Nucleic acids research.
Extra large 
|
|
|
Figure 2 Ligase is less strongly associated to replication sites than PCNA (A) Time-lapse in situ salt extraction of non-S- and S-phase cells expressing GFP-Ligase and RFP-PCNA showing that GFP-Ligase is largely extracted after short permeabilization with Triton X-100, while RFP–PCNA remains associated at RF for many minutes (for quantification, see diagram). (B) Identical results were obtained for endogenous Ligase and PCNA, which were detected by immunofluorescence. Scale bar, 10 µm. (C) C2C12 cells were cotransfected with both GFP-PCNA and GFP-Ligase to ensure the same number of transfected cells and the same percentage of S-phase cells. Cells were permeabilized and extracted, and equal amounts of the cell extract and the insoluble cell pellet were analyzed by SDS–PAGE followed by western blotting. Whereas most of the endogenous Ligase and the respective GFP fusion protein were found in the soluble fraction, under the same conditions, endogenous PCNA and GFP-PCNA were only partially extracted from the pellet and found equally distributed in both fractions. In this direct comparison, PCNA showed a stronger, label-independent association with nuclear structures than Ligase.
- For extraction experiments (Figure 2), C2C12 cells doubly transfected with GFP-Ligase and GFP-PCNA were extracted with a buffer (50 mM Tris–HCl, pH 8, 120 mM NaCl and 0.5% NP40) containing protease inhibitors by freezing and thawing three times followed by a 30 min incubation on ice.
- For the triple immunostaining (Figure 2B), the following primary antibodies were used: mouse monoclonal anti-PCNA antibody (clone PC10, Dako), rabbit affinity purified anti-DNA Ligase I antibody (10) and a rat monoclonal anti-BrdU antibody (clone BU1/75, SeraLab).
- For nuclear distribution analysis of PCNA and Ligase fusions (Figures 1 and 2), mean fluorescence intensities (FIs) of pixels were calculated in each image of a z-stack after thresholding to select only pixels representing nucleoplasmic fluorescence or RF (LSM 510 software).
- To investigate whether the differential distribution of PCNA and Ligase reflects different association properties of the two proteins, salt extractions of endogenous PCNA and Ligase and the respective fusion proteins were performed (Figure 2).
- Live cells coexpressing GFP-Ligase and RFP-PCNA were permeabilized for 1 min directly on the microscope stage followed by extraction with phosphate buffer containing 500 mM NaCl (Figure 2A) for several minutes.
- This differential extraction of Ligase and PCNA was also observed for the endogenous non-tagged proteins (Figure 2B).
- Both proteins were extracted in non-S-phase nuclei (Figure 2B, arrow) while PCNA, in contrast to Ligase, remained associated at RF in S-phase cells (identified by BrdU incorporation; Figure 2B, arrowhead) even after 5 min extraction (data not shown).
- Similarly, extraction experiments monitored by western blot analysis (Figure 2C) showed that both endogenous Ligase and GFP-Ligase are more readily extracted than PCNA and GFP-PCNA.
Nucleic acids research.
Extra large 
|
|
|
Figure 1 Ligase has a higher nucleoplasmic fraction than PCNA (A) Confocal images of live C2C12 cells in S-phase transiently or stably expressing GFP-Ligase or GFP-PCNA. (B) Quantification of NP and RF-bound protein (green/red in the false color image) by determining their mean FI (n = 5 cells with 5–7 z slices). Scale bar, 5 µm.
- For nuclear distribution analysis of PCNA and Ligase fusions (Figures 1 and 2), mean fluorescence intensities (FIs) of pixels were calculated in each image of a z-stack after thresholding to select only pixels representing nucleoplasmic fluorescence or RF (LSM 510 software).
- Although Ligase (10,13) and PCNA (14,15) have both been shown to spread uniformly throughout the nucleus in non-S-phase cells and to redistribute to sites of active DNA replication during S-phase, a closer look revealed quantitative differences in their distribution in live (Figure 1A) and fixed (data not shown) S-phase cells.
- After a threshold-based assignment of pixels to NP or RF, mean FI and the ratio between nucleoplasmic and RF-bound fluorescence were determined (Figure 1B).
- In both stably and transiently transfected cells, the fraction of Ligase in the NP during S-phase was 2-fold higher as compared with PCNA (Figure 1B).
Nucleic acids research.
Extra large 
|
|
|
Figure 3 Ligase and Fen1 have a shorter residence time at RF than PCNA (A) Half of the nucleus (BA) was bleached by one single, 12 s bleach pulse in live cells expressing GFP-Ligase or GFP-Fen1 and RFP-PCNA, resulting in an almost complete depletion of Ligase and Fen1 from the unbleached nucleoplasm and RF, in contrast to PCNA. (B) Different residence times of PCNA, Ligase and Fen1 at RF shown by loss of fluorescence from unbleached RF immediately after bleaching (16–20 RF from five cells were analyzed in each case). (C) Fast equilibration of the remaining Ligase and Fen1 between bleached and unbleached half of the nucleus within a few seconds in contrast to a slow equilibration of PCNA over minutes (three cells each). Scale bar, 5 µm.
- Curves describing the FDP datasets (Figure 3 and Supplementary Figure S4) were generated in KaleidaGraph 3.5.
- In live S-phase cells coexpressing GFP-Ligase or GFP-Fen1 and RFP-PCNA (for characterization see Supplementary Figure S1) about half of the nucleus was bleached for 12–15 s (Figure 3A).
- Further controls with cells coexpressing GFP-PCNA and RFP-PCNA ruled out that the observed differences on the dynamics were caused by the fluorescent label (Figure 3B and Supplementary Figure S4A).
- The FI was quantified to evaluate the amount of PCNA, Ligase and Fen1 fluorescence depleted due to dissociation from RF and diffusion into the bleach area (Figure 3B).
- While 70–75% of the initial PCNA remained at the original RF, only 20–30% of the initial Ligase or Fen1 did so (see Figure 3B), indicating a higher dissociation rate of the latter proteins.
- The equilibration of Ligase and Fen1 between bleached and unbleached half of the nucleus was achieved within seconds after the bleach interval while PCNA slowly redistributed during the following 10–20 min (Figure 3C).
Nucleic acids research.
Extra large 
|
|
|
Figure 4 Fast reassociation of Ligase and Fen1 at active RF contrasts to slow assembly of PCNA at newly established RF (A) In S-phase cells expressing GFP-Ligase or GFP-Fen1 and RFP-PCNA, one RF was bleached (BA). Ligase and Fen1 showed recovery at the bleached RF within a few seconds, while reappearance of PCNA fluorescence occurred several minutes later (for quantification, see diagrams). (B) Detailed spatial (insets, top panel) and time overlay analyses (insets, lower panel) showing the fast recovery of Ligase (top panel, mid) at previously bleached RF (lower panel, middle). However, the shape of the recovered Ligase focus changes over time (lower panel, right). In contrast, delayed recovery of PCNA occurs only at a small portion of the Ligase-labeled RF (top panel, middle and right) and is mostly adjacent to the bleached RF (lower panel, left). (C) Schematic interpretation of the data with fast exchanging Ligase/Fen1 continuously labeling active and newly assembled replisomes, in contrast with PCNA reassembling mostly at newly established replisomes. (D) Model of PCNA (red) as a stationary loading platform for the sequential loading of replication enzymes (here, represented by Fen1 in green and Ligase in blue) at the lagging strand synthesis. For simplicity, only one replicating lagging strand and one Okazaki fragment are shown. Scale bar, 5 µm.
- Images shown in Figure 4B were generated using the same threshold per channel.
- To test this further, we performed traditional FRAP experiments with PCNA and the PCNA-binding proteins but simultaneously measured the recovery of two fluorescently labeled proteins at the same RF (Figure 4, dual color FRAP).
- After bleaching one RF labeled with GFP-Ligase and RFP-PCNA, Ligase recovered within seconds while recovery of PCNA occurred only after a few minutes (Figure 4A, upper panel).
- Similar observations were made in experiments comparing the recovery of GFP-Fen1 and RFP-PCNA (Figure 4A, lower panel), at other S-phase stages (data not shown) and in cell lines stably expressing GFP-Ligase or GFP-PCNA (Supplementary Figure S4B).
- Since RF change constantly throughout S-phase, we compared the recovery/reassembly sites of Ligase and PCNA with their prebleach sites by time overlay analysis (Figure 4B, lower panel).
- This analysis indicates that the Ligase continuously exchanges at the active RF, while bleached PCNA remains at the bleached focus, and reassembly occurs at newly initiated RF (Figure 4C).
- This argues strongly for an alternative model, in which Ligase or Fen1 and PCNA are independently loaded at the replication fork and stay for different times (Figure 4D).
- Instead, PCNA appears to act as a stationary loading platform (Figure 4D) that is reused over multiple Okazaki fragments with PCNA-binding proteins associating transiently and subsequently dissociating rather than being part of one stable, multifunctional, processive replication machinery.