Report |
Address correspondence to E.S. Knudsen, Dept. of Cell Biology, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Ave., Cincinnati, OH 45267. Tel.: (513) 558-8885. Fax: (513) 558-4454. email: erik.knudsen{at}uc.edu; or D.A. Solomon, email: das67{at}georgetown.edu
Abstract
Components of the DNA replication machinery localize into discrete subnuclear foci after DNA damage, where they play requisite functions in repair processes. Here, we find that the replication factors proliferating cell nuclear antigen (PCNA) and RPAp34 dynamically exchange at these repair foci with discrete kinetics, and this behavior is distinct from kinetics during DNA replication. Posttranslational modification is hypothesized to target specific proteins for repair, and we find that accumulation and stability of PCNA at sites of damage requires monoubiquitination. Contrary to the popular notion that phosphorylation on the NH2 terminus of RPAp34 directs the protein for repair, we demonstrate that phosphorylation by DNA-dependent protein kinase enhances RPAp34 turnover at repair foci. Together, these findings support a dynamic exchange model in which multiple repair factors regulated by specific modifications have access to and rapidly turn over at sites of DNA damage.
Key Words: proliferating cell nuclear antigen; replication protein A; cisplatin; FRAP; foci
Abbreviations used in this paper: APH, aphidicolin; CDDP, cisplatin or cis-diamminedichloroplatinum II; DNA-PK, DNA-dependent protein kinase; HU, hydroxyurea; PCNA, proliferating cell nuclear antigen.
Introduction
The cellular response to DNA damage requires a wide range of protein factors to ensure the fidelity of genetic material passed on to daughter cells. These factors include detection and signaling proteins to warn that damage has occurred, checkpoint response proteins to inhibit cell division allowing time for repair, and the plethora of proteins that function in the repair of genetic lesions (Rouse and Jackson, 2002). Apart from its function in the replication of the genome, the DNA replication machinery plays an essential role in the repair of damaged DNA. The heterotrimeric replication protein A (RPA) complex has been implicated in the damage detection process, whereas factors including proliferating cell nuclear antigen (PCNA), DNA polymerases, and DNA ligases are known to be important in the resynthesis of excised nucleotide sequences (Shivji et al., 1992; He et al., 1995; Zou and Elledge, 2003). Though DNA replication and repair both involve DNA synthesis, we speculated that classical replication proteins might behave distinctly during these two processes and that discrete signaling pathways might underlie these differences.
Results and discussion
To investigate the action of specific replication factors during DNA replication and repair in living cells, we generated Rat-1 and U2OS cell lines stably expressing GFP fused to the replication proteins PCNA and RPAp34 (the 34-kD subunit of the replication protein A complex, also termed RPA2). The GFP-PCNA fusion protein colocalized with sites of BrdU incorporation in S-phase cells and was homogeneously distributed throughout the nucleoplasm during other phases of the cell cycle (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200312048/DC1). GFP-RPAp34 displayed similar localization but was more difficult to detect at sites of replication until the formation of large replication clusters during late S-phase (Fig. S1). This finding is likely due to the transient role of RPA in DNA replication and rapid turnover at replication sites (Sporbert et al., 2002). Similar localization and behavior of these GFP-fusion proteins during replication in other cell systems has been documented (Leonhardt et al., 2000; Sporbert et al., 2002). As such, these cell lines provide an effective model for studying the action of the replication machinery in the DNA damage response.
Numerous proteins have been identified to relocalize within the nucleus in response to genotoxic insult. For example, ionizing radiation induces the formation of nuclear foci enriched in proteins including BRCA1, 53BP1, and the Rad50/Mre11/Nbs1 complex (Maser et al., 1997; Wang et al., 2002). Therefore, the localization of PCNA and RPAp34 in response to DNA damage was examined (Fig. 1). Upon treatment of the stable lines with cis-diamminedichloroplatinum II (CDDP), a chemotherapeutic agent that induces platinum-DNA (Pt-DNA) adducts (Siddik, 2003), the GFP-fusion proteins accumulated into discrete subnuclear foci (Fig. 1 A). GFP-RPAp34 and GFP-PCNA also accumulated into numerous subnuclear foci in response to acute UV irradiation (Fig. 1 B and not depicted), consistent with reports on the function of these replication factors in the nucleotide excision repair of pyrimidine dimers (He et al., 1995; Riedl et al., 2003). Focus formation of GFP-RPAp34 and GFP-PCNA was restricted to microdomains of the nucleus that were irradiated through pores in a polycarbonate filter, indicating that the nucleation of these factors in response to UV irradiation is not a pan-nuclear event (Fig. 1 C and Fig. S1). Although GFP-RPAp34 was largely diffuse in replicating cells, it assembled into discrete foci when DNA replication was stalled with hydroxyurea (HU) or aphidicolin (APH; Fig. 1 D). These foci presumably represent the accumulation of RPA at regions of single-stranded DNA occurring at stalled or collapsed replication forks, as appearance of these foci preceded the development of strand breaks (Fig. 1 E) indicated by the accumulation of H2AX phosphorylation (-H2AX; Ward and Chen, 2001). The topoisomerase inhibitor camptothecin also induced the formation of GFP-RPAp34 foci that colocalized with
-H2AX foci (Fig. 1 F). Insult with CDDP or UV irradiation induced the formation of GFP-RPAp34 foci irrespective of ongoing cell cycle progression (Fig. 1 G). In contrast, challenge with HU only induced GFP-RPAp34 and GFP-PCNA foci formation in cells progressing into S-phase (Fig. 1 G and not depicted). Together, these results demonstrate that replication factors are recruited to sites of genetic insult, independent of ongoing DNA replication.
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Several studies have described specific signaling pathways that regulate the biological activity of replication factors. Specifically, phosphorylation of RPA subunits after genotoxic insult has been observed (Shao et al., 1999; Wang et al., 2001; Barr et al., 2003). These reports have speculated that phosphorylation enhances RPA affinity for damaged or single-stranded DNA, effectively stabilizing a denatured DNA structure that RPA has been predicted to play a static role in maintaining. Our results strongly question this model. RPAp34 was observed to rapidly exchange at repair foci with significantly faster kinetics than other repair factors (i.e., PCNA). Contrary to expectation, we found that phosphorylation of RPAp34 by DNA-PK was not required for focus formation. Rather, this phosphorylation event destabilized RPAp34 at sites of damage. The use of increasing the turnover of RPAp34 at repair foci is at present unknown. However, several tantalizing possibilities exist, such as a role for phosphorylated RPA in signaling for the recruitment of additional factors from the nucleoplasm or to facilitate accessibility of repair factors to the region of damage. Alternatively, phosphorylation by DNA-PK may be required for efficient functioning of RPAp34 in repair processes. Cells deficient in DNA-PK activity have defects in DNA repair and V(D)J recombination, and an inability to rapidly modulate RPADNA interactions may explain these defects. Additionally, we have delineated a posttranslational mechanism by which PCNA is targeted to or stabilized at repair foci. Although there appears to be no significant modification of human PCNA during replication or other phases of the cell cycle, modification by ubiquitin after DNA damage is essential for the stable assembly of PCNA at repair foci. This result provides an explanation for the lack of viability in yeast harboring the K164R allele after DNA damage (Hoege et al., 2002). Notably, ubiquitination also plays a role in targeting the Fanconi anemia protein FANCD2 to foci after DNA damage (Garcia-Higuera et al., 2001). It will be interesting to further examine the critical role of ubiquitination in the DNA damage response.
The dynamic behavior of repair factors in living cells was first demonstrated by Houtsmuller et al. (1999) who observed that the endonuclease complex ERCC1-XPF rapidly exchanges at sites of UV-induced damage. However, not all proteins behave identically at sites of damage, as Nbs1 is largely tethered whereas Chk2 remains highly mobile (comparable to our observations with PCNA and RPAp34, respectively; Lukas et al., 2003). In this context, our data support a dynamic exchange model in which multiple repair factors have access to and rapidly turn over at sites of genetic insult. Further analysis of the DNA damage response in living cells will continue to provide powerful insight into the complex process of repair essential for the maintenance of genomic stability.
Materials and methods
Plasmids and mutagenesis
Expression plasmids encoding GFP-PCNA, GFP-RPAp34, and GFP-histone H2B have been described previously (Leonhardt et al., 2000; Phair and Misteli, 2000; Sporbert et al., 2002). Lys-164 of GFP-PCNA was mutated to Arg, and multiple NH2-terminal phosphorylation sites of GFP-RPAp34 (Thr-21, Ser-23, Ser-29, and Ser-33) were mutated to Ala and confirmed by sequencing.
Cell culture and generation of stable cell lines
Rat-1, U2OS, and M059 cells were grown in DME supplemented with 10% FBS. Rat-1 and U2OS cells were transfected with GFP-PCNA, GFP-RPAp34, and GFP-histone H2B along with pBABE-Puro using FuGene 6 (Roche), and multiple independent stable cell lines were established after puromycin selection. Transient transfection of M059 cells was performed using FuGene 6. Clinical grade CDDP was obtained from Bristol Oncology. All other drugs were obtained from commercially available sources.
Immunoprecipitation and immunoblotting
Cells were lysed in NET-N supplemented with protease inhibitors, and clarified lysate was immunoprecipitated with a monoclonal GFP antibody (B-2; Santa Cruz Biotechnology, Inc.). Proteins were detected with the following antibodies: PCNA (FL-261; Santa Cruz Biotechnology, Inc.), RPA (Ab-3; Oncogene Research Products), and ubiquitin (U5379; Sigma-Aldrich).
BrdU labeling and immunofluorescence
Asynchronously growing cells were pulsed with BrdU for 15 min, and sites of BrdU incorporation were visualized as described previously (Angus et al., 2003). For detection of specific DNA lesions, cells were fixed in 3.7% formaldehyde and probed with antibodies to cyclobutane pyrimidine dimers (T. Matsunaga, Osaka University, Osaka, Japan) or Pt-DNA adducts (M. Tilby, University of Newcastle upon Tyne, Newcastle, UK). For all other immunofluorescence, cells were fixed in cold methanol for 20 min and probed with antibodies to RPA (Ab-3; Oncogene Research Products), MSH2 (N-20; Santa Cruz Biotechnology, Inc.), ERCC1 (FL-297; Santa Cruz Biotechnology, Inc.), or phospho-histone H2A.X Ser-139 (JBW301; Upstate Biotechnology). Imaging was performed on a microscope (model Microphot-FX; Nikon) using a plan-apochromat 60x 1.4 NA objective.
UV microirradiation
UVC radiation was delivered using a low-pressure mercury lamp with maximal output at 254 nm. Polycarbonate filters containing either 3- or 5-µm diameter pores were gently laid over the cells before UV irradiation.
Dot-blot assay of Pt-DNA adduct levels
4 µg of genomic DNA isolated from U2OS cells after CDDP damage was extensively boiled and sheared by sonication, transferred onto a nylon membrane, and probed with the Pt-DNA adduct antibody.
Live-cell imaging and FRAP
Live-cell imaging and photobleaching analysis was performed at 37°C on a confocal microscope (model LSM510 Axiovert; Carl Zeiss MicroImaging, Inc.) using the 488-nm line of an argon laser (25 mW nominal output, photomultiplier detection through a pinhole diameter of 96 µm and a 505-nm long-pass filter) as described previously (Phair and Misteli, 2000; Angus et al., 2003).
Online supplemental material
Three supplemental figures and accompanying figure legends are available online at http:www.jcb.org/cgi/conent/full/jcb.200312048/DC1.
Acknowledgments
We thank Nancy Kleene, Robert Hennigan, Michael Tilby, Tsukasa Matsunaga, Yoli Sanchez, and Kathleen Dixon for kindly providing reagents and technical assistance. We thank Andre Nussenzweig, Yoli Sanchez, Peter Stambrook, and Karen Knudsen for critical reading of the manuscript.
M.C. Cardoso is funded by the Deutsche Forschungsgemeinschaft, and E.S. Knudsen is funded by the American Cancer Society (grant RSG-01-254-ECG) and National Cancer Institute (grant CA-106471).
Submitted: 5 December 2003
Accepted: 10 June 2004
Angus, S.P., D.A. Solomon, L. Kuschel, R.F. Hennigan, and E.S. Knudsen. 2003. Retinoblastoma tumor suppressor: analyses of dynamic behavior in living cells reveal multiple modes of regulation. Mol. Cell. Biol. 23:81728188.
Barr, S.M., C.G. Leung, E.E. Chang, and K.A. Cimprich. 2003. ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation. Curr. Biol. 13:10471051.[CrossRef][Medline]
Dutta, A., and B. Stillman. 1992. cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 11:21892199.[Abstract]
Garcia-Higuera, I., T. Taniguchi, S. Ganesan, M.S. Meyn, C. Timmers, J. Hejna, M. Grompe, and A.D. D'Andrea. 2001. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell. 7:249262.[Medline]
He, Z., L.A. Henricksen, M.S. Wold, and C.J. Ingles. 1995. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature. 374:566569.[CrossRef][Medline]
Hoege, C., B. Pfander, G.L. Moldovan, G. Pyrowolakis, and S. Jentsch. 2002. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 419:135141.[CrossRef][Medline]
Hoogstraten, D., A.L. Nigg, H. Heath, L.H. Mullenders, R. van Driel, J.H. Hoeijmakers, W. Vermeulen, and A.B. Houtsmuller. 2002. Rapid switching of TFIIH between RNA polymerase I and II transcription and DNA repair in vivo. Mol. Cell. 10:11631174.[Medline]
Houtsmuller, A.B., S. Rademakers, A.L. Nigg, D. Hoogstraten, J.H. Hoeijmakers, and W. Vermeulen. 1999. Action of DNA repair endonuclease ERCC1/XPF in living cells. Science. 284:958961.
Izzard, R.A., S.P. Jackson, and G.C. Smith. 1999. Competitive and noncompetitive inhibition of the DNA-dependent protein kinase. Cancer Res. 59:25812586.
Leonhardt, H., H.P. Rahn, P. Weinzierl, A. Sporbert, T. Cremer, D. Zink, and M.C. Cardoso. 2000. Dynamics of DNA replication factories in living cells. J. Cell Biol. 149:271280.
Lukas, C., J. Falck, J. Bartkova, J. Bartek, and J. Lukas. 2003. Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat. Cell Biol. 5:255260.[CrossRef][Medline]
Maser, R.S., K.J. Monsen, B.E. Nelms, and J.H. Petrini. 1997. hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol. Cell. Biol. 17:60876096.[Abstract]
Meijer, L., A. Borgne, O. Mulner, J.P. Chong, J.J. Blow, N. Inagaki, M. Inagaki, J.G. Delcros, and J.P. Moulinoux. 1997. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 243:527536.[Abstract]
Niu, H., H. Erdjument-Bromage, Z.Q. Pan, S.H. Lee, P. Tempst, and J. Hurwitz. 1997. Mapping of amino acid residues in the p34 subunit of human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro. J. Biol. Chem. 272:1263412641.
Phair, R.D., and T. Misteli. 2000. High mobility of proteins in the mammalian cell nucleus. Nature. 404:604609.[CrossRef][Medline]
Riedl, T., F. Hanaoka, and J.M. Egly. 2003. The comings and goings of nucleotide excision repair factors on damaged DNA. EMBO J. 22:52935303.
Rouse, J., and S.P. Jackson. 2002. Interfaces between the detection, signaling, and repair of DNA damage. Science. 297:547551.
Sarkaria, J.N., E.C. Busby, R.S. Tibbetts, P. Roos, Y. Taya, L.M. Karnitz, and R.T. Abraham. 1999. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 59:43754382.
Shao, R.G., C.X. Cao, H. Zhang, K.W. Kohn, M.S. Wold, and Y. Pommier. 1999. Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J. 18:13971406.
Shivji, K.K., M.K. Kenny, and R.D. Wood. 1992. Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 69:367374.[Medline]
Siddik, Z.H. 2003. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene. 22:72657279.[CrossRef][Medline]
Sporbert, A., A. Gahl, R. Ankerhold, H. Leonhardt, and M.C. Cardoso. 2002. DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters. Mol. Cell. 10:13551365.[Medline]
Svejstrup, J.Q., Z. Wang, W.J. Feaver, X. Wu, D.A. Bushnell, T.F. Donahue, E.C. Friedberg, and R.D. Kornberg. 1995. Different forms of TFIIH for transcription and DNA repair: holo-TFIIH and a nucleotide excision repairosome. Cell. 80:2128.[Medline]
Tilby, M.J., C. Johnson, R.J. Knox, J. Cordell, J.J. Roberts, and C.J. Dean. 1991. Sensitive detection of DNA modifications induced by cisplatin and carboplatin in vitro and in vivo using a monoclonal antibody. Cancer Res. 51:123129.[Abstract]
Vassin, V.M., M.S. Wold, and J.A. Borowiec. 2004. Replication protein A (RPA) phosphorylation prevents RPA association with replication centers. Mol. Cell. Biol. 24:19301943.
Waga, S., and B. Stillman. 1998. The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67:721751.[CrossRef][Medline]
Walker, E.H., M.E. Pacold, O. Perisic, L. Stephens, P.T. Hawkins, M.P. Wymann, and R.L. Williams. 2000. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol. Cell. 6:909919.[Medline]
Wang, B., S. Matsuoka, P.B. Carpenter, and S.J. Elledge. 2002. 53BP1, a mediator of the DNA damage checkpoint. Science. 298:14351438.
Wang, H., J. Guan, A.R. Perrault, Y. Wang, and G. Iliakis. 2001. Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase. Cancer Res. 61:85548563.
Ward, I.M., and J. Chen. 2001. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276:4775947762.
Zou, L., and S.J. Elledge. 2003. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science. 300:15421548.