such as BRCA1, RAD51 and FANCD2 form nuclear foci [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] RS Tibbetts, 2000, Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress, Genes Dev source

      Not checked in the free run.

41 of 41

  • The paper relies on this without support.

    ATM is phosphorylated only after its recruitment to damage sites.

    “For detection of ATM, we used pATM antibody as ATM is phosphorylated only after its recruitment to the damage sites.”

  • The paper relies on this without support.

    In G1 phase, only UV lesions are present (no replication forks).

    “during G1 phase when only the UV lesions are present.”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Micropore filter size is given in incompatible units

    Minor

    The Fig 1 caption describes the filter as 5 μM, a concentration, while the methods and other figure captions give its pore size as 5 μm, a length.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. KA Cimprich, 2008, ATR: an essential regulator of genome integrity, Nat Rev Mol Cell Biol, 9, 616, 10.1038/nrm2450 · DOI · Paper page
  2. M Wakasugi, 2014, Nucleotide excision repair-dependent DNA double-strand break formation and ATM signaling activation in mammalian quiescent cells, J Biol Chem, 289, 28730, 10.1074/jbc.M114.589747 · DOI · Paper page

Cited by

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Related

Authors
Ray, A., Blevins, C., Wani, G., & Wani, A. A.
Affiliations
  • The Ohio State University (US)
  • The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (US)
Corresponding author
  • Ray, Alo
  • Wani, Altaf A.
ORCID
Ray, Alo
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2016-07-21
Volume
11
Issue
7
Pages
e0159344
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0159344
PMC
PMC4956099
OpenAlex
W2495200680
Cited by
44
References
72
Citations by year
2026: 2 · 2025: 2 · 2024: 5 · 2023: 2 · 2022: 3 · 2021: 6 · 2020: 3 · 2019: 5 · 2018: 5 · 2017: 7 · 2016: 4
FWCI
3.64
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
National Institutes of Health (ES002388, ES12991)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Effects of Radiation Exposure
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Structure and Function
Keywords
ATR kinase, ATM kinase, DNA damage response, nucleotide excision repair, DDB2, XPC, XPA, ultraviolet radiation, G1 phase, S phase, cell cycle checkpoints, H2AX phosphorylation, single-stranded DNA, replication fork stalling
MeSH terms
Cell Line, DNA Damage, DNA Repair, DNA-Binding Proteins, Histones, Humans, Models, Biological, Phosphorylation, Substrate Specificity, Ultraviolet Rays, G1 Phase, S Phase, Xeroderma Pigmentosum Group A Protein, Cell Cycle Checkpoints, Ataxia Telangiectasia Mutated Proteins

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Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

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