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• W2010160165 abstract "Both DNA and chromatin need to be duplicated during each cell division cycle. Replication happens in the context of defects in the DNA template and other forms of replication stress that present challenges to both genetic and epigenetic inheritance. The replication machinery is highly regulated by replication stress responses to accomplish this goal. To identify important replication and stress response proteins, we combined isolation of proteins on nascent DNA (iPOND) with quantitative mass spectrometry. We identified 290 proteins enriched on newly replicated DNA at active, stalled, and collapsed replication forks. Approximately 16% of these proteins are known replication or DNA damage response proteins. Genetic analysis indicates that several of the newly identified proteins are needed to facilitate DNA replication, especially under stressed conditions. Our data provide a useful resource for investigators studying DNA replication and the replication stress response and validate the use of iPOND combined with mass spectrometry as a discovery tool.Background: DNA replication and the replication stress response require the coordinated actions of many proteins.Results: iPOND coupled with mass spectrometry identified 290 proteins associated with active, stalled, or collapsed replication forks.Conclusion: iPOND-MS is a useful discovery tool.Significance: The data increase our understanding of the network of proteins involved in DNA replication and the replication stress response. Both DNA and chromatin need to be duplicated during each cell division cycle. Replication happens in the context of defects in the DNA template and other forms of replication stress that present challenges to both genetic and epigenetic inheritance. The replication machinery is highly regulated by replication stress responses to accomplish this goal. To identify important replication and stress response proteins, we combined isolation of proteins on nascent DNA (iPOND) with quantitative mass spectrometry. We identified 290 proteins enriched on newly replicated DNA at active, stalled, and collapsed replication forks. Approximately 16% of these proteins are known replication or DNA damage response proteins. Genetic analysis indicates that several of the newly identified proteins are needed to facilitate DNA replication, especially under stressed conditions. Our data provide a useful resource for investigators studying DNA replication and the replication stress response and validate the use of iPOND combined with mass spectrometry as a discovery tool. Background: DNA replication and the replication stress response require the coordinated actions of many proteins. Results: iPOND coupled with mass spectrometry identified 290 proteins associated with active, stalled, or collapsed replication forks. Conclusion: iPOND-MS is a useful discovery tool. Significance: The data increase our understanding of the network of proteins involved in DNA replication and the replication stress response. Chromosomal replication requires the coordinated action of a large molecular machine, called the replisome, consisting of multiple subunits, including helicases, polymerases, histone chaperones, and chromatin-modifying enzymes. The replisome must work with speed and precision to replicate the DNA and chromatin during each cell division cycle. Damage to the DNA template from endogenous and environmental genotoxins, depletion of nucleotide precursors, and even difficult-to-replicate DNA sequences can impede replication fork progression. Multiple mechanisms respond to this stress to repair the damaged DNA, signal checkpoint activation, ensure the completion of DNA replication, and maintain genome stability. Defects in replication stress response mechanisms cause diseases that are characterized by developmental abnormalities, premature aging, and cancer predisposition. The ataxia-telangiectasia- and Rad3-related (ATR) 2The abbreviations used are: ATRataxia-telangiectasia- and Rad3-relatediPONDisolation of proteins on nascent DNAEdU5-ethynyl-2′-deoxyuridinePCNAproliferating cell nuclear antigenssDNAsingle-stranded DNARFCreplication factor CRPAreplication protein A. protein kinase signaling pathway is a primary regulator of the replication stress response (1.Cimprich K.A. Cortez D. ATR. An essential regulator of genome integrity.Nat. Rev. Mol. Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1320) Google Scholar). A complex of ATR and its obligate partner ATRIP is activated by interactions with TOPBP1 when DNA polymerase and helicase activities at the replication fork are uncoupled (2.Cortez D. Guntuku S. Qin J. Elledge S.J. ATR and ATRIP. Partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (748) Google Scholar, 3.Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. TopBP1 activates the ATR-ATRIP complex.Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar, 4.Mordes D.A. Glick G.G. Zhao R. Cortez D. TopBP1 activates ATR through ATRIP and a PIKK regulatory domain.Genes Dev. 2008; 22: 1478-1489Crossref PubMed Scopus (260) Google Scholar, 5.Byun T.S. Pacek M. Yee M.C. Walter J.C. Cimprich K.A. Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.Genes Dev. 2005; 19: 1040-1052Crossref PubMed Scopus (569) Google Scholar). Activated ATR stabilizes the stalled fork, promotes fork restart, and regulates cell cycle checkpoints to ensure completion of DNA synthesis prior to mitosis. If ATR is not functional, then forks collapse into double-strand breaks because of the action of unregulated fork remodeling and nuclease activities (6.Couch F.B. Bansbach C.E. Driscoll R. Luzwick J.W. Glick G.G. Bétous R. Carroll C.M. Jung S.Y. Qin J. Cimprich K.A. Cortez D. ATR phosphorylates SMARCAL1 to prevent replication fork collapse.Genes Dev. 2013; 27: 1610-1623Crossref PubMed Scopus (267) Google Scholar). ataxia-telangiectasia- and Rad3-related isolation of proteins on nascent DNA 5-ethynyl-2′-deoxyuridine proliferating cell nuclear antigen single-stranded DNA replication factor C replication protein A. The continued high rate of discovery of new replication stress response proteins suggests that our inventory of replication regulators remains incomplete. Thus, identifying proteins that function at active and damaged replication forks and characterizing how they work in a coordinated fashion to maintain genome integrity remain critically important research goals. We recently developed a technology called isolation of proteins on nascent DNA (iPOND) that can be used to track protein recruitment to active and damaged replication forks as well as study the processes of chromatin deposition and maturation (7.Sirbu B.M. Couch F.B. Cortez D. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA.Nat. Protoc. 2012; 7: 594-605Crossref PubMed Scopus (129) Google Scholar, 8.Sirbu B.M. Couch F.B. Feigerle J.T. Bhaskara S. Hiebert S.W. Cortez D. Analysis of protein dynamics at active, stalled, and collapsed replication forks.Genes Dev. 2011; 25: 1320-1327Crossref PubMed Scopus (300) Google Scholar). Importantly, the technique provides high resolution and sensitivity and is compatible with unbiased approaches such as mass spectrometry. iPOND uses the nucleoside analog 5-ethynyl-2′-deoxyuridine (EdU) and click chemistry (8.Sirbu B.M. Couch F.B. Feigerle J.T. Bhaskara S. Hiebert S.W. Cortez D. Analysis of protein dynamics at active, stalled, and collapsed replication forks.Genes Dev. 2011; 25: 1320-1327Crossref PubMed Scopus (300) Google Scholar). EdU is rapidly incorporated into newly synthesized DNA when added to cell culture medium and does not interfere with replication or cause detectable DNA damage when used in short term cell culture (8.Sirbu B.M. Couch F.B. Feigerle J.T. Bhaskara S. Hiebert S.W. Cortez D. Analysis of protein dynamics at active, stalled, and collapsed replication forks.Genes Dev. 2011; 25: 1320-1327Crossref PubMed Scopus (300) Google Scholar, 9.Salic A. Mitchison T.J. A chemical method for fast and sensitive detection of DNA synthesis in vivo.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2415-2420Crossref PubMed Scopus (1329) Google Scholar). An alkyne functional group on EdU can be reacted with an azide linked to biotin using click chemistry. This facilitates a streptavidin-biotin method of purification of the EdU-labeled nascent DNA with associated proteins. Fixation of cells with a reversible cross-linking agent prior to click chemistry and cell lysis permits purification under denaturing conditions, making a single-step isolation procedure possible. Cross-link reversal separates the proteins from the DNA fragments, which can then be detected by immunoblotting or mass spectrometry. Here we coupled iPOND to unbiased shotgun proteomics to probe the changes in replisome composition at active, stalled, and collapsed replication forks. iPOND was performed largely as described previously (7.Sirbu B.M. Couch F.B. Cortez D. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA.Nat. Protoc. 2012; 7: 594-605Crossref PubMed Scopus (129) Google Scholar), with the following modifications. 500 ml of logarithmically growing (3.3 × 106 cells/ml) suspension of 293T cells (a total of 1.6 × 109 cells) were labeled with 12 μm EdU for 15 min. An EdU labeling period of this length may label ∼15–20 kb of DNA depending on the rate of polymerization and how rapidly EdU is imported into the cell and phosphorylated by thymidine kinase (8.Sirbu B.M. Couch F.B. Feigerle J.T. Bhaskara S. Hiebert S.W. Cortez D. Analysis of protein dynamics at active, stalled, and collapsed replication forks.Genes Dev. 2011; 25: 1320-1327Crossref PubMed Scopus (300) Google Scholar). Following EdU incorporation, the stalled fork sample was incubated in 3 mm of hydroxyurea for 2 h, and the collapsed fork sample was treated with 3 mm hydroxyurea and 3 μm of ATR inhibitor for 2 h to induce fork collapse (10.Reaper P.M. Griffiths M.R. Long J.M. Charrier J.D. Maccormick S. Charlton P.A. Golec J.M. Pollard J.R. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR.Nat. Chem. Biol. 2011; 7: 428-430Crossref PubMed Scopus (460) Google Scholar). After EdU labeling, the thymidine chase sample was centrifuged at 1000 rpm for 4 min, medium was decanted, and cells were resuspended in medium equilibrated for temperature and pH containing 10 μm thymidine. The thymidine chase was conducted for 60 min. All samples were fixed with 1% formaldehyde for 20 min at room temperature, followed by 5-min incubation with 0.125 m glycine to quench the formaldehyde. Fixed samples were split evenly into six 50-ml conical tubes, centrifuged at 2000 rpm at 4 °C for 6 min, washed three times with PBS, and frozen at −80 °C. Five of the six tubes were processed independently on a scale of 2.7 × 108 cells/sample for iPOND purifications. Briefly, click chemistry reactions were performed to conjugate biotin to the EdU-labeled DNA. Streptavidin beads were used to capture the biotin-conjugated DNA-protein complexes. Captured complexes were washed extensively using SDS and high-salt wash buffers. Purified replication fork proteins were eluted under reducing conditions by boiling in 2× SDS sample buffer for 25 min. One-sixth of the eluted protein sample was resolved 1 cm into a 10% Novex precast gel (Invitrogen), excised from the gel slice, alkylated, and in-gel trypsin-digested using standard procedures. Recovered tryptic peptides were subjected to two-dimensional LC-MS/MS (multidimensional protein identification technology) separation as described previously (11.MacCoss M.J. McDonald W.H. Saraf A. Sadygov R. Clark J.M. Tasto J.J. Gould K.L. Wolters D. Washburn M. Weiss A. Clark J.I. Yates 3rd., J.R. Shotgun identification of protein modifications from protein complexes and lens tissue.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 7900-7905Crossref PubMed Scopus (548) Google Scholar). Briefly, digested peptides were separated by a combined strong cation exchange and reversed-phase chromatographic strategy. Subsets of peptides were eluted from the strong cation exchange onto the reverse phase using a series of ammonium acetate pulses of increasing concentration. This was performed for eight steps, each followed by a 105-min aqueous to organic separation on the reversed-phase column. Eluted peptides were directly nanoelectrospray-ionized and introduced into an LTQ-XL mass spectrometer (Thermo Fisher Scientific) where peptide tandem mass spectra (MS/MS) were collected in a data-dependent manner. The peptide spectral data were searched against the canonical human proteome subset of UniProtKB (v. 155) using the Myrimatch (v. 1.6.75) (12.Tabb D.L. Fernando C.G. Chambers M.C. MyriMatch. Highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis.J. Proteome Res. 2007; 6: 654-661Crossref PubMed Scopus (447) Google Scholar), Sequest (v. 27) (13.Yates 3rd, J.R. Eng J.K. McCormack A.L. Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1110) Google Scholar), and Myrimatch and Sequest (14.Chen Y.Y. Dasari S. Ma Z.Q. Vega-Montoto L.J. Li M. Tabb D.L. Refining comparative proteomics by spectral counting to account for shared peptides and multiple search engines.Anal. Bioanal. Chem. 2012; 404: 1115-1125Crossref PubMed Scopus (6) Google Scholar) database search engines. Protein groups were assembled using IDPicker, which uses parsimony to report the minimum number of confident protein identifications (15.Ma Z.Q. Dasari S. Chambers M.C. Litton M.D. Sobecki S.M. Zimmerman L.J. Halvey P.J. Schilling B. Drake P.M. Gibson B.W. Tabb D.L. IDPicker 2.0. Improved protein assembly with high discrimination peptide identification filtering.J. Proteome Res. 2009; 8: 3872-3881Crossref PubMed Scopus (268) Google Scholar). Matched peptides were filtered at a 5% peptide and protein false discovery rate, and each protein required a minimum of two independent peptides for identification. Protein identifiers were converted to EntrezID unique identifiers using the UniProt ID mapping database (16.UniProt C. Reorganizing the protein space at the Universal Protein Resource (UniProt).Nucleic Acids Res. 2012; 40: D71-D75Crossref PubMed Scopus (1100) Google Scholar) and the DAVID bioinformatics database (17.Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25496) Google Scholar). To determine fold enrichments of proteins relative to the negative controls, spectral count data were imported into the statistical software program QuasiTel (18.Li M. Gray W. Zhang H. Chung C.H. Billheimer D. Yarbrough W.G. Liebler D.C. Shyr Y. Slebos R.J. Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.J. Proteome Res. 2010; 9: 4295-4305Crossref PubMed Scopus (86) Google Scholar) for pairwise comparisons. QuasiTel applies a quasi-likelihood model to raw spectral count data and reports protein fold enrichment and statistical significance as a quasi p value. Spectral count data are normalized for each multidimensional protein identification technology run using the total number of spectra reported for the run. The threshold for spectral counts was set at an average of one spectral count per experimental sample. For example, when comparing the five replicates from the replication fork sample to the five replicates from the chromatin chase sample, a minimum of 10 total spectral counts was required from the 10 samples for QuasiTel comparisons. Furthermore, to be considered a protein significantly enriched on nascent DNA, the filtering criteria required a minimum of 1.5-fold enrichment above both of the negative controls and a quasi p value of ≤ 0.05. These filtering criteria were applied to proteins identified using each of the three protein identification search algorithms (Myrimatch plus Sequest, Myrimatch alone, and Sequest alone). Therefore, three lists of enriched proteins were generated independently. The final data reported in supplemental Tables S1–S3 represent the union of all three lists and report the median fold enrichment relative to the chromatin-bound negative control, median p value, and median spectral counts. The median p value, in some cases, is > 0.05 because three independent p values were calculated by QuasiTel for each protein identified by the three different search algorithm methods. If any one of the analyses yielded a p value < 0.05, that protein is reported in supplemental Tables S1–S3 along with the median p value from the three analyses. It should also be noted that when no spectra were detected in the thymidine chase negative control, QuasiTel calculated relative fold enrichment using a small, non-zero value in the denominator. This factor may lead to an overestimation of protein enrichment. Although these values are included in supplemental Tables S1–S3, they are omitted from FIGURE 2, FIGURE 3, FIGURE 4.FIGURE 3iPOND-MS identifies proteins at stalled replication forks. A, the fold enrichment relative to the thymidine chase negative control, the p value, and the spectral count data are depicted for the proteins listed in supplemental Table S2. B, proteins that contain PCNA-interacting motifs (24.Gilljam K.M. Feyzi E. Aas P.A. Sousa M.M. Müller R. Vågbø C.B. Catterall T.C. Liabakk N.B. Slupphaug G. Drabløs F. Krokan H.E. Otterlei M. Identification of a novel, widespread, and functionally important PCNA-binding motif.J. Cell Biol. 2009; 186: 645-654Crossref PubMed Scopus (119) Google Scholar) or ATM/ATR phosphorylation sites (25.Matsuoka S. Ballif B.A. Smogorzewska A. McDonald 3rd, E.R. Hurov K.E. Luo J. Bakalarski C.E. Zhao Z. Solimini N. Lerenthal Y. Shiloh Y. Gygi S.P. Elledge S.J. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.Science. 2007; 316: 1160-1166Crossref PubMed Scopus (2361) Google Scholar, 26.Bensimon A. Schmidt A. Ziv Y. Elkon R. Wang S.Y. Chen D.J. Aebersold R. Shiloh Y. ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage.Sci. Signal. 2010; 3: rs3Crossref PubMed Scopus (227) Google Scholar) or that cause increased DNA damage signaling when silenced by siRNA (27.Lovejoy C.A. Xu X. Bansbach C.E. Glick G.G. Zhao R. Ye F. Sirbu B.M. Titus L.C. Shyr Y. Cortez D. Functional genomic screens identify CINP as a genome maintenance protein.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19304-19309Crossref PubMed Scopus (44) Google Scholar, 28.Paulsen R.D. Soni D.V. Wollman R. Hahn A.T. Yee M.C. Guan A. Hesley J.A. Miller S.C. Cromwell E.F. Solow-Cordero D.E. Meyer T. Cimprich K.A. A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability.Mol. Cell. 2009; 35: 228-239Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar) are listed.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 2iPOND-MS identifies proteins enriched at active replication forks. A, the fold enrichment relative to the thymidine chase negative control, the p value, and the spectral count data are depicted for the proteins listed in supplemental Table S1. The dot size indicates the total number of MS spectra counted in the normal replication fork samples from the five replicate purifications. The dot color represents the median p value as calculated using QuasiTel. B, protein network analyses used GeneMANIA predictions to probe the physical interactions within the normal replication fork dataset. Gene not in query refers to proteins known to physically interact with other proteins in the depicted physical interaction network but that were not identified in the iPOND-MS screen. C, proteins that contain PCNA-interacting motifs (24.Gilljam K.M. Feyzi E. Aas P.A. Sousa M.M. Müller R. Vågbø C.B. Catterall T.C. Liabakk N.B. Slupphaug G. Drabløs F. Krokan H.E. Otterlei M. Identification of a novel, widespread, and functionally important PCNA-binding motif.J. Cell Biol. 2009; 186: 645-654Crossref PubMed Scopus (119) Google Scholar) or ATM/ATR phosphorylation sites (25.Matsuoka S. Ballif B.A. Smogorzewska A. McDonald 3rd, E.R. Hurov K.E. Luo J. Bakalarski C.E. Zhao Z. Solimini N. Lerenthal Y. Shiloh Y. Gygi S.P. Elledge S.J. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage.Science. 2007; 316: 1160-1166Crossref PubMed Scopus (2361) Google Scholar, 26.Bensimon A. Schmidt A. Ziv Y. Elkon R. Wang S.Y. Chen D.J. Aebersold R. Shiloh Y. ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage.Sci. Signal. 2010; 3: rs3Crossref PubMed Scopus (227) Google Scholar) or that cause increased DNA damage signaling when silenced by siRNA (27.Lovejoy C.A. Xu X. Bansbach C.E. Glick G.G. Zhao R. Ye F. Sirbu B.M. Titus L.C. Shyr Y. Cortez D. Functional genomic screens identify CINP as a genome maintenance protein.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19304-19309Crossref PubMed Scopus (44) Google Scholar, 28.Paulsen R.D. Soni D.V. Wollman R. Hahn A.T. Yee M.C. Guan A. Hesley J.A. Miller S.C. Cromwell E.F. Solow-Cordero D.E. Meyer T. Cimprich K.A. A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability.Mol. Cell. 2009; 35: 228-239Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar) are listed.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Proteins identified at elongating, stalled, and collapsed replication forks were classified on the basis of gene ontology using ToppGene (19.Chen J. Bardes E.E. Aronow B.J. Jegga A.G. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization.Nucleic Acids Res. 2009; 37: W305-W311Crossref PubMed Scopus (1785) Google Scholar). To display median fold enrichment relative to the thymidine chase negative control, median quasi p value, and median spectral counts from the experimental sample were graphed using R. Protein network modeling was performed using the GeneMANIA prediction server (20.Warde-Farley D. Donaldson S.L. Comes O. Zuberi K. Badrawi R. Chao P. Franz M. Grouios C. Kazi F. Lopes C.T. Maitland A. Mostafavi S. Montojo J. Shao Q. Wright G. Bader G.D. Morris Q. The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function.Nucleic Acids Res. 2010; 38: W214-W220Crossref PubMed Scopus (2444) Google Scholar). The antibodies used were as follows: H2A, H2B, MSH2, and SNF2H (Abcam); H1 (Millipore); PCNA (Santa Cruz Biotechnology); SNF2L (Cell Signaling Technology); BAZ1B (Novus). Four individual siRNAs for each of the genes arrayed in 384-well dishes were transfected into U2OS cells at 10 nm final siRNA concentrations. Three days after transfection, cells were treated with 2 mm hydroxyurea for 24 h. Hydroxyurea was removed, and cells were incubated with 10 μm EdU for 4 h. Cells were then fixed with paraformaldehyde and processed with Alexa Fluor 488-coupled biotin azide followed by labeling with antibodies to γH2AX as described previously (21.Bansbach C.E. Bétous R. Lovejoy C.A. Glick G.G. Cortez D. The annealing helicase SMARCAL1 maintains genome integrity at stalled replication forks.Genes Dev. 2009; 23: 2405-2414Crossref PubMed Scopus (174) Google Scholar). Images were obtained on a PerkinElmer Life Sciences Opera automated microscope, and the intensities of EdU and γH2AX per nucleus were quantitated by Columbus image analysis software. The ratio of γH2AX to EdU intensities was used as the final scoring criterion. Samples with elevated ratios were identified using the Wilcoxon rank-sum test requiring a false discovery rate-adjusted p value of < 0.001 and a ratio of at least 2.0. As a comparison, the average ratio for the negative control siRNA was 1.07 with an S.E. of 0.026. To identify proteins associated with nascent DNA at active, stalled, and collapsed replication forks, we coupled iPOND purifications to mass spectrometry. Five samples were prepared for iPOND-MS (Fig. 1A). For all samples, cells were treated for 15 min with EdU to label nascent DNA. To examine proteins at active replication forks, EdU-labeled cells were collected immediately. To monitor proteins associated with stalled replication forks, EdU-labeled cells were treated with 3 mm hydroxyurea for 2 h to arrest fork movement and induce a replication stress response. To identify proteins associated with fork collapse, cells were treated with hydroxyurea and a selective ATR inhibitor (10.Reaper P.M. Griffiths M.R. Long J.M. Charrier J.D. Maccormick S. Charlton P.A. Golec J.M. Pollard J.R. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR.Nat. Chem. Biol. 2011; 7: 428-430Crossref PubMed Scopus (460) Google Scholar) for 2 h. These conditions elicit fork collapse, including accumulation of double-strand breaks and excess single-stranded DNA (ssDNA) at the replication fork (6.Couch F.B. Bansbach C.E. Driscoll R. Luzwick J.W. Glick G.G. Bétous R. Carroll C.M. Jung S.Y. Qin J. Cimprich K.A. Cortez D. ATR phosphorylates SMARCAL1 to prevent replication fork collapse.Genes Dev. 2013; 27: 1610-1623Crossref PubMed Scopus (267) Google Scholar). EdU remained in the growth media during the hydroxyurea treatments. The specificity of replication fork protein purifications was tested relative to two negative controls. The first were cells treated identically to the normal replication fork sample, but the biotin azide was omitted during the iPOND procedure. Proteins purified in this “no click reaction” sample represent those that interact nonspecifically with streptavidin-conjugated beads. For the second negative control, cells labeled with EdU were washed and then incubated with medium containing a small amount of thymidine for 1 h. This procedure allows replication to continue without additional EdU incorporation. The small concentration of thymidine does not interfere with replication but is used to compete for whatever EdU is left in the cell after removing it from the growth medium. Thus, this negative control monitors proteins bound to mature chromatin that are no longer close to the replication fork. Proteins detected in this “thymidine chase” sample represent chromatin-bound proteins that are not specifically enriched at replisomes (8.Sirbu B.M. Couch F.B. Feigerle J.T. Bhaskara S. Hiebert S.W. Cortez D. Analysis of protein dynamics at active, stalled, and collapsed replication forks.Genes Dev. 2011; 25: 1320-1327Crossref PubMed Scopus (300) Google Scholar). To test the relative enrichment of replication proteins in the samples submitted for mass spectrometry analyses, iPOND purifications were examined for PCNA levels. As observed previously, PCNA was detected at elongating replication forks, and its levels decreased in the thymidine chase sample (Fig. 1B). Although still detectable, PCNA levels at stalled and collapsed replication forks were also decreased compared with the active fork sample, likely because of unloading of PCNA from the mature Okazaki fragments (8.Sirbu B.M. Couch F.B. Feigerle J.T. Bhaskara S. Hiebert S.W. Cortez D. Analysis of protein dynamics at active, stalled, and collapsed replication forks.Genes Dev. 2011; 25: 1320-1327Crossref PubMed Scopus (300) Google Scholar). The equal level of histone H2B detected on isolated chromatin (Fig. 1B) indicates that an equivalent amount of EdU-labeled DNA was purified in each sample. The five experimental samples were purified independently five times each using the iPOND procedure (Fig. 1C). Eluted proteins were analyzed using two-dimensional liquid chromatography coupled with tandem mass spectrometry (multidimensional protein identification technology). The MS/MS spectra were matched to the human protein database using the Myrimatch and Sequest search engines (12.Tabb D.L. Fernando C.G. Chambers M.C. MyriMatch. Highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis.J. Proteome Res. 2007; 6: 654-661Crossref PubMed Scopus (447) Google Scholar, 13.Yates 3rd, J.R. Eng J.K. McCormack A.L. Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.Anal. Chem. 1995; 67: 1426-1436Crossref PubMed Scopus (1110) Google Scholar, 14.Chen Y.Y. Dasari S. Ma Z.Q. Vega-Montoto L.J. Li M. Tabb D.L. Refining comparative proteomics by spectral counting to account for shared peptides and multiple search engines.Anal. Bioanal. Chem. 2012; 404: 1115-1125Crossref PubMed Scopus (6) Google Scholar). QuasiTel was used to compute fold enrichment values of each experimental sample relative to both of the negative control samples (18.Li M. Gray W. Zhang H. Chung C.H. Billheimer D. Yarbrough W.G. Liebler D.C. Shyr Y. Slebos R.J. Comparative shotgun proteomics using spectral count data and quasi-likelihood modeling.J. Proteome Res. 2010; 9: 4295-4305Crossref PubMed Scopus (86) Google Scholar). The final lists include proteins enriched at least 1.5-fold (relative to both negative controls) with p values from at least one of the search engines yielding a p value ≤ to 0.05 as computed by QuasiTel. These filtering criteria led to the identification of a total of 29" @default.
• W2010160165 created "2016-06-24" @default.
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• W2010160165 date "2013-11-01" @default.
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• W2010160165 title "Identification of Proteins at Active, Stalled, and Collapsed Replication Forks Using Isolation of Proteins on Nascent DNA (iPOND) Coupled with Mass Spectrometry" @default.
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