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• W1972300957 abstract "Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as “TIPs”) during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends. Chromosome ends contain nucleoprotein structures known as telomeres. Damage to chromosome ends during interphase elicits a DNA damage response (DDR) resulting in cell cycle arrest. However, little is known regarding the signaling from damaged chromosome ends (designated here as “TIPs”) during mitosis. In the present study, we investigated the consequences of DNA damage induced at a single TIP in mitosis. We used laser microirradiation to damage mitotic TIPs or chromosome arms (non-TIPs) in PtK2 kidney epithelial cells. We found that damage to a single TIP, but not a non-TIP, delays anaphase onset. This TIP-specific checkpoint response is accompanied by differential recruitment of DDR proteins. Although phosphorylation of H2AX and the recruitment of several repair factors, such as Ku70-Ku80, occur in a comparable manner at both TIP and non-TIP damage sites, DDR factors such as ataxia telangiectasia mutated (ATM), MDC1, WRN, and FANCD2 are specifically recruited to TIPs but not to non-TIPs. In addition, Nbs1, BRCA1, and ubiquitin accumulate at damaged TIPs more rapidly than at damaged non-TIPs. ATR and 53BP1 are not detected at either TIPs or non-TIPs in mitosis. The observed delay in anaphase onset is dependent on the activity of DDR kinases ATM and Chk1, and the spindle assembly checkpoint kinase Mps1. Cells damaged at a single TIP or non-TIP eventually exit mitosis with unrepaired lesions. Damaged TIPs are segregated into micronuclei at a significantly higher frequency than damaged non-TIPs. Together, these findings reveal a mitosis-specific DDR uniquely associated with chromosome ends. Unrepaired DNA damage can lead to mutation, chromosomal fragmentation, and genomic rearrangements (1.Berns M.W. Directed chromosome loss by laser microirradiation.Science. 1974; 186: 700-705Crossref PubMed Scopus (22) Google Scholar, 2.Phillips J.W. Morgan W.F. Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.Mol. Cell. 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Mechanisms of DNA double-strand break repair and their potential to induce chromosomal aberrations.Mutagenesis. 2000; 15: 289-302Crossref PubMed Scopus (345) Google Scholar, 5.Noon A.T. Goodarzi A.A. 53BP1-mediated DNA double strand break repair: insert bad pun here.DNA Repair. 2011; 10: 1071-1076Crossref PubMed Scopus (83) Google Scholar, 6.Takai H. Smogorzewska A. de Lange T. DNA damage foci at dysfunctional telomeres.Curr. Biol. 2003; 13: 1549-1556Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 7.Xiao W. Samson L. In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells.Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 2117-2121Crossref PubMed Scopus (168) Google Scholar, 8.Shigenaga M.K. Gimeno C.J. Ames B.N. Urinary 8-hydroxy-2′-deoxyguanosine as a biological marker of in vivo oxidative DNA damage.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 9697-9701Crossref PubMed Scopus (698) Google Scholar). The DNA damage response (DDR) pathways recognize DNA lesions and recruit proteins to these sites to promote repair. The ends of linear chromosomes, which contain telomeres (also referred to as “TIPs” in this work), can also be recognized as damaged DNA. Telomeres are normally protected from recognition as DNA damage or inappropriate repair processes by a nucleoprotein structure composed of repetitive DNA and the shelterin protein complex (9.Blackburn E.H. Switching and signaling at the telomere.Cell. 2001; 106: 661-673Abstract Full Text Full Text PDF PubMed Scopus (1761) Google Scholar, 10.de Lange T. Protection of mammalian telomeres.Oncogene. 2002; 21: 532-540Crossref PubMed Google Scholar, 11.Greider C.W. Telomeres do D-loop-T-loop.Cell. 1999; 97: 419-422Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 12.Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1384) Google Scholar, 13.Wright W.E. Shay J.W. Telomere-binding factors and general DNA repair.Nat. Genet. 2005; 37: 116-118Crossref PubMed Scopus (38) Google Scholar). Removal of this protein complex from chromosome ends results in local activation of the DDR and recruitment of repair proteins to the telomeres (6.Takai H. Smogorzewska A. de Lange T. DNA damage foci at dysfunctional telomeres.Curr. Biol. 2003; 13: 1549-1556Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 12.Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1384) Google Scholar, 14.Hockemeyer D. Sfeir A.J. Shay J.W. Wright W.E. de Lange T. POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end.EMBO J. 2005; 24: 2667-2678Crossref PubMed Scopus (234) Google Scholar, 15.Bailey S.M. Meyne J. Chen D.J. Kurimasa A. Li G.C. Lehnert B.E. Goodwin E.H. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 14899-14904Crossref PubMed Scopus (349) Google Scholar). double-stranded break chromosome end/telomere DNA damage response ataxia telangiectasia mutated spindle assembly checkpoint near-infrared ubiquitin non-homologous end joining proliferating cell nuclear antigen enhanced yellow fluorescent protein enhanced green fluorescent protein. Shelterin proteins TIN2, TRF1, RAP1, TRF2, TPP1, and POT1 act to prevent activation of the canonical DDR. Specifically, TRF2 binds to double-stranded DNA ends to prevent its recognition as a DSB and subsequent activation of the ataxia telangiectasia mutated (ATM) kinase. On the other hand, POT1 binds to single-stranded DNA in the telomere, preventing ATR kinase (ATM and Rad3-related) activation (16.Yang Q. Zheng Y.L. Harris C.C. POT1 and TRF2 cooperate to maintain telomeric integrity.Mol. Cell. Biol. 2005; 25: 1070-1080Crossref PubMed Scopus (136) Google Scholar). DNA lesions and uncapping of the telomere during interphase of the cell cycle both activate ATM and ATR kinases, phosphorylation of downstream kinases Chk1 and Chk2, and the transcription factor p53, which leads ultimately to cell cycle arrest with a persistent DDR (17.d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (2043) Google Scholar, 18.Verdun R.E. Crabbe L. Haggblom C. Karlseder J. Functional human telomeres are recognized as DNA damage in G2 of the cell cycle.Mol. Cell. 2005; 20: 551-561Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 19.Fumagalli M. Rossiello F. Clerici M. Barozzi S. Cittaro D. Kaplunov J.M. Bucci G. Dobreva M. Matti V. Beausejour C.M. Herbig U. Longhese M.P. d'Adda di Fagagna F. Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation.Nat. Cell Biol. 2012; 14: 355-365Crossref PubMed Scopus (526) Google Scholar). In vertebrate cells, uncapped telomeres induce G2 arrest through the inactivation and degradation of cyclin B by activating the phosphatase Cdc25C (20.Thanasoula M. Escandell J.M. Suwaki N. Tarsounas M. ATM/ATR checkpoint activation downregulates CDC25C to prevent mitotic entry with uncapped telomeres.EMBO J. 2012; 31: 3398-3410Crossref PubMed Scopus (58) Google Scholar) as well as the up-regulation of the Cdk inhibitor p21 (17.d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (2043) Google Scholar). In addition to activation of DDR kinases, unprotected or damaged telomeres are also marked by phosphorylation of H2AX (γ-H2AX) and recruit a number of repair proteins, such as MDC1 and 53BP1, which form telomere dysfunction-induced foci (17.d'Adda di Fagagna F. Reaper P.M. Clay-Farrace L. Fiegler H. Carr P. Von Zglinicki T. Saretzki G. Carter N.P. Jackson S.P. A DNA damage checkpoint response in telomere-initiated senescence.Nature. 2003; 426: 194-198Crossref PubMed Scopus (2043) Google Scholar, 18.Verdun R.E. Crabbe L. Haggblom C. Karlseder J. Functional human telomeres are recognized as DNA damage in G2 of the cell cycle.Mol. Cell. 2005; 20: 551-561Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Our understanding of how the cell responds to telomere damage in mitosis is limited. Cells with defective ATM or p53 escape from G2 arrest and enter mitosis with persisting telomere dysfunction-induced foci on mitotic chromosomes (20.Thanasoula M. Escandell J.M. Suwaki N. Tarsounas M. ATM/ATR checkpoint activation downregulates CDC25C to prevent mitotic entry with uncapped telomeres.EMBO J. 2012; 31: 3398-3410Crossref PubMed Scopus (58) Google Scholar). However, recent studies indicated that DDR is attenuated in mitosis compared with interphase (21.Giunta S. Belotserkovskaya R. Jackson S.P. DNA damage signaling in response to double-strand breaks during mitosis.J. Cell Biol. 2010; 190: 197-207Crossref PubMed Scopus (231) Google Scholar, 22.Peterson S.E. Li Y. Chait B.T. Gottesman M.E. Baer R. Gautier J. Cdk1 uncouples CtIP-dependent resection and Rad51 filament formation during M-phase double-strand break repair.J. Cell Biol. 2011; 194: 705-720Crossref PubMed Scopus (54) Google Scholar, 23.Orthwein A. Fradet-Turcotte A. Noordermeer S.M. Canny M.D. Brun C.M. Strecker J. Escribano-Diaz C. Durocher D. Mitosis inhibits DNA double-strand break repair to guard against telomere fusions.Science. 2014; 344: 189-193Crossref PubMed Scopus (228) Google Scholar), and thus cells may respond differently to telomere damage induced during mitosis (24.Cesare A.J. Hayashi M.T. Crabbe L. Karlseder J. The telomere deprotection response is functionally distinct from the genomic DNA damage response.Mol. Cell. 2013; 51: 141-155Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). A recent study demonstrated that the forced mitotic arrest results in telomere uncapping with γ-H2AX focus formation and ATM activation (25.Hayashi M.T. Cesare A.J. Fitzpatrick J.A. Lazzerini-Denchi E. Karlseder J. A telomere-dependent DNA damage checkpoint induced by prolonged mitotic arrest.Nat. Struct. Mol. Biol. 2012; 19: 387-394Crossref PubMed Scopus (129) Google Scholar). However, the cellular response to telomere damage in mitosis under normal cell cycling conditions has not been explored in detail. In this study, we asked how cells respond to damage induced specifically at telomere-containing chromosome ends (TIPs) in comparison with damage induced at chromosome arms (non-TIPs) in mitosis using laser microirradiation. We performed systematic comparison of the DDR and repair factor recruitment at TIP and non-TIP damage sites and the effect on the progression of mitosis. Laser microirradiation has been shown to produce predominantly DSBs, akin to ionizing radiation, eliciting the DDR in mammalian interphase cells, mitotic chromosomes, and anaphase telomeres of PtK2 cells (26.Ferrando-May E. Tomas M. Blumhardt P. Stöckl M. Fuchs M. Leitenstorfer A. Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.Front. Genet. 2013; 4: 135Crossref PubMed Scopus (27) Google Scholar, 27.Gomez-Godinez V. Wu T. Sherman A.J. Lee C.S. Liaw L.H. Zhongsheng Y. Yokomori K. Berns M.W. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.Nucleic Acids Res. 2010; 38: e202Crossref PubMed Scopus (37) Google Scholar, 28.Kong X. Mohanty S.K. Stephens J. Heale J.T. Gomez-Godinez V. Shi L.Z. Kim J.S. Yokomori K. Berns M.W. Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells.Nucleic Acids Res. 2009; 37: e68Crossref PubMed Scopus (168) Google Scholar, 29.Botchway S.W. Reynolds P. Parker A.W. O'Neill P. Use of near infrared femtosecond lasers as sub-micron radiation microbeam for cell DNA damage and repair studies.Mutat. Res. 2010; 704: 38-44Crossref PubMed Scopus (49) Google Scholar, 30.Träutlein D. Deibler M. Leitenstorfer A. Ferrando-May E. Specific local induction of DNA strand breaks by infrared multi-photon absorption.Nucleic Acids Res. 2010; 38: e14Crossref PubMed Scopus (44) Google Scholar, 31.Mari P.O. Florea B.I. Persengiev S.P. Verkaik N.S. Brüggenwirth H.T. Modesti M. Giglia-Mari G. Bezstarosti K. Demmers J.A. Luider T.M. Houtsmuller A.B. van Gent D.C. Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 18597-18602Crossref PubMed Scopus (313) Google Scholar, 32.Hartlerode A.J. Guan Y. Rajendran A. Ura K. Schotta G. Xie A. Shah J.V. Scully R. Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.PLoS One. 2012; 7: e49211Crossref PubMed Scopus (43) Google Scholar, 33.Silva B.A. Stambaugh J.R. Berns M.W. Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.J. Biomed. Opt. 2013; 18 (095003)Crossref Scopus (5) Google Scholar). Previous studies have shown that laser-induced DNA breaks on mitotic chromosomes do not result in chromosome fragmentation, and DSBs are only introduced at the laser focal spot (27.Gomez-Godinez V. Wu T. Sherman A.J. Lee C.S. Liaw L.H. Zhongsheng Y. Yokomori K. Berns M.W. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.Nucleic Acids Res. 2010; 38: e202Crossref PubMed Scopus (37) Google Scholar, 33.Silva B.A. Stambaugh J.R. Berns M.W. Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.J. Biomed. Opt. 2013; 18 (095003)Crossref Scopus (5) Google Scholar). The spatio-temporal control of laser ablation permits the generation of damaged chromosome tips without genetic perturbation or long term mitotic arrest. Moreover, cells from the rat kangaroo (Potorous tridactylus kidney, PtK2) are ideally suited to study cell division and checkpoint signaling because they have a small number of large metacentric chromosomes (2n = 12) (34.Altmann S.C.A. Ellery M.E.W. The chromosomes of four species of marsupials.Q. J. Microsc. Sci. 1925; 69: 463-469Google Scholar), and close sequence identity with humans, mice, and rats (80–90%) (35.Stout J.R. Rizk R.S. Kline S.L. Walczak C.E. Deciphering protein function during mitosis in PtK cells using RNAi.BMC Cell Biol. 2006; 7: 26Crossref PubMed Scopus (41) Google Scholar). Additionally, a single chromosome tip-containing telomere can be targeted by laser microirradiation in these cells (33.Silva B.A. Stambaugh J.R. Berns M.W. Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.J. Biomed. Opt. 2013; 18 (095003)Crossref Scopus (5) Google Scholar, 36.Baker N.M. Zeitlin S.G. Shi L.Z. Shah J. Berns M.W. Chromosome tips damaged in anaphase inhibit cytokinesis.PLoS One. 2010; 5: e12398Crossref PubMed Scopus (11) Google Scholar). This facilitates the investigation of signaling and protein recruitment on a single damaged chromosome tip. Here, we report that laser-induced damage at TIPs recruits a distinct set of DDR factors compared with damage at non-TIPs. Remarkably, the damage to a single TIP results in a delay in the transition from metaphase to anaphase. This delay was found to be dependent on the DNA damage checkpoint kinases ATM and Chk1 and the spindle assembly checkpoint (SAC) kinase Mps1. Despite the damage-induced delay, cells with a single damaged chromosome tip eventually exited mitosis with persistent DNA lesions forming micronuclei in the G1 phase. Thus, our results uncover a mitotic DDR specifically associated with telomere-containing chromosome ends. Long nosed potoroo (rat kangaroo), P. tridactylus (PtK2 (male) and PtK1 (female)) kidney epithelial cells (American Type Culture Collection (ATCC), CCL 56 and CCL 35) and TRF2-AID-EYFP PtK2 (where AID represents an auxin-inducible degron that has been shown to degrade the AID-tagged target protein upon the addition of a plant hormone; in these experiments, activation of AID was not induced) (37.Holland A.J. Fachinetti D. Han J.S. Cleveland D.W. Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: E3350-E3357Crossref PubMed Scopus (179) Google Scholar) were grown in Invitrogen Advanced Minimum Essential Medium (Invitrogen) supplemented with l-glutamine, 4% fetal bovine serum (FBS), and antibiotics. PtK1 cells stably expressing a green fluorescent protein (GFP)-tagged Nbs1 previously generated (27.Gomez-Godinez V. Wu T. Sherman A.J. Lee C.S. Liaw L.H. Zhongsheng Y. Yokomori K. Berns M.W. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.Nucleic Acids Res. 2010; 38: e202Crossref PubMed Scopus (37) Google Scholar) were incubated with Advanced F-12/DMEM supplemented with l-glutamine, 4% FBS, and antibiotics. All cell types were incubated at 37 °C with 5% CO2. Three days before experiments, cells were trypsinized (TrypLETM Express, Invitrogen) and plated on round 35-mm gridded imaging dishes (MatTek, Ashland, MA) at ∼20,000 cells/dish as shown previously (33.Silva B.A. Stambaugh J.R. Berns M.W. Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.J. Biomed. Opt. 2013; 18 (095003)Crossref Scopus (5) Google Scholar). Before laser microirradiation, the medium was replaced with Hanks' balanced salt solution (1×) to prevent the absorption of the laser light by the phenol red and to facilitate the monitoring of the cells after damage via live fluorescence imaging. To generate PtK2 cells stably expressing eGFP-53BP1 or TRF2-AID-EYFP, we transiently transfected retroviral plasmids eGFP-53BP1 pLPC (kindly donated by the Denchi laboratory, Scripps Research Institute) and pBABEneo TRF2-AID-EYFP (kindly donated by the Cleveland laboratory, University of California, San Diego) (37.Holland A.J. Fachinetti D. Han J.S. Cleveland D.W. Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: E3350-E3357Crossref PubMed Scopus (179) Google Scholar) into Phoenix amphotropic packing cell line, using Effectene transfection reagent (Qiagen) according to the manufacturer's instructions. Viral particles were generated using a modified protocol (38.Dimitrova N. Chen Y.C. Spector D.L. de Lange T. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility.Nature. 2008; 456: 524-528Crossref PubMed Scopus (442) Google Scholar, 39.Shah J.V. Botvinick E. Bonday Z. Furnari F. Berns M. Cleveland D.W. Dynamics of centromere and kinetochore proteins: implications for checkpoint signaling and silencing.Curr. Biol. 2004; 14: 942-952Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). For 53BP1 and TRF2 infections, PtK2 cells were plated in growth medium containing 4 μg ml−1 Polybrene (Sigma) and viruses. Cells were infected at a multiplicity of infection of 3. Forty-eight hours after infection, cells were split and were incubated with medium containing 2 mg ml−1 puromycin for 53BP1 and 2 mg ml−1 neomycin for TRF2. Cells were selected for 5 days. Stable cell lines were further selected using fluorescence-activated cell sorting (FACS) (City of Hope, Duarte, CA). The partial PtK2 ATM sequence was identified by high-throughput sequencing (Illumina) of a commercially generated PtK2 cDNA library (Express Genomics). Duplexes targeting ATM PtK protein were designed and synthesized (Invitrogen). The sequences of the duplexes for ATM siRNA were as follows: 1) sense (5′-GCAGCUUGGUUAAAUACUUTT-3′) and antisense (5′-AAGUAUUUAACCAAGCUGCTT-3′); 2) sense (5′-GCUACUUAUGGAGCGGAUUTT-3′) and antisense (5′-AAUCCGCUCCAUAAGUAGCTT-3′). Scramble siRNA was used as a control. Both siRNAs (1 and 2) were transfected into PtK2 cells using Lipofectamine RNAiMAX (Invitrogen). To obtain 70% transfection efficiency, two consecutive rounds of two-siRNA duplex (1 and 2) transfection were carried out according to the manufacturer's protocol. After 48 h, transfected PtK2 cells were microirradiated and monitored for 15–30 min. Cells were fixed and subjected to immunofluorescent staining using antibodies specific for ATM and γ-H2AX. A previously described custom RoboLase ablation system was used in these studies (27.Gomez-Godinez V. Wu T. Sherman A.J. Lee C.S. Liaw L.H. Zhongsheng Y. Yokomori K. Berns M.W. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.Nucleic Acids Res. 2010; 38: e202Crossref PubMed Scopus (37) Google Scholar, 33.Silva B.A. Stambaugh J.R. Berns M.W. Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.J. Biomed. Opt. 2013; 18 (095003)Crossref Scopus (5) Google Scholar, 40.Duquette M.L. Zhu Q. Taylor E.R. Tsay A.J. Shi L.Z. Berns M.W. McGowan C.H. CtIP is required to initiate replication-dependent interstrand crosslink repair.PLoS Genet. 2012; 8: e1003050Crossref PubMed Scopus (28) Google Scholar). Briefly, the system uses a femtosecond pulsed titanium-sapphire near-infrared (NIR) laser (Coherent Inc., Santa Clara, CA) coupled to a motorized inverted Zeiss microscope (Axiovert 200 M) with a 37 °C culture dish stage (Warner Instruments, LLC). LabView software was developed for use with the automated microscope system and laser (41.Botvinick E.L. Berns M.W. Internet-based robotic laser scissors and tweezers microscopy.Microsc. Res. Tech. 2005; 68: 65-74Crossref PubMed Scopus (49) Google Scholar). Single chromosome tips (TIPs) and chromosome arms (non-TIPs) of live unsynchronized metaphase cells were microirradiated using the 200-fs pulse width NIR emission wavelength of 800 nm with a diffraction-limited (0.7-μm diameter) focal spot as shown previously (33.Silva B.A. Stambaugh J.R. Berns M.W. Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.J. Biomed. Opt. 2013; 18 (095003)Crossref Scopus (5) Google Scholar). Due to the ultrashort pulse duration, damage was confined to the focal spot (26.Ferrando-May E. Tomas M. Blumhardt P. Stöckl M. Fuchs M. Leitenstorfer A. Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling.Front. Genet. 2013; 4: 135Crossref PubMed Scopus (27) Google Scholar, 27.Gomez-Godinez V. Wu T. Sherman A.J. Lee C.S. Liaw L.H. Zhongsheng Y. Yokomori K. Berns M.W. Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation.Nucleic Acids Res. 2010; 38: e202Crossref PubMed Scopus (37) Google Scholar). Individual laser exposure (irradiation) to single TIP and non-TIP of cells was performed at a dose range of 2.43–2.65 ×10+11 watts/cm2. DNA breaks were assayed by using that DSB marker H2AX, which becomes phosphorylated on serine 139 (γ-H2AX) upon damage. In addition, to check the recruitment of DDR and repair factors at microirradiated DNA, eGFP-53BP1 PtK2 cells were used as controls. 5B. Alcarez Silva, J. R. Stambaugh, K. Yokomori, J. V. Shah, and M. W. Berns, unpublished data. DNA breaks were created in interphase cells. However, at irradiances above 2.65 ×10+11 watts/cm2, 53BP1 protein was not recruited, which may be due to optical breakdown and microscopic thermoelastic stress waves (28.Kong X. Mohanty S.K. Stephens J. Heale J.T. Gomez-Godinez V. Shi L.Z. Kim J.S. Yokomori K. Berns M.W. Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells.Nucleic Acids Res. 2009; 37: e68Crossref PubMed Scopus (168) Google Scholar). Thus, the lower irradiance (2.43 ×10+11 watts/cm2) was used for optimal mitotic protein recruitment and kinetics and has recently been shown to produce DSBs (33.Silva B.A. Stambaugh J.R. Berns M.W. Targeting telomere-containing chromosome ends with a near-infrared femtosecond laser to study the activation of the DNA damage response and DNA damage repair pathways.J. Biomed. Opt. 2013; 18 (095003)Crossref Scopus (5) Google Scholar). Cells grown on gridded dishes were fixed with 3% formaldehyde Tris-buffered saline (TBS) for 10 min at room temperature and placed on ice after microirradiation. Cells were washed three times in PBS and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Cells were later washed twice with PBS for 5 min at room temperature and incubated with blocking solution (10% calf serum, 1% BSA in PBS) for 1 h at room temperature. Cells were later washed once in PBS for 5 min at room temperature. Next, cells were incubated with primary antibodies in PBS with 3% BSA. After the incubation, cells were washed twice in PBS, 0.05% Tween 20 for 5 min at room temperature and incubated with secondary antibodies (1:1,000; Invitrogen) for 1 h at room temperature. Cells were washed twice with PBS, 0.05% Tween 20 for 5 min at room temperature, and DNA was stained with 4,6-diamidino-2-phenylindole (1:500 in PBS) for 5 min at room temperature. A final wash was performed with PBS for 5 min. Samples were visualized on a Zeiss inverted microscope (Axiovert 200 M) equipped with a Hamamatsu Orca cooled CCD camera. Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). Immunofluorescent staining was repeated at least five times for each antibody in TIP- and non-TIP-damaged cells, and consistent localization results were obtained with all of the cells examined (n > 5). Localization of the DDR signal at damaged TIPs was observed in all of the cells tested. The following antibodies were used: anti-γ-H2AX (07-164, Millipore) anti-Nbs1 (NB100-143, Novus Biological), anti-Mre11 (NB1000-142, Novus Biologicals), anti-ATM (NB100-104, Novus Biologicals), anti-FANCD2 (NB100-182, Novus Biologicals), anti-MDC1 (NB100-395, Novus Biologicals), anti-XRCC1 (NB100-532, Novus Biologicals), anti-WRN (ab200, Abcam), anti-CtIP (ab70163, Abcam), RNF8 (ab4183, Abcam), RNF168 (ab58063, Abcam), anti-γ-H2AX (9718, Cell Signaling), phospho-Chk1 Ser-345 (2348, Cell Signaling), phospho-Chk2 Thr-68 (2661, Cell Signaling), anti-proliferating cell nuclear antigen (PCNA) (2586, Cell Signaling), anti-GFP (2956, Cell Signaling), anti-Ku70-Ku80 (sc-71471, Santa Cruz Biotechnology, Inc.), phospho-p53 Ser-15 (sc-101762, Santa Cruz Biotechnology), anti-PARP1 (42.Heale J.T. Ball Jr., A.R. Schmiesing J.A. Kim J.S. Kong X. Zhou S. Hudson D.F. Earnshaw W.C. Yokomori K. Condensin I interacts with the PARP-1-XRCC1 complex and functions in DNA single-strand break repair.Mol. Cell. 2006; 21: 837-848Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), anti-ubiquitin (Ub) (spa-205, StressGen), and anti-BRCA1 (GTX50692, GeneTex). GFP-Nbs1 PtK1 mitotic cells grown in coverglass bottom 35-mm dishes (World Precision Instruments, Inc. Sarasot" @default.
• W1972300957 created "2016-06-24" @default.
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• W1972300957 date "2014-08-01" @default.
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• W1972300957 title "DNA Damage to a Single Chromosome End Delays Anaphase Onset" @default.
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