To measure the dynamics of polη in human cells, we used a cell line in which N-terminally tagged eGFP-polη was expressed in XP30RO cells (Kannouche et al., 2001
). These cells contain a truncation mutation in the POLH
gene close to the N terminus (Johnson et al., 1999a
) and can be considered as polη null mutants. We previously showed that the eGFP-polη expressed in this cell line was able to correct the typical sensitivity of XP30RO to UV followed by treatment with caffeine (Kannouche et al., 2001
). Using fluorescence-activated cell sorting, we selected a subpopulation of the cells in which the level of eGFP-polη was similar to that of endogenous polη in normal MRC5 cells. A shows a Western blot of polη in this cell line, compared with that in the normal cell line MRC5. Polη expression levels remained stable over several weeks. There was no evidence, either by Western blotting or by the appearance of cytoplasmic autofluorescence, for any free eGFP protein (data not shown). We conclude that the quantitative fluorescence measurements described in the following sections were derived from cells that express full-length and biologically active GFP-tagged polη.
Figure 1. Dynamics of eGFP-polη in living cells. (A) Western blot of the XP30RO-eGFP-polη cell line used in this study (lane 3), compared with MRC5 (lane 1) and XP30RO (lane 2). (B) Comparison of FRAP curves (relative fluorescence recovery plotted (more ...)
In previous work, we showed that eGFP-polη transfected into human fibroblasts was uniformly distributed throughout the nucleus outside S phase but that it accumulated in bright foci representing replication factories in S phase cells. In cells treated with 15 Jm−2
UV-irradiation and incubated for 7 h or with 1 mM HU for 24 h, the number of cells in which polη was located in foci increased substantially, partly or wholly because of the accumulation of S phase cells after these treatments (Kannouche et al., 2001
). Supplemental Figure S1 shows stills from a confocal time-lapse series in which the stable cell line was UV-irradiated through a micropore filter to generate localized damage within the nucleus (Volker et al., 2001
). Using this procedure, proteins involved in processing of DNA damage accumulate at the sites of the localized irradiation. We found that, throughout S phase, eGFP-polη accumulated at the sites of local damage. Within the damaged area polη accumulated in a focal pattern because of the stalling of replication forks (Kannouche et al., 2001
). In contrast, in G2 the eGFP-polη neither accumulated at the local damage nor was it in bright foci, but it became uniformly distributed through the nucleus. These data confirm the S phase-specific function of polη.
We have used FRAP to measure the mobility of polη under different conditions. We photobleached a small square of the nucleus and measured the rate of recovery of fluorescence within the square. Polη that was uniformly distributed in the nucleus (i.e., in G1 or G2 cells) relocalized into the bleached area extremely rapidly with a t0.5 of 0.15 s (B, polη untreated-diffuse), indicating that it is highly mobile within the nucleus.
We next photobleached the eGFP-polη within a focus in an S phase nucleus by aligning the square over a visible focus in S phase cells (Box-FRAP). We used a square as small as possible so that the focus filled almost the whole area of the square. In this situation, the recovery rate was reduced about two-fold (B). The t0.5
was still very short, 0.33 s. The mobility of polη in foci generated in cells irradiated with UV-irradiation or following HU treatment was indistinguishable from that in an unperturbed S phase (B). Thus, surprisingly, even when associated with microscopically visible structures, the majority of the polη molecules within the focus remained highly mobile. Examination of the curves in B at later times (up to 15 s; see inset) suggests that at most only 7% of the molecules were immobilized for a long period (see Materials and Methods
for definitions of t0.5
and immobile fraction). In contrast to the highly dynamic association of eGFP-polη, we observed a relatively large (~60%) fraction of eGFP-PCNA (C), in which proteins were significantly immobilized for long periods (see Materials and Methods
for calculation of long-lasting immobile fractions), in line with previous studies (Sporbert et al., 2002
; Essers et al., 2005
).This demonstrates that our system was capable of detecting immobilized proteins.
To determine whether the catalytic activity of polη might affect its mobility, we generated an XP30RO cell line expressing eGFP-polη in which amino acids (aa) D115 and E116, shown to be vital for catalytic activity (Johnson et al., 1999b
), were mutated to alanines. This mutation allows the incoming dNTP to bind but cannot support the formation of the phosphodiester bond (Li et al., 1998
). The mobility of this “pol dead” polη mutant, when distributed uniformly in the nucleus, was identical to that of wild-type polη (data not shown), but interestingly, its mobility in foci was about twofold lower than that of wild-type polη, with a t0.5
of ~0.67 s and a long-lasting immobile fraction of 15% (C).
As an alternative methodology, we have also used FLIP-FRAP in which we bleached half the nucleus. We then measured both the rate of reduction in fluorescence intensity of the unbleached half (FLIP) and the rate of recovery in the bleached half of the nucleus (FRAP). With this technique, we are able to analyze the overall mobility in the whole of the nucleus, providing the collective mobility of polη in a large number of foci, in contrast to the mobility within a single focus in the experiments described above. As with bleaching of a small square, we observed rapid redistribution of polη. The difference between FLIP and FRAP immediately after bleaching was normalized to 1, and, in D, at different times after bleaching, the normalized difference between the FLIP and FRAP is presented on a log scale. With nuclei in which polη was uniformly distributed, polη had returned to 90% of the prebleach distribution (i.e., normalized fluorescence = 0.1) in 25 s (D). Using this FLIP-FRAP analysis, we have examined the effect of different doses of UV on the mobility of polη in nuclei containing focal polη. We compared polη mobility in these cells with its mobility when diffusely distributed in untreated cells. A UV dose response was observed, with increasing delay in polη redistribution due to transient immobilization to subnuclear structures (D). Higher UV doses resulted in a more pronounced delay in redistribution, reaching a maximum after irradiation with 16 Jm−2, with a redistribution time of about 45 s, compared with ~25 s in untreated cells not exhibiting foci. This approximate doubling of the redistribution time agrees well with the approximately two-fold decrease in mobility in the Box-FRAP data presented in B. These data suggest that with increasing UV-doses, as expected, more substrate sites (i.e., stalled forks) were created that transiently bind a larger pool of the resident polη molecules but that the average binding time within a single focus is not affected by an increasing number replication blocks.
In a further variation, we UV-irradiated cells through a micropore filter to produce localized damage in the nucleus (Volker et al., 2001
). Five hours after irradiation, we selected cells in which polη had accumulated in foci at the sites of local damage (examples shown in E), and we bleached the entire site of local damage. As control, we bleached an identical area in a nucleus in which no local damage had been inflicted. Relocalization into the bleached damaged site was again approximately two-fold slower than into undamaged areas (F). We conclude from these different photobleaching studies that polη is highly mobile within the nucleus and that its mobility is only slightly reduced within replication foci.
Role of PCNA-Ubiquitination
Polη has a PIP box binding motif for interaction with PCNA (Haracska et al., 2001
; Kannouche et al., 2001
), and it is likely that PCNA plays a role in assisting polη to find its substrate. After exposure of cells to UV-irradiation or other agents that block progression of the replication fork, PCNA becomes mono-ubiquitinated on lysine-164 at the sites of stalled forks, a reaction mediated by the Rad6–Rad18 ubiquitination system (Hoege et al., 2002
; Kannouche et al., 2004
; Watanabe et al., 2004
). It is widely assumed, but without direct evidence, that ubiquitination of PCNA is required for localization of polη in replication foci. Dantuma et al. (2006)
reported that treatment of cells with the general proteasome inhibitor MG132 induced a depletion of the free ubiquitin pool and a concomitant reduction of mono-ubiquitinated target proteins such as ubiquitinated histones. We observed similar effects on UV-irradiation–induced PCNA mono-ubiquitination when cells were treated with either MG132 (data not shown) or with another proteasome inhibitor epoxomicin (A). Remarkably, polη accumulated in foci to a similar extent in UV-irradiated MRC5 cells treated with or without epoxomicin (B), indicating that ubiquitination of PCNA is not essential for polη foci formation.
Figure 2. Ubiquitination of PCNA and polη mobility. (A) MRC5 cells were UV-irradiated (15 Jm−2) and incubated for 6 h with epoxomicin. PCNA was analyzed by Western blotting. (B) MRC5 cells transfected with eGFP-polη and mRFP-PCNA were UV (more ...)
These findings do not however rule out the possibility that ubiquitination of PCNA affects the dynamics of polη in foci. Because inhibition of the proteasome is likely to have many pleiotropic effects, it would be difficult to interpret dynamic experiments making use of this inhibitor. An alternative way of preventing PCNA ubiquitination is by depletion of Rad18, by using siRNA (Kannouche et al., 2004
). However, Rad18 interacts physically with polη and is required for the accumulation of polη in foci, independently from its role in PCNA ubiquitination (Watanabe et al., 2004
); so, this approach also could not be used. Instead, we looked at the effect of overexpressing Rad18 in our eGFP-polη–expressing cells and measured the mobility of polη, both uniformly distributed and in foci. Overexpression of Rad18 has been reported to cause increased PCNA ubiquitination (Huang et al., 2006
; Davies et al., 2008
). To test whether this was also the case in our experimental system, we contransfected His-PCNA and Rad18. The use of His-PCNA was needed because the low transfection efficiency of our cell lines made it impossible to detect any changes in endogenous PCNA. In the overexpressing cells, there was an increase in the level of ubiquitination of His-tagged PCNA, especially after UV-irradiation (C, compare lanes 4 and 2).
Overexpression of Rad18 (together with mRFP-α-tubulin, used as transfection marker) had no effect on the mobility of uniformly distributed polη (D). In contrast, there was a decrease in the mobility of polη in foci (D). To determine whether this effect of Rad18 was mediated by ubiquitination of PCNA or by binding to polη, we mutated the RING finger motif of Rad18 that is required for its ubiquitin ligase activity and the ubiquitination of PCNA but is not involved in direct interaction of Rad18 with polη (Watanabe et al., 2004
). Using the Rad18-C28F mutation (Tateishi et al., 2000
), in which the E3 ubiquitin ligase activity is inactivated, levels of ubiquitinated PCNA were the same as in mock-transfected cells (C, lane 6), and the reduction in mobility of focal polη was abolished (D).
USP1 is a deubiquitinating enzyme (DUB), which removes the ubiquitin from ubiquitinated PCNA (Huang et al., 2006
). Depletion of USP1 by using siRNA results in increased levels of ubiquitinated PCNA in undamaged cells (Huang et al., 2006
; E, bottom, lane 3). In these USP1-depleted cells, the mobility of uniformly distributed GFP-polη was slightly reduced; in foci in HU-treated cells, it was reduced to a similar level to that in the cells overexpressing Rad18 (F). (Note that we could not use UV in these experiments as UV-irradiation results in disappearance of USP1 from the cell. This is not seen after HU treatment; Huang et al., 2006
and our unpublished data.) Together, these results suggest that although ubiquitination of PCNA is not required for accumulation of polη into replication factories, it results in an increased residence time of polη in the factories.
Mobility of polι
Polι is a paralogue of polη (Tissier et al., 2000
), but its precise function remains to be established. In previous work, we showed that polη could physically interact with polι, although we could not demonstrate such an interaction in human cell lysates (Kannouche et al., 2003
). Polι accumulates in replication foci in an identical manner to polη, and this accumulation is substantially dependent on the presence of polη, because it was greatly reduced in XP-V cells (Kannouche et al., 2003
). To investigate the intracellular relationship between polη and ι further, we established stable MRC5 and XP-V XP30RO cell lines expressing eGFP-polι. The levels of polι expression are shown in A and are approximately 4 times the endogenous level (see Supplemental Figure S3 for calculation). This is the minimum level of expression that enables us to visualize the eGFP-polι foci. However, by comparing cells expressing different levels of eGFP-polι, we ascertained that the mobility of polι was independent of its expression level. We compared the mobility of polι with that of polη. We found that polι was even more mobile than polη, with a t0.5
of only 90 ms when uniformly distributed, this mobility being similar in MRC5 and XP30RO cells and therefore independent of the presence of polη (B). As with polη, the mobility of polι was somewhat decreased in replication foci (t0.5
of 200 ms), but it remained more mobile than polη (C). These data do not support the idea that the two polymerases exist in the same complex within the cell (although they do not rule out the possibility that a small subfraction might be associated).
Figure 3. Polι is more mobile than polη. (A) Western blot with anti-polι of lysates from stable cell lines expressing eGFP-polι. (Note that the slow mobility band is the previously reported ubiquitinated form of polι; Bienko (more ...)
Because polη and ι have very similar molecular weights, if they exist in the cell as freely diffusible monomers, their redistribution kinetics should be very similar. There are two possible explanations for the different kinetics. The first possibility is that when uniformly distributed, both polymerases are components of protein complexes that are freely diffusible within the cell and the polη complex is larger than the polι complex. Alternatively, the polymerases spend a proportion of their time transiently immobilized. To distinguish between these alternatives, we have applied Monte Carlo simulations to the redistribution kinetics of uniformly distributed polη and ι. The best fits to the data are shown in Supplemental Figure S2 and , and they are derived from a model in which both polymerases diffuse through the cell but are transiently immobilized. As shown in , the diffusion coefficients of the two polymerases inside the cell are quite similar, but it is the proportion of transiently immobilized polη (48%) that is much greater than that of polι (17.5%) and accounts for the slower redistribution of polη than polι. The immobilization time is ~150 ms for both.
Mobility parameters of polη and i
To explore further the relationship between polη and polι inside cells, we have fractionated cell lysates by both gel filtration and glycerol gradient centrifugation and analyzed the fractions for the polymerases by immunoblotting. Gel filtration separates proteins on the basis of their size and shape, whereas glycerol gradient fractionates on the basis of sedimentation coefficient, which is determined by mass, size, and shape (see Materials and Methods). Using gel filtration (A), we found that polη and polι were associated with complexes of different Stokes radii, and interestingly the exclusion of polη increased following UV-irradiation. On the glycerol gradients (B), both polymerases sedimented at approximately the same rate and this was independent of UV-irradiation. Putting these data together (C) suggests that polη and ι are in complexes of 112 and 130 kDa, respectively, somewhat greater than the molecular weights of the polymerases themselves (78 kDa).
Figure 4. Fractionation of polη and polι from cell lysates. (A) Lysates from unirradiated or UV-irradiated MRC5 cells were fractionated on a Superdex 200 gel filtration column, and fractions were analyzed by immunoblotting for polη and polι. (more ...)
Combining the biochemical with the cell biological data, we conclude that the majority of polη and ι molecules diffuse independently in the cell, possibly complexed with other proteins, but the major difference in their mobilities results from the larger fraction of transiently immobilized polη than polι.
Effect of Chromatin Structure on Mobility of Polymerases
To gain further insight into factors affecting the intracellular mobilities of the polymerases, we looked for ways of disrupting chromatin structure to expose the DNA. We made use of the intercalating agent DRAQ5, which binds to DNA with selectivity for A-T base pairs (Njoh et al., 2006
). DRAQ5 has recently been shown to disrupt chromatin structure (Wojcik and Dobrucki, 2008
), and we have shown that the immobile fraction of transcription factor TFIIH becomes mobilized on treatment of cells with DRAQ5 (Giglia-Mari and Vermeulen, unpublished data). We measured the effect of DRAQ5 on the mobility of the core histone H2B. Histones are normally completely immobile in chromatin, but remarkably, 20% of H2B became mobile within minutes of DRAQ5 treatment (A). This result is consistent with findings of Wojcik and Dobrucki (2008)
. After 1 h in DRAQ5, the original immobility was restored (data not shown). These data suggest that DRAQ5 causes a temporary opening up of the chromatin structure. We next exposed cells to DRAQ5 and measured the effects on the mobilities of polη and ι. Strikingly, we found that treatment of cells in which polη is uniformly distributed resulted in a long-lasting immobilization of 25% of the total polη population within 3 min (A). In contrast, the effect on the mobility of polι was much smaller (A), with just a slightly reduced mobility and <5% increase in the long-lasting immobile fraction. The effect of DRAQ5 on polη was temporary, and normal mobility was restored within 1 h (data not shown), consistent with the reimmobilization of H2B. We interpret these data as follows: DRAQ5 loosens chromatin structure resulting in release of histones and exposure of the DNA to nucleoplasmic proteins. Polη is then able to bind to DNA and becomes immobilized for a long time (in contrast to the very transient immobilization seen under normal conditions). We can exclude the possibility that DRAQ5 generates a DNA damage response that somehow accounts for the observed changes in mobility, because DRAQ5 treatment does not result in either ubiquitination of PCNA or activation of a DNA damage checkpoint (Verbiest, Mari, Gourdin, Sabbioneda, Wijgers, Dinant, Lehmann, Vermeulen, and Giglia-Mari, unpublished data).
Figure 5. Effects of DRAQ5 on the mobilities of polη and ι. (A) Effect of DRAQ5 on the mobility of eGFP-histone H2B, eGFP-pol η, and eGFP-polι. Cells were treated with or without DRAQ5 for 3 min and then subjected to FRAP analysis. (more ...)
Polι has a lower affinity for DNA than polη and remains mobile. Consistent with the idea that polι is more loosely associated with nuclear structures than polη, we confirmed our earlier findings (Kannouche and Lehmann, 2004
) that polη localized in foci was resistant to extraction with triton, whereas polι was quantitatively extracted under identical conditions (B).