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Identification of Proteins at Active, Stalled, and Collapsed Replication Forks Using Isolation of Proteins on Nascent DNA (iPOND) Coupled with Mass Spectrometry*

Open AccessPublished:September 18, 2013DOI:https://doi.org/10.1074/jbc.M113.511337
      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.

      Introduction

      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)
      The abbreviations used are: ATR
      ataxia-telangiectasia- and Rad3-related
      iPOND
      isolation of proteins on nascent DNA
      EdU
      5-ethynyl-2′-deoxyuridine
      PCNA
      proliferating cell nuclear antigen
      ssDNA
      single-stranded DNA
      RFC
      replication factor C
      RPA
      replication protein A.
      protein kinase signaling pathway is a primary regulator of the replication stress response (
      • Cimprich K.A.
      • Cortez D.
      ATR. An essential regulator of genome integrity.
      ). 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 (
      • Cortez D.
      • Guntuku S.
      • Qin J.
      • Elledge S.J.
      ATR and ATRIP. Partners in checkpoint signaling.
      ,
      • Kumagai A.
      • Lee J.
      • Yoo H.Y.
      • Dunphy W.G.
      TopBP1 activates the ATR-ATRIP complex.
      ,
      • Mordes D.A.
      • Glick G.G.
      • Zhao R.
      • Cortez D.
      TopBP1 activates ATR through ATRIP and a PIKK regulatory domain.
      ,
      • 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.
      ). 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 (
      • 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.
      ).
      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 (
      • 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.
      ,
      • 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.
      ). 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 (
      • 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.
      ). 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 (
      • 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.
      ,
      • Salic A.
      • Mitchison T.J.
      A chemical method for fast and sensitive detection of DNA synthesis in vivo.
      ). 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.

      DISCUSSION

      Coupling iPOND with two-dimensional LC-MS/MS is a powerful discovery tool. We identified 290 proteins at active, stalled, and collapsed forks. Providing validation of the approach, the dataset is highly enriched in proteins known to function in DNA damage responses, cell cycle control, DNA repair, and replication. For example, at normally elongating replication forks, 15 of the top 20 proteins, as measured by fold enrichment and p value, are established replisome components and chromatin replication factors. These include the replicative polymerases, PCNA, the replication-loading complex RFC (RFC1–5), and the chromatin assembly factors CAF1A and CAF1B. The stalled fork dataset enriched for DNA damage response proteins above a random chance of occurrence. Collapsed replication forks exhibited strong enrichment of RPA and RPA-interacting proteins, double-strand break repair proteins, and fork-remodeling helicases.
      While this manuscript was in preparation, the Fernadez-Capetillo group (
      • Lopez-Contreras A.J.
      • Ruppen I.
      • Nieto-Soler M.
      • Murga M.
      • Rodriguez-Acebes S.
      • Remeseiro S.
      • Rodrigo-Perez S.
      • Rojas A.M.
      • Mendez J.
      • Muñoz J.
      • Fernandez-Capetillo O.
      A proteomic characterization of factors enriched at nascent DNA molecules.
      ) completed an iPOND-MS study only looking at proteins enriched at active forks. They identified many of the same replisome components, including ATAD5, BAZ1B, CHAF1A, CHAF1B, DNMT1, EXO1, LIG1, MSH2, MSH3, MSH6, PCNA, POLD1, POLE, RFC1–5, UHRF1, and WIZ. They also identified the MCM helicase complex, which was not enriched in our datasets. In immunoblotting experiments we have observed variable results in detecting the MCM proteins. We suspect that the differences are due to how much cleavage of the ssDNA at the fork happens during the iPOND processing. The MCM proteins function to unwind parental DNA and are not directly associated with newly synthesized, EdU-labeled DNA. Thus, detection of the MCM helicase would rely on purifying larger fragments of DNA containing both nascent and parental strands. Other differences in methodologies may further explain differences in the datasets. Most notably, we used ∼10-fold less cells in our samples and examined hydroxyurea-stalled and hydroxyurea/ATR inhibitor-induced collapsed forks in addition to active forks. The decreased amount of starting material may also explain why many known replication and stress response proteins were not identified. Nonetheless, both datasets provide useful resources for investigators interested in replication and replication stress responses. Finally, we would caution that although we applied stringent criteria for protein identification and enrichment, further validation of the candidate proteins is required, especially in cases with higher p values and lower enrichment scores.
      The mismatch repair proteins MSH2, MSH3, and MSH6 were some of the most highly enriched proteins at unperturbed replication forks. The high level of enrichment of mismatch repair proteins is unlikely to be due to the need to remove true mismatches because the polymerase error rate is low. More likely, the mismatch repair proteins are scanning for errors in conjunction with replication, as shown recently for the yeast MMR system (
      • Hombauer H.
      • Srivatsan A.
      • Putnam C.D.
      • Kolodner R.D.
      Mismatch repair, but not heteroduplex rejection, is temporally coupled to DNA replication.
      ), or possibly involved in removing ribonucleotides from the DNA (
      • Lujan S.A.
      • Williams J.S.
      • Clausen A.R.
      • Clark A.B.
      • Kunkel T.A.
      Ribonucleotides are signals for mismatch repair of leading-strand replication errors.
      ). It is also possible that the MMR proteins may recognize EdU-labeled DNA. However, any DNA damage because of EdU incorporation does not activate a DNA damage signaling pathway in the time frame of these experiments, and very little (if any) of the EdU is removed from the DNA because we do not observe a decrease in chromatin capture after growing cells for hours after the EdU labeling (
      • 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.
      ).
      The FANCI and FANCD2 proteins are highly enriched at stalled and collapsed replication forks. FANCI was also detected at active forks. FANCI and FANCD2 function during interstrand cross-link repair (
      • Ciccia A.
      • Elledge S.J.
      The DNA damage response. Making it safe to play with knives.
      ). These lesions are some of the most difficult-to-repair substrates, requiring specialized repair mechanisms governed by genes mutated in patients with Fanconi anemia as well as components of nucleotide excision and double-strand break repair (
      • Kim H.
      • D'Andrea A.D.
      Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway.
      ,
      • Sirbu B.M.
      • Cortez D.
      DNA damage response. Three levels of DNA repair regulation.
      ). FANCD2 is ubiquitylated in response to hydroxyurea and even as cells enter a normal S phase. Thus, the FANCI/FANCD2 may recognize DNA structures generated during replication stress, such as ssDNA-dsDNA junctions (
      • Joo W.
      • Xu G.
      • Persky N.S.
      • Smogorzewska A.
      • Rudge D.G.
      • Buzovetsky O.
      • Elledge S.J.
      • Pavletich N.P.
      Structure of the FANCI-FANCD2 complex. Insights into the Fanconi anemia DNA repair pathway.
      ), and these proteins may have functions outside of cross-link repair. Indeed, FANCD2 promotes the restart of aphidicolin-stalled replication forks (
      • Chaudhury I.
      • Sareen A.
      • Raghunandan M.
      • Sobeck A.
      FANCD2 regulates BLM complex functions independently of FANCI to promote replication fork recovery.
      ). Alternatively, it is possible that the FANCI and FANCD2 proteins were identified because of a small amount of continued EdU incorporation during the beginning of the formaldehyde fixation. If this were the case, it might create protein cross-links to the DNA that could be recognized by the Fanconi proteins (
      • Langevin F.
      • Crossan G.P.
      • Rosado I.V.
      • Arends M.J.
      • Patel K.J.
      Fancd2 counteracts the toxic effects of naturally produced aldehydes in mice.
      ).
      The high-level enrichment of the heterotrimeric ssDNA-binding protein RPA is a striking feature of stalled replication forks that collapse after ATR inhibition. Concomitant with RPA accumulation, we observed enrichment of the disease-associated helicases BLM, CHD1L, SMARCAL1, and WRN as well as many other RPA interacting proteins. These data are consistent with the recent observation that ATR inhibition causes the extensive production of nascent-strand ssDNA at replication forks through a process involving fork reversal, enzymatic cleavage, and end resection (
      • 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.
      ).
      Finally, our data confirm important functions for chromatin remodeling enzymes, including SNF2L and SNF2H, at replication forks. The highly related SNF2H and SNF2L chromatin remodelers are the motor enzymes of ISWI complexes (
      • Struhl K.
      • Segal E.
      Determinants of nucleosome positioning.
      ). In complex with BAZ1B, SNF2H is recruited to replication forks via an interaction with PCNA to maintain the chromatin landscape through DNA replication (
      • Poot R.A.
      • Bozhenok L.
      • van den Berg D.L.
      • Steffensen S.
      • Ferreira F.
      • Grimaldi M.
      • Gilbert N.
      • Ferreira J.
      • Varga-Weisz P.D.
      The Williams syndrome transcription factor interacts with PCNA to target chromatin remodelling by ISWI to replication foci.
      ). The activity of SNF2L at replication forks has not been described, but our data indicate that it must have an important non-redundant function that, perhaps, is especially needed in the context of replication stress.
      Collectively, our data indicate that iPOND can be combined with mass spectrometry to provide a powerful discovery approach. In addition to analyzing normal, stalled, and collapsed forks, there are many other instances in which iPOND-MS analysis would be useful to understand DNA repair, replication, and chromatin biology.

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