Dynamics of DNA Replication Factories in Living Cells

1 The Rockefeller University Press, 0021-9525/2000/04/271/9 $ Journal of Cell Biology, Volume 149, Number 2, April 17, 2000 271 279 Dynamics of DNA Replication Factories in Living Cells Heinrich Leonhardt,* Hans-Peter Rahn,* Peter Weinzierl, Anje Sporbert,* Thomas Cremer, Daniele Zink, and M. Cristina Cardoso* *Max Delbr ck Center for Molecular Medicine, D-13125 Berlin, Germany; and Institute for Anthropology and Human Genetics, LMU, D-80336 Munich, Germany Abstract. DNA Replication occurs in microscopically visible complexes at discrete sites ( Replication foci) in the nucleus. These foci consist of DNA associated with Replication machineries, , large protein complexes in-volved in DNA Replication . To study the Dynamics of these nuclear Replication foci in Living Cells , we fused proliferating cell nuclear antigen (PCNA), a central component of the Replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that repli-cation foci are heterogeneous in size and lifetime.

2 Time-lapse studies revealed that Replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses sug-gested that Replication Factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, as-sembly and disassembly throughout S phase. Key words: cell cycle DNA Replication foci green fluorescent protein nuclear organization proliferat-ing cell nuclear antigen Introduction Over the last decade it has become clear that the mamma-lian nucleus, despite the absence of intranuclear mem-branes, is organized into functional domains or foci wherenuclear processes like DNA Replication , ribosome biogene-sis, transcription, and RNA processing take place (Spector,1993; Xing and Lawrence, 1993; Scheer and Weisenberger,1994). These compartments have been identified in somecases by phase-contrast microscopy, by in situ hybridizationtechniques, and more commonly by detection of incorpo-rated nucleotides or associated proteins.

3 Different proteinsparticipating in the same process are organized together inone functional compartment, which is often referred to asfunctional organization of the nucleus (Leonhardt and Car-doso, 1995). In the case of Replication foci, these proteins in-clude not only Replication factors, but also other proteinsthat are not directly involved in DNA Replication , such asthe cell cycle regulators cyclin A (Cardoso et al., 1993; Sob-czak-Thepot et al., 1993) and cdk2 (Cardoso et al., 1993),uracil-DNA glycosylase (UNG2; Otterlei et al., 1999) in-volved in base excision repair, and also DNA methyltrans-ferase (Dnmt1; Leonhardt et al., 1992). These Replication machines and Factories are obviouslybound to the DNA they are replicating, but they also seemto be tethered to an underlying framework called the nu-clear matrix or skeleton (Tubo and Berezney, 1987; Hozaket al., 1993). The molecular mechanism underlying the cellcycle-dependent association of a growing list of proteins withsubnuclear Replication foci is mostly unknown.

4 In the case oftwo of these factors, Dnmt1 and DNA ligase I, specific pro-tein sequences were identified that are separated from thecatalytic domains and are necessary and sufficient for associ-ation with Replication foci (Leonhardt et al., 1992; Cardosoet al., 1997). These targeting sequences seem to position thedifferent factors at the right place at the right time to assem-ble a processive Replication -methylation machinery workinglike an assembly-line (Cardoso and Leonhardt, 1998).The existence of Replication foci, which had been shownin fixed and stained Cells , was recently established in livingmammalian Cells (Cardoso et al., 1997). It has often beenproposed, but not directly shown, that Replication foci pat-terns undergo reproducible changes in individual cellsthroughout S phase. Furthermore, it is not known howthese different patterns form and possibly change during Sphase. To address these questions, we set out to establish acellular system to investigate the Dynamics of DNA repli-cation Factories in Living Cells during the cell cycle, makinguse of translational fusions of Replication factors with the green fluorescent protein (GFP) 1.

5 We chose proliferating Dr. Peter Weinzierl died on 13 October correspondence to Cardoso, Franz Volhard Clinic, Wilt-bergstr. 50, 13125 Berlin, Germany. Tel.: 49-30-9417-2341. Fax: 49-30-9417-2336. E-mail: 1 Abbreviations used in this paper: 4D, four-dimensional; BrdU, 5-bromo-2 9 -deoxyuridine; GFP, green fluorescent protein; PCNA, proliferating cellnuclear antigen. The Journal of Cell Biology, Volume 149, 2000272 cell nuclear antigen (PCNA) as a marker for replicationfactories since it is highly conserved from yeast to mam-malian Cells , has no known enzymatic activity, but is nev-ertheless a central and essential factor for DNA replica-tion (Jaskulski et al., 1988; Bauer and Burgers, 1990;Waseem et al., 1992). PCNA was first identified as the pro-cessivity factor for DNA polymerase delta (Bravo et al.,1987; Prelich et al., 1987). It forms a homotrimeric ringaround the DNA serving as a sliding clamp that tethers thepolymerases, as well as many other Replication associatedfactors, to the DNA ensuring high processivity (Wymanand Botchan, 1995; Jonsson and Hubscher, 1997; Kelmanand Hurwitz, 1998).

6 Historically, PCNA was the first pro-tein identified at Replication foci during S phase (Celis andCelis, 1985; Bravo and Macdonald-Bravo, 1987) and issince widely used as a marker for Replication coiled bodies (Boudonck et al., 1999; Sleemanand Lamond, 1999) and speckles (Misteli et al., 1997),which have been recently shown to move and merge, theresults presented here indicate that Replication foci visual-ized in Living Cells by GFP-PCNA do not seem to move,merge, or divide, but instead assemble and disassemble inan asynchronous manner throughout S phase. Materials and Methods GFP-PCNA Expression Plasmid Expression of the fusion protein is driven by the CMV promoter and thetranslation signals of the thymidine kinase gene, both provided by thepEVRF expression vector (Matthias et al., 1989). The GFP-PCNAL2 fu-sion protein contains a SV40 nuclear localization signal at the NH 2 termi-nus followed by an enhanced mutant GFP gene that is fused to the humanPCNA by a flexible and hydrophilic linker (GEGQGQGQGPGR-GYAYRS).

7 The GFP gene uses a humanized codon usage for better ex-pression in mammalian Cells and mutations confering improved spectralproperties (enhanced GFP; Clontech) and thermal stability (Siemering etal., 1996). Generation of Mammalian Cell Lines ExpressingGFP-PCNA Fusion Protein C2C12 mouse myoblast Cells were grown in DME supplemented with20% FCS. To generate GFP-PCNA expressing cell lines, linearized plas-mid DNA coding for the fusion protein GFP-PCNAL2 was cotransfectedwith the plasmid pSV2neo (at a ratio of 20:1) into C2C12 Cells by the lipo-fectamine method (GIBCO BRL). 24 h after transfection, the GFP ex-pressing Cells were sorted by flow cytometry (FACSort; Becton Dickin-son), replated at clonal density, and grown for 9 d in media containing 600 m g/ml G418 (Geneticin; GIBCO BRL). GFP-PCNA expressing cell cloneswere selected under the fluorescence microscope, expanded, and furthersubcloned by the limiting dilution method to ensure their clonal nature.

8 Western Blot Analysis For immunoblot analysis, 5 3 10 5 COS7 Cells , transiently transfected bythe DEAE dextran pretreatment method, and the same number of mousemyoblast Cells were extracted and immunoblots were performed, both asdescribed before (Cardoso et al., 1997). Blots were probed with the mousemonoclonal anti-PCNA antibody (clone PC10; Dako). Flow Cytometry Analysis To analyze the intracellular DNA content, Cells were trypsinized, washedin PBS, and fixed in 100% methanol. Fixed Cells were subsequentlystained in a solution containing 50 m g/ml propidium iodide, mg/mlRNAse, NP-40, and trisodium citrate. Samples were thenwashed in PBS and DNA content estimated by measuring propidium io-dide fluorescence with a flow cytometer (FACSort; Becton Dickinson) us-ing the 600-nm long pass filter. Data were analyzed using the ModFit LTsoftware (Becton Dickinson). Immunofluorescence Analyses Indirect immunofluorescence stainings were carried out as previously de-scribed (Cardoso et al.)

9 , 1997). The following primary antibodies wereused: mouse monoclonal anti-PCNA antibody (clone PC10; Dako), rabbitpolyclonal anti-Dnmt1 antiserum (Leonhardt et al., 1992), and affinity-purified rabbit polyclonal anti-DNA ligase I antibody (Cardoso et al., 1997).C2C12 Cells were transfected with plasmid DNA coding for the fusionprotein GFP-PCNAL2 by the calcium phosphate precipitation method,and 24 h later pulse-labeled for 20 min with 10 m M 5-bromo-2'-deoxyuri-dine (BrdU). Cells were then formaldehyde-fixed, followed by acid dena-turation of the DNA, and staining with anti-BrdU antibody (Becton Dick-inson) as described (Zink et al., 1998). Optical sections were acquired witha Leica TCS four-dimensional (4D) confocal microscope. Live Cell Microscopy and Image Analysis For live cell observation, Cells were plated onto 40-mm glass coverslipsand allowed to attach overnight. They were then assembled into a FCS2live cell microscopy chamber (Bioptechs) set to 37 8 C that was mounted ona Zeiss Axiovert inverted microscope.

10 For most observations, a 63 3 Plan apochromat oil immersion objective heated to 37 8 C and a band-pass fluorescein filter set (excitation 450 490 nm, dichroic 510 nm, emis-sion 515 565 nm; Zeiss) were used. Shorter excitation wavelengths provedto be phototoxic to the Cells . Time of exposure per image varied 4 s. In most experiments, stacks of 21 images at m m Z incrementswere collected at each time point. Images were acquired with a cooledCCD camera, Sensicam (1,280 3 1,024 pixels; m m pixel size) using theQuanticell software (VisiTech) or, in the case of Fig. 3 B, with a Micro-Max camera (1,317 3 1,035 pixels, m m pixel size) using MetaMorph (Universal Imaging Corp.). Images were assembled and anno-tated using NIH Image , Adobe Photoshop , and Adobe software on Power MacIntosh of image stacks collected at different time points were first ana-lyzed for movements of the nucleus itself. After correcting for cellularmovements and focal drift, we examined Replication foci over time formovements, assembly, or disassembly using the neighboring foci and nu-cleoli as reference points.