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Genes to Cells (2006) 11, 731-744. doi:10.1111/j.1365-2443.2006.00974.x
© 2006 Blackwell Publishing or its licensors
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A human DNA polymerase {eta} complex containing Rad18, Rad6 and Rev1; proteomic analysis and targeting of the complex to the chromatin-bound fraction of cells undergoing replication fork arrest

Mayumi S. Yuasa1,2, Chikahide Masutani1, Akihiko Hirano1, Martin A. Cohn3, Masaru Yamaizumi4, Yoshihiro Nakatani3 and Fumio Hanaoka1,5,*

1 Graduate School of Frontier Biosciences, Osaka University, and SORST, Japan Science and Technology Agency, 1-3 Yamada-Oka, Suita, Osaka 565-0871, Japan
2 Graduate School of Medicine, Osaka University, 2-2 Yamada-Oka, Suita, Osaka 565-0871, Japan
3 Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115 USA
4 Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan
5 Celluler Physiology Laboratory, RIKEN Discovery Research Institute, Wako-shi, Saitama 351-0198, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
DNA polymerase eta (Pol{eta}) is responsible for efficient translesion synthesis (TLS) past cis-syn cyclobutane thymine dimers (TT dimers), the major DNA lesions induced by UV irradiation. Loss of human Pol{eta} leads to xeroderma pigmentosum variant syndrome, clearly indicating that Pol{eta} plays a vital role in preventing skin cancer caused by exposure to sunlight. To further examine Pol{eta} functions and the mechanisms that regulate this important protein, Pol{eta} complexes were purified from HeLa cells over-expressing epitope-tagged Pol{eta}, and polypeptides associated with Pol{eta}, including Rad18, Rad6 and Rev1, were identified by a combination of mass spectrometry and Western blot analysis. The chromatin-bound fractions of cells subjected to UV irradiation, S phase synchronization, or S phase arrest were specifically enriched in such complexes. These results suggest that arrested replication forks strengthen interactions among Pol{eta}, Rad18/Rad6 and Rev1, consistent with the requirement for effective TLS by Pol{eta} at sites of DNA lesions.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Replicative DNA polymerases cannot insert nucleotides opposite damaged sites on template DNA due to their high fidelity and stringency of nucleotide incorporation, and replication is stalled (Echols & Goodman 1991). Cells have several processes that prevent acute cell death caused by replication blockage. A major pathway that counteracts replication blockage due to DNA damage is translesion synthesis (TLS), in which specialized DNA polymerases, such as the Y family of DNA polymerases (Ohmori et al. 2001), are thought to be involved in TLS past various DNA lesions. Higher eukaryotic cells have four Y family members, DNA polymerase {eta} (Pol{eta}) (Johnson et al. 1999; Masutani et al. 1999), Pol{iota} (McDonald et al. 1999), Pol{kappa} (Gerlach et al. 1999; Ogi et al. 1999) and Rev1, which has a DNA-dependent deoxycytidyl transferase activity (Gibbs et al. 2000). In vitro analyses indicate that Y family members lack 3'-5' proofreading exonuclease activities and that they replicate undamaged DNA with low fidelity and in a distributive manner (Boudsocq et al. 2002; Friedberg et al. 2002; Goodman 2002; Kunkel et al. 2003). Since they do not strictly sense if Watson-Crick pairing is correctly formed, their high misincorporation rate enables them to catalyze TLS past a variety of lesions, and their low processivity restricts their activity to damaged sites. Among these TLS polymerases, Pol{eta} is noteworthy since deficiencies in the human protein have been found to be responsible for the variant complementation group of xeroderma pigmentosum (XP-V) (Johnson et al. 1999; Masutani et al. 1999), which is characterized by sunlight sensitivity and a cancer-prone syndrome (Kraemer et al. 1994; Berneburg & Lehmann 2001). Pol{eta} efficiently carries out TLS past the TT dimer (Johnson et al. 1999; Masutani et al. 1999), which is the most abundant and most slowly repaired DNA lesion induced by UV light. In most cases TLS is achieved by inserting two adenines opposite the dimer, as has been shown in vitro (Johnson et al. 2000; Masutani et al. 2000). Cells from XP-V patients are impaired in replicating UV-damaged DNA and exhibit UV-induced hypermutability (Lehmann et al. 1975; Maher et al. 1976; Wang et al. 1991, 1993; Waters et al. 1993; McGregor et al. 1999). The ectopic expression of Pol{eta} in XP-V cells restores UV-induced mutagenesis to normal levels (Stary et al. 2003; King et al. 2005). Pol{eta} knockdown cells generated by siRNA also show an increased mutation frequency (Choi & Pfeifer 2005). These observations indicate that the other DNA polymerases bypass this lesion inaccurately in the absence of Pol{eta}, and that Pol{eta} is biologically important because it confers protection from UV-induced mutagenesis in vivo. Given the error-prone activity of Pol{eta} on undamaged templates in vitro, it is likely that its activity is tightly restricted to lesion sites in vivo, but the cellular mechanisms that regulate Pol{eta} have not been elucidated. In addition, while the enzymatic properties of TLS polymerases in the presence of various lesions have been identified by in vitro analyses, how they specialize for different lesions in vivo is unknown. Recently, it was reported that Pol{eta} is recruited to lesion sites in stalled replication forks through interaction with Rad18 and that the interaction of Pol{eta} with mono-ubiquitinated PCNA may mediate polymerase switching (Kannouche et al. 2004; Watanabe et al. 2004). In addition, we have shown that the binding properties of Pol{eta} with respect to certain DNA structures enable the enzyme to carry out faithful TLS past TT dimers and allow it to replace replicative polymerases (Kusumoto et al. 2004). Furthermore, protein–protein interactions involving TLS proteins have been reported, including interactions of Rev1 with the TLS polymerases Pol{eta}, Pol{kappa}, Pol{iota} (Guo et al. 2003; Ohashi et al. 2004; Tissier et al. 2004) and the interaction of Pol{eta} with Pol{iota} (Kannouche et al. 2003), suggesting the existence of a TLS multicomplex that includes Rev1 as a platform to allow switching between TLS polymerases at stalled replication forks (Friedberg et al. 2005; Lehmann 2005; Prakash et al. 2005).

To better understand how Pol{eta} is regulated in vivo, we purified it as multimeric protein complexes from HeLa cells over-expressing an epitope-tagged version of the protein. Mass spectrometry of the polypeptides associated with Pol{eta} identified Rad18 and Rad6 and Western blot analyses identified Rev1 in addition to Rad18 and Rad6 in these complexes. Like Rev1, Rad18 was also previously reported to interact with Pol{eta}, and Rad18 was reported to bind to Rad6, thus validating our approach. In this study, we describe the purification of Pol{eta} complexes including Rad18, Rad6 and Rev1 and we demonstrate that similar complexes accumulate in chromatin-bound fractions after UV irradiation. We also show that these increased interactions between Pol{eta} and Rad18, Rad6 and Rev1 are caused by replication fork blockage and/or other cellular responses and we discuss the biological significance of Pol{eta} complexes.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Identification of Pol{eta}-interacting proteins

To identify proteins that interact with human Pol{eta}, we generated HeLa/Pol{eta} cells that stably express Pol{eta} tagged at the C-terminus with the FLAG-HA-epitope (e-Pol{eta}) by using a retroviral transduction system. The tagged protein was expressed over tenfold more abundantly than endogenous Pol{eta}. The UV sensitivities of XP-V cells were corrected by expression of the tagged protein (data not shown), indicating that the tags did not disturb essential functions of Pol{eta}. e-Pol{eta} was purified from nuclear extracts of HeLa/Pol{eta} cells by sequential immunoprecipitations with anti-FLAG and anti-HA antibodies as a major band of 86 kDa (Fig. 1A), as confirmed by Western analysis with anti-HA antibodies (Fig. 2B). Coomassie staining of the gel showed that many polypeptides copurified with e-Pol{eta} (Fig. 1A, lane 2). Few polypeptides were detected in mock-purified control extracts from untransduced HeLa cells (Fig. 1A, lane 1), indicating that Pol{eta}-interacting proteins were specifically copurified. Some smaller polypeptides were recognized by antibodies specific to the tags or to Pol{eta} (data not shown), indicating that these were degraded Pol{eta} polypeptides (Fig. 1A, asterisks).


Figure 1
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  		Figure 1  Purification of Pol {eta} complexes. (A) FLAG-HA-epitope-tagged Pol{eta} (e-Pol{eta}) was purified from HeLa cells by immunoprecipitation with the anti-FLAG antibody followed by the anti-HA antibody. The eluted fraction was resolved by 4–12% Bis-Tris NuPAGE (Invitrogen) as described in Experimental procedures and visualized by Coomassie Brilliant Blue staining (lane 2). As a control, untransduced HeLa cells were mock purified (lane 1). e-Pol{eta}, Rad18, tubulin, and Rad6 were identified from mass spectrometric analyses of major bands. Rev1 was identified by Western blot analysis. (B) 2D gel electrophoresis was performed to separate two isoforms of Rad6 (HR6A and HR6B) in the nuclear extract (NE, upper panel) and in the purified e-Pol{eta} complexes (lower panel). NE and purified Pol{eta} complexes not subjected to immunoprecipitation were loaded on the left of the second dimensional gel (In). HR6A and HR6B were detected by Western analysis with anti-Rad6 antibodies.

 

Figure 2
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  		Figure 2  Separation of Pol{eta} complexes by glycerol gradient sedimentation. Purified Pol{eta} complexes were separated on a 10–40% glycerol density gradient by centrifugation. Marker proteins: myoglobin (17 kDa, myo); ovalbumin (44 kDa, oval); {gamma}-globulin (158 kDa, {gamma}-glob) and thyroglobulin (670 kDa, thyro) were separated as standards. Input (In) and fractions (1–25; top to bottom) were resolved by (A) Nu-PAGE and visualized by silver staining and (B) immunoblot analysis with an anti-HA antibody (to detect e-Pol{eta}) and anti-Rad18, anti-Rad6 and anti-Rev1 antibodies.

 
To identify each polypeptide in the affinity-purified Pol{eta} fraction, the region of the gel containing these polypeptides was subdivided into four slices that were subjected to mass spectrometric analysis (Supplementary Fig. 1A). Mock-purified samples were included as negative controls. We identified many polypeptides specific to e-Pol{eta} complexes (Supplementary Table S1). Several major bands detected in the affinity-purified Pol{eta} fraction were individually excised from the gel, treated with trypsin, and subjected to mass spectrometric analysis (Supplementary Fig. 1B). From a gel slice containing 70 kDa bands, nine polypeptides estimated to be derived from human Rad18 were identified (Supplementary Table S2). Also, polypeptides corresponding to Rad18 sequences were identified in the gel slice containing 80 kDa bands (Supplementary Table S2), whereas Rad18 was never identified in mock-purified samples. Both the 70 kDa and 80 kDa polypeptides in e-Pol{eta} complexes were recognized by anti-Rad18 antibodies (Fig. 2B). Two forms of Rad18 have been observed in human cells, one non-ubiquitinated and the other mono-ubiquitinated (Miyase et al. 2005). Thus, the two forms of Rad18 in e-Pol{eta} complexes are likely to be the non-ubiquitinated and mono-ubiquitinated proteins. Polypeptides derived from human Rad6 were identified in a gel slice containing the 17 kDa band (Supplementary Table S2). Human cells have two Rad6 homologs, designated HR6A and HR6B, which share about 95% identity (Koken et al. 1991), and we could not determine which was present in the Pol{eta} fraction. It has been reported that HR6A is more abundant in HeLa cells and that its isoelectric point is more basic than that of HR6B (Koken et al. 1996). We therefore subjected a portion of the affinity-purified fraction to 2D gel electrophoresis and detected Rad6 by Western analysis. As shown in Fig. 1B, spots corresponding to an abundant basic protein and a less abundant acidic protein, probably HR6A and HR6B, respectively, were observed in e-Pol{eta} complexes as well as in nuclear extracts, suggesting that both Rad6 homologs are present in these complexes. The 50 kDa bands proved to be a mixture of {alpha}-tubulin and ß-5 tubulin (Fig. 1A, lane 2). Although tubulins were prominent components of the purified complexes, we consider that their associations with e-Pol{eta} are not biologically significant at present.

We failed to find Rev1 sequences in the affinity-purified Pol{eta} fraction by mass spectrometric analysis. However, in separate experiments, we identified Pol{eta} sequences in the affinity-purified Rev1 fraction from HeLa/Rev1 cells over-expressing FLAG-HA-tagged Rev1 protein by mass spectrometric analysis (data not shown). We therefore probed the Pol{eta} fraction purified from HeLa/Pol{eta} cells with an anti-Rev1 antibody and found that Rev1 was indeed present in the fraction (Fig. 2B). The other two Y-family polymerases, Pol{iota} and Pol{kappa}, were not identified by our present analyses. Here we focus on interactions between Pol{eta} and the Rad18, Rad6 and Rev1 proteins, which are all involved in DNA damage tolerance pathways.

Separation of Pol{eta} complexes by glycerol density gradient sedimentation

To further separate the multiple Pol{eta} complexes, we developed the affinity-purified Pol{eta} fraction on a 10–40% glycerol gradient by ultracentrifugation and resolved the resulting fractions by PAGE. Silver staining (Fig. 2A) and Western blot analysis with the anti-HA antibody (Fig. 2B) showed that e-Pol{eta} was present in fractions throughout the gradient, suggesting that it was abundant and/or that it interacted with other proteins. e-Pol{eta} in fraction 3 could be a monomeric form unassociated with other proteins, as judged from its low sedimentation value together with the observation that there was no other protein of similar abundance in this fraction (Fig. 2A).

Western blot analysis (Fig. 2B) revealed that the bulk of Rad18 and Rad6 cosedimented in fraction 5. Judging from the Coomassie staining (Fig. 1A) and silver staining (Fig. 2A) profiles, almost equivalent amounts of Rad6 and Rad18 proteins were present in this fraction. All of the Rad6 protein in Pol{eta} complexes cosedimented with Rad18, although human cells have more Rad6 than Rad18, suggesting that Rad6 complexed with Rad18 was specifically copurified with Pol{eta}. The estimated molecular weight of the proteins sedimented in fraction 5 (100–140 kDa) was lower than the sum of the molecular weights calculated for e-Pol{eta}, Rad18 and Rad6 together (161 kDa). However, immunoprecipitation of e-Pol{eta} from the Rad18/Rad6 peak fractions of the glycerol gradient with the anti-FLAG antibody revealed that most Rad18/Rad6 in these fractions co-immunoprecipitated with e-Pol{eta} (Supplementary Fig. 2). Thus, we conclude that a major complex consists of the e-Pol{eta}, Rad18, and Rad6 proteins, which can be detected in fraction 5 of the glycerol gradient. In addition, trace amounts of Rad18 and Rad6 were observed from fractions 5 through at least 15, suggesting the presence of larger complexes that include Pol{eta}, Rad18/Rad6, and other proteins. Rev1 sedimented in fractions 9–19, and its peak of abundance in fraction 13 corresponded to about 670 kDa. The faster sedimentation of Rev1 in Pol{eta} complexes than expected for the total molecular mass of Rev1 and e-Pol{eta} (219 kDa) may indicate that purified Pol{eta} complexes containing Rev1 also include Rad18/Rad6 and/or other proteins.

Rad18/Rad6 and Rev1 may simultaneously complex with Pol{eta}

Since we detected Rad18/Rad6 and Rev1 in purified Pol{eta} complexes, we then asked whether Pol{eta} interacts with Rad18/Rad6 and Rev1 simultaneously. To examine this point, the affinity-purified Pol{eta} fraction (corresponding to input, Fig. 2) was further immunoprecipitated with anti-Rev1 antibody-conjugated Protein G-Sepharose beads (Fig. 3). Rev1 in the Pol{eta} complexes was immunoprecipitated by the antibody while only low levels of Rev1 were precipitated by mock treatment with protein G-Sepharose beads (compare lanes 4 and 5, Fig. 3). Low but detectable levels of Pol{eta} and Rad18 were observed in the Rev1-bound fraction (lane 4) whereas only a slight signal corresponding to Pol{eta} was detected for the mock precipitates (lane 5). The low efficiency of co-precipitation of these proteins is consistent with observations that much more e-Pol{eta} (and also Rad18) than Rev1 is included in the complexes, as demonstrated in Figs 1 and 2. Rad6 was also detectable in the Rev1-bound fraction, but the signal was often faint, suggesting that Rad6 readily dissociates from Rad18 in Rev1-containing Pol{eta} complexes. From these results, we conclude that Pol{eta}/Rev1 complexes associate with Rad18 (and Rad6), although we do not exclude the possibility that there are Pol{eta}/Rev1 complexes that do not include Rad18.


Figure 3
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  		Figure 3  Pol{eta}/Rev1 complexes including Rad18/Rad6. FLAG-HA-purified Pol{eta} complexes were immunoprecipitated with anti-Rev1 antibody-conjugated Protein G-Sepharose (lane 4) or control Protein G-Sepharose (lane 5) and immunoblotted with anti-Rev1 antibodies. The blot at left was also probed with anti-Rad18, anti-Rad6 and anti-Pol{eta} antibodies. In, input; UB, unbound fraction; C, control immunoprecipitation with Protein G-Sepharose beads.

 
The formation of Pol{eta} complexes on chromatin is enhanced after UV irradiation

In addition to proteomic analysis, we performed biochemical fractionation experiments with HeLa/Pol{eta} cells and examined the behavior of e-Pol{eta} complexes. e-Pol{eta} was detected in all fractions, and UV irradiation only moderately altered the distribution of e-Pol{eta} in each fraction, as shown in Fig. 4A. Rad18 was detected mainly in the nuclear extract (NE) and micrococcal nuclease (MNase)-soluble fractions and also in the insoluble fractions (wash and ppt). The band of highest mobility (60 kDa) was likely to be a degradation byproduct generated by these procedures, because it was rarely observed in whole cell lysates prepared by direct lysis with SDS (data not shown). Rev1 was also detected mainly in the NE and MNase-soluble fractions, and Rad6 was abundant in soluble fractions and it was also present in the NE and MNase-soluble fractions. These observations coincide with previous reports that Rad18 and Rev1 localize predominantly in nuclei (Tissier et al. 2004; Miyase et al. 2005) and that Rad6 is present in both the cytoplasmic and nuclear compartments (Lyakhovich & Shekhar 2003). After UV irradiation, the levels of Rad18 slightly increased in the MNase-soluble and -insoluble fractions (Fig. 4A, compare lanes 7, 10, 13 with 9, 12, 15), suggesting that Rad18 accumulates in chromatin bound/nuclear matrix fractions after UV irradiation. The levels of Rev1 in the MNase-soluble and -insoluble fractions were not significantly changed by UV irradiation. Changes in the distribution of Rad6 and Rad18 after UV irradiation did not coincide, reflecting the excess Rad6 protein freed from Rad18. It has been reported that PCNA is mono-ubiquitinated by Rad18/Rad6 following UV irradiation and that mono-ubiquitinated PCNA tightly associates with chromatin (Kannouche et al. 2004; Watanabe et al. 2004). In our system, non-ubiquitinated PCNA was found in all fractions. In addition to the major PCNA signal, faint bands with lower mobilities were observed. Among them was a relatively clear signal whose intensity increased in a time-dependent manner after UV irradiation and which was observed in the MNase-soluble and -insoluble fractions but almost undetectable in the NE fractions (Fig. 4A, arrow). As suggested by previous reports (Kannouche et al. 2004; Watanabe et al. 2004), this band likely represents a mono-ubiquitinated form of PCNA.


Figure 4
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  		Figure 4  Pol{eta} complexes after UV irradiation. (A) HeLa/Pol{eta} cells were irradiated with 15 J/m2 UV and incubated in fresh media for the indicated time. Cells were fractionated as described in Experimental procedures and total fractions were subjected to immunoblot analysis with anti-HA (e-Pol{eta}) and other antibodies as indicated. In the Rad18 panel, * shows a degradation product. In the PCNA panel, the arrow and the sharp show PCNA and nonspecific bands, respectively. NE, nuclear extract; MNase sol., MNase-soluble. (B) e-Pol{eta} in nuclear extracts (material corresponding to Fig. 4A) was immunoprecipitated with an anti-FLAG antibody. Nuclear extracts prepared from untransduced HeLa cells were subjected to mock immunoprecipitation with the same antibody (NC, negative control; lanes 10). The same blot was probed with the indicated antibodies. * in the Rad18 panel shows a degradation product. (C) The MNase-soluble and wash fractions were combined as a chromatin-bound fraction. e-Pol{eta} was immunoprecipitated from the chromatin-bound fractions with the anti-FLAG antibody and probed with the indicated antibodies. * shows degraded proteins derived from Rad18 or Rev1 in each panel. (D) HeLa/Pol{eta} cells were irradiated with different UV doses and incubated for 0.5 h. e-Pol{eta} was immunoprecipitated from the chromatin-bound fractions with the anti-FLAG antibody and probed with the indicated antibodies. * shows degraded proteins derived from Rad18 or Rev1 in each panel.

 
We next examined Pol{eta} complexes in the NE (Fig. 4B) and MNase-soluble fractions (Fig. 4C). As shown in Fig. 4B. e-Pol{eta} was efficiently recovered from NE fractions by the anti-FLAG antibody (lanes 7–9), while no band was detected for NE from untransduced HeLa cells (lane 10). A portion of the Rad18 and Rev1 population and lesser amounts of Rad6 were co-immunoprecipitated with e-Pol{eta} (lanes 7–9), reflecting the greater abundance of Rad6 than Rad18 in nuclear extracts. Importantly, the levels of proteins co-precipitated with e-Pol{eta} were similar irrespective of UV irradiation, although the recovery of Rad18 and Rev1 from extracts prepared 8 h after UV irradiation was slightly higher than for unirradiated cells.

Figure 4C shows an anlaysis of MNase-soluble fractions. Very low levels of Rad18, Rad6 and Rev1 co-precipitated with e-Pol{eta} from MNase-soluble fractions prepared from unirradiated cells (lane 5), suggesting that Pol{eta} associated with chromatin in unirradiated cells does not form stable complexes with Rad18/Rad6 and Rev1. Interestingly, and in contrast, the levels of Rad18, Rad6 and Rev1 co-precipitated with e-Pol{eta} from UV-irradiated cells were significantly increased, 5–7-fold, 6–10-fold and 6–7-fold, respectively (compare lane 5 with lanes 6–8), as could be observed 0.5 h after UV-irradiation, and these increased levels were maintained for at least 8 h. Although the levels of Rad18 and also Rev1 and Rad6 in MNase-soluble fractions (inputs) increased 1–2-fold after UV irradiation (compare lane 1 and lanes 2–4), the differences between irradiated and unirradiated samples were greater for Pol{eta}-bound fractions than for inputs. These results suggest that interactions of Pol{eta} with Rad18/Rad6 and Rev1 on chromatin are induced relatively soon after UV irradiation. The similar kinetic profiles of Rad18/Rad6 and Rev1 suggest that the interactions of Pol{eta} with these proteins on chromatin share a common mechanism. An enhanced co-immunoprecipitation from MNase-soluble fractions was also observed in the presence of ethidium bromide (100 µg/mL) (data not shown), suggesting that these proteins interact directly rather than indirectly via DNA.

We also examined PCNA in co-immunoprecipitated samples from MNase-soluble fractions and found that very small but detectable populations of PCNA co-precipitated with e-Pol{eta} (Fig. 4C, lanes 5–8). The levels of PCNA that co-precipitated with e-Pol{eta} were greater for UV-irradiated than for unirradiated cells (lanes 5–8, longer exposure), suggesting that interactions between Pol{eta} and PCNA on chromatin are also enhanced by UV irradiation. The more slowly migrating band, which probably represents mono-ubiquitinated PCNA, accumulated in the MNase-soluble fractions in a time-dependent manner (lanes 2–4) and co-precipitated with e-Pol{eta} (lanes 6–8). This profile may reflect the presence of arrested replication forks. The different kinetic profiles of PCNA with Rad18/Rad6 and Rev1 in Pol{eta} interactions may suggest that Pol{eta} interacts with Rad18/Rad6 and/or Rev1 prior to replication fork arrest and mono-ubiquitination of PCNA. The ratio of non-ubiquitinated to ubiquitinated PCNA protein in inputs and co-immunoprecipitated fractions was not constant. Specifically, when the same levels of ubiquitinated PCNA were observed in inputs and co-precipitated fractions (e.g. compare lane 4 with lane 8, longer exposure), the levels of non-ubiquitinated PCNA were much greater in the input than in the precipitated fraction, suggesting that Pol{eta} preferentially interacts with mono-ubiquitinated PCNA, consistent with previous reports (Kannouche et al. 2004; Watanabe et al. 2004). The interactions of Pol{eta} with Rad18/Rad6 and Rev1 increased again after UV irradiation in a dose-dependent manner up to 15 J/m2 and reached a plateau at 50 J/m2 (Fig. 4D). In addition, mono-ubiquitinated PCNA increased after UV irradiation in a dose-dependent manner up to 15 J/m2 and reached a plateau at 50 J/m2, and the levels of PCNA co-precipitated with Pol{eta} also increased with similar kinetics (Fig. 4D).

Replication arrest enhances the formation of Pol{eta} complexes

To examine whether the increased interactions between e-Pol{eta} and Rad18/Rad6, Rev1 and PCNA in chromatin fractions after UV irradiation were associated with DNA replication blocks or other cellular responses, co-immunoprecipitation experiments were performed with MNase-soluble fractions prepared from synchronized cells. HeLa/Pol{eta} cells were synchronized at early S phase by the double thymidine block method and released into a synchronous cell cycle. The excess thymidine depletes cells of dCTP so that the incorporation of nucleotides into DNA is substantially reduced, causing replication fork progression to slow (Bjursell & Reichard 1973). Two hours after release from the thymidine block, cells were irradiated with 15 J/m2 UV or mock-irradiated and incubated for an additional 0.5 h. HeLa/Pol{eta} cells were effectively synchronized in early S phase (0 h), and 2.5 h after release, the majority of the cells were in S phase (Fig. 5A). The level of Rad18 in MNase-soluble fractions (inputs) from synchronized cells at early S phase (0 h) was increased 1.5-fold compared to that from asynchronous samples (Fig. 5B, compare lanes 1 and 2), and e-Pol{eta}, Rad6 and Rev1 behaved similarly. Mono-ubiquitinated PCNA was also increased in inputs from 0 h samples compared to that from asynchronous samples (compare lanes 1 and 2). Thus, synchronization was characterized by the association of these proteins with chromatin. At 2.5 h after the release from the thymidine block, the levels of these proteins in MNase-soluble fractions (inputs) decreased (compare lanes 2 and 3). The levels of Rad18 and mono-ubiquitinated PCNA slightly increased again after UV irradiation (compare lanes 3 and 4), although this effect was not so clear for Rad6, Rev1 and e-Pol{eta}. These results suggest that the accumulation of these proteins in chromatin fractions is associated with replication fork arrest caused by nucleotide depletion or DNA lesions.


Figure 5
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  		Figure 5  Interactions between e-Pol{eta} and Rad18, Rad6, Rev1 proteins are increased by blocking replication forks. (A) HeLa/Pol{eta} cells were synchronized by the double thymidine block method. Cells were harvested immediately after release from double thymidine block (0 h). Two hours after release, cells were irradiated with 15 J/m2 UV or mock irradiated and incubated in fresh media for an additional 0.5 h and harvested (2.5 h, 2 h+ UV0.5 h). The cell cycle profiles of these samples were analyzed by flow cytometry and representative DNA histograms of these samples are shown. (B) e-Pol{eta} in chromatin-bound fractions prepared from the indicated samples was immunoprecipitated with the anti-FLAG antibody and immunoblotted with the indicated antibodies. * shows degraded proteins derived from Rad18. AS, asynchronous cells. (C) HeLa/Pol{eta} cells were treated with 1 mM HU for 12 h and released into fresh media, and cells were harvested at 0 h and 4 h after release. DNA histograms analyzed by flow cytometry are shown. (D) e-Pol{eta} in chromatin-bound fractions prepared from the indicated samples was immunoprecipitated with the anti-FLAG antibody and immunoblotted with the indicated antibodies. * shows degraded proteins derived from Rev1. The sharp shows nonspecific bands.

 
The recovery of co-immunoprecipitated Rad18, Rad6, Rev1 and non- and mono-ubiquitinated PCNA from synchronized cells at early S phase was dramatically increased compared with that from asynchronous cells (compare lanes 5 and 6). Differences between synchronized and asynchronous cells were clearly greater for bound fractions than for inputs (compare lanes 5 and 6 with 1 and 2), indicating that interactions between Pol{eta} and these proteins are increased by synchronization. Interestingly, after release from the thymidine block, the levels of Rad18, Rad6, Rev1 and PCNA co-precipitating with Pol{eta} diminished rapidly in S phase cells (compare lanes 6 and 7), suggesting that the complexes collapsed or were destabilized soon after replication re-start. The amount of proteins co-precipitating with Pol{eta} was increased again by UV irradiation of S phase cells 2 h after release (compare lanes 7 and 8). However, the recovery of co-precipitated proteins from these S phase cells was lower than that from unirradiated synchronized cells in early S phase (compare lanes 6 and 8). These results suggest that UV-induced complex formations might be promoted by UV lesion-induced replication arrest in S phase cells.

We also examined the effects of hydroxyurea (HU), which depletes cells of deoxyribonucleotides by inhibiting ribonucleotide reductase activities, resulting in an accumulation of stalled replication forks. HeLa/Pol{eta} cells treated with HU (12 h) were harvested immediately and 4 h after release. Cells were arrested in S phase at 0 h after release, and by 4 h, most cells were in S and G2/M phases (Fig. 5C). As shown in Fig. 5D, co-immunoprecipitated Rad18, Rad6, Rev1 and non- and mono-ubiquitinated PCNA proteins were significantly increased in HU-treated cells compared to asynchronous cells (compare lanes 4 and 5). Four hours after release, this enhancement was almost undetectable. These results strongly suggest that arrested replication forks in S phase cells can trigger interactions between Pol{eta} and Rad18/Rad6, Rev1, and mono-ubiquitinated and non-ubiquitinated PCNA.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Pol{eta} protein complexes

In this paper we have identified human Pol{eta} complexes including Rad18/Rad6 and Rev1 in nuclear extracts of HeLa/Pol{eta} cells. Coomassie staining and glycerol gradient analysis of purified Pol{eta} complexes (Figs 1 and 2) indicate that there are multiple cellular Pol{eta} complexes. Rad18/Rad6 and Rev1, the focus of our investigations, were observed in at least two Pol{eta} complexes. One is a slowly sedimenting complex that consists of Pol{eta} and Rad18/Rad6, and the other is a larger complex that includes Rev1. From the observation that Rad18/Rad6 as well as Pol{eta} co-immunoprecipitate from the e-Pol{eta} complexes with Rev1 (Fig. 3), we conclude that Pol{eta} associates with Rad18/Rad6 and Rev1 simultaneously, although the Rev1-containing complex is less abundant than the Pol{eta}/Rad18/Rad6 complex. Yeast-two hybrid analysis has indicated that two Pol{eta} regions (amino acids 370–492 and 509–557) are involved in the interaction with Rev1 (Ohashi et al. 2004; Tissier et al. 2004), and pull down analysis has shown that the C-terminal portion of Pol{eta} (amino acids 556–713) interacts with Rad18 (Watanabe et al. 2004). Thus, the Pol{eta} residues that interact with Rev1 differ from those that interact with Rad18. Direct physical interactions between Rad18 and Rev1 have not been reported so far. These results suggest that Rad18 and Rev1 form a complex through Pol{eta}. Interactions between Rad18 and Pol{eta}, and also between Rad18 and Rad6, have been reported to be required for recruiting Pol{eta} to replication stalling sites (Watanabe et al. 2004). The interaction of Rev1 with Pol{eta} may contribute to TLS polymerase switching (Friedberg et al. 2005; Lehmann 2005; Prakash et al. 2005), although the full significance of this interaction remains unclear. Thus, our finding that Pol{eta} simultaneously interacts with Rad18/Rad6 and Rev1 suggests that there are mechanisms that coordinate DNA polymerase recruitment and switching for TLS. While we have focused here on proteins that have been found to interact with Pol{eta} by other methods, analyses of other Pol{eta} complexes, which are ongoing, may give new clues about TLS regulatory mechanisms.

Formation of Pol{eta} complexes on chromatin after UV irradiation

By biochemical fractionation of HeLa/Pol{eta} cells coupled with immunoprecipitation, we found that Pol{eta} interacts with Rad18/Rad6 and/or Rev1 proteins in chromatin fractions after UV irradiation. The complexes in the chromatin fractions are not necessarily equivalent to those in NE fractions, which are only moderately affected by irradiation, although they may be structurally related. As we over-expressed Pol{eta} in HeLa cells, it is possible that the complexes in NE fractions may be due to unphysiological concentrations of Pol{eta} and that only the complexes in chromatin fractions are physiologically relevant.

The similar kinetic profiles of Rad18/Rad6 and Rev1 in association with Pol{eta} on chromatin strongly suggest that these associations result from concerted actions, although Rev1 was less abundant than Rad18 in complexes, as estimated from Western analysis with purified proteins (data not shown). As UV irradiation increased the proportions of these proteins in chromatin fractions, one possible explanation is that complexes in NE fractions translocate to chromatin after UV irradiation. However, the increases in Pol{eta}-associated Rad18/Rad6 and Rev1 proteins after UV irradiation were greater than those accounted for by translocation to chromatin (Fig. 4C). In addition, similar dynamics of translocation of Rad18 protein have been observed in UV-irradiated XP2SA cells lacking Pol{eta} (data not shown) as well as in HeLa/Pol{eta} cells, although UV-induced dynamics of Rev1 translocation were not obvious in either cell type. This observation reinforces the idea that at least Rad18 is recruited to damaged chromatin largely independently of Pol{eta}. It has been shown by immunofluorescence analysis that Rad18 disperses rapidly (within 20 min) after UV irradiation from discrete irregular dots in the nucleus, resulting in a rapid increase in a diffuse form of Rad18 (Watanabe et al. 2004; Masuyama et al. 2005). Such rapid responses might be regulated by post-translational protein modifications such as phosphorylation. Consistent with this, non-ubiquitinated Rad18 is observed as broad multiple bands, as can be seen in Fig. 4, although we do not yet have evidence that these bands reflect post-translational modifications of the protein. After dispersion, Rad18 co-localizes with eGFP-Pol{eta} in foci within several hours (Watanabe et al. 2004). Pol{eta} focus formation increases gradually in a time-dependent manner up to several hours after UV irradiation, probably reflecting the accumulation of arrested replication forks (Kannouche et al. 2001). Combining these observations with our present result that Pol{eta} complexes form relatively soon (0.5 h) after irradiation in chromatin, we propose that the dispersion of Rad18 increases the likelihood of interaction with Pol{eta}, which may result in increased interactions between Rad18 with Pol{eta} on chromatin prior to Pol{eta} focus formation. We also speculate that there are unknown mechanisms that increase interactions between these proteins, in a coordinated way, in addition to Rad18 dispersion. A more complete elucidation of the biological significance and molecular bases of UV-induced complex formation on chromatin require the purification and identification of Pol{eta} complexes from the chromatin of UV-irradiated cells.

Triggers for Pol{eta} complex formation

We found that cellular synchronization by the double thymidine block method or by hydroxyurea treatment drastically enhances the formation of Pol{eta} complexes in chromatin (Fig. 5). These treatments perturb ongoing replication, resulting in the accumulation of arrested replication forks. The release of cells from these stressors diminishes interactions of Pol{eta} with Rad18/Rad6 and Rev1, suggesting that arrested replication forks closely correlate with interactions among these proteins. Taking these results together, we assume that increased protein interactions after UV irradiation are triggered by arrested replication forks as a result of DNA damage. Indeed, we also observed increased interactions of Pol{eta} with Rad18/Rad6 and Rev1 0.5 h after UV irradiation in G2/M phase cells, but the increases were smaller than those observed in asynchronous and S phase cells (data not shown). Closer scrutiny to discriminate between DNA damage responses and replication arrests in UV-irradiated cells is required to determine the cause of the increased interactions of Pol{eta} with these proteins. Although we noted that interactions of Pol{eta} with Rad18/Rad6 increased prior to the accumulation of the mono-ubiquitinated form of PCNA after UV irradiation, Rad18/Rad6 bound to Pol{eta} is not essential for PCNA mono-ubiquitination, since mutant Rad18 lacking the Pol{eta} binding site can mono-ubiquitinate PCNA after UV irradiation in vivo (Watanabe et al. 2004). The biological significance of the increased interactions of Pol{eta} with Rad18/Rad6 and Rev1 remains to be resolved.

Our data provide an attractive model to describe the cooperation between normal DNA replication and Pol{eta} complex formation: replication arrest causes an increase in interactions between Pol{eta}, Rad18/Rad6 and Rev1, which probably provides an opportunity for Pol{eta} to function at sites containing lesions and for effective TLS. The UV-induced DNA damage response may also increase interactions of Pol{eta} with these proteins, and the release of stalled replication forks may diminish interactions among Pol{eta} and Rad18, Rad6, and Rev1.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Purification and identification of Pol{eta} complexes

HeLa cells stably expressing Pol{eta} tagged at the C-terminus with the FLAG-HA-epitope (e-Pol{eta}, HeLa/Pol{eta} cells) were established with a retroviral transduction system as previously described (Ogawa et al. 2002; Nakatani & Ogryzko 2003). Briefly, HeLa cells were transduced with a recombinant retrovirus expressing a bicistronic mRNA encoding FLAG-HA tagged human Pol{eta} (e-Pol{eta}) linked to an interleukin-2 receptor {alpha} as a cell surface marker. The transduced subpopulation of cells was purified by repeated cycles of affinity sorting. Resulting HeLa/Pol{eta} cells were grown to a final volume of 12 L with a density of ~1 x 106 cells/mL in Dulbecco's modified Eagle's medium containing 5% calf serum. Nuclear extracts were prepared as previously described (Dignam et al. 1983a,b; Tagami et al. 2004). e-Pol{eta} was affinity purified from nuclear extracts with an anti-FLAG M2 antibody affinity agarose gel (SIGMA) followed by anti-HA antibody (12CA5)-conjugated Protein A-Sepharose 4 Fast Flow (Amersham Biosciences) or anti-HA antibody affinity agarose gels (SIGMA, HA7) as previously described (Ikura et al. 2000; Ogawa et al. 2002; Nakatani & Ogryzko 2003; Tagami et al. 2004). Briefly, 15 mL nuclear extracts (98 mg protein) were incubated with 0.6 mL of the bead suspension (50% slurry) of anti-FLAG-conjugated beads at 4 °C for 3 h, washed with wash buffer (20 mM Tris-HCl [pH 8.0], 100 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 0.1% Tween 20, 2 mM ß-mercaptoethanol, 0.2 mM PMSF), and bound materials were eluted with 400 µg/mL FLAG peptide. The eluate was incubated with 0.3 mL of the bead suspension (50% slurry) of anti-HA-conjugated beads at 4 °C for 3 h, washed with wash buffer, and bound materials were eluted with 500 µg/mL HA peptide. Proteins were reduced and separated on 4–12% Bis-Tris Nu-PAGE gels (Invitrogen) formulated for denaturing gel electrophoresis in MOPS buffer according to the Invitrogen instruction manual. For the identification of all proteins in Pol{eta} complexes, proteins were treated with iodoacetoamide and subjected to electrophoresis on 10% Bis-Tris Nu-PAGE gels. The run was stopped when the buffer front had migrated 1.8 cm into the gel, and proteins were stained with Coomassie Brilliant Blue (Supplementary Fig. 1A). The entire gel was then cut into four regions which were digested in-gel with trypsin and analyzed by MALDI-TOF mass spectrometry and/or by LC-MS/MS mass spectrometry at the Taplin Biological Mass Spectrometry Facility at the Harvard Medical School.

For the identification of several major proteins in the Pol{eta} complexes, major protein bands detected by silver staining (Silver stain MS kit, Wako) (Supplementary Fig. 1B) were individually cut out from gels, digested by trypsin in situ and analyzed by MALDI-TOF mass spectrometry and/or by LC-MS/MS mass spectrometry at the APRO Life Science Institute, Inc.

Glycerol density gradient sedimentation

A portion (50 µL) of the affinity-purified fraction from nuclear extracts was loaded on to a 5 mL 10–40% glycerol gradient in HA elution buffer (100 mM Tris-HCl [pH 6.8], 100 mM KCl, 0.2 mM EDTA, 0.1% Tween 20, 2 mM ß-mercaptoethanol, 0.2 mM PMSF), centrifuged at 55 000 r.p.m. in a Beckman SW55Ti rotor for 4 h at 4 °C, and 200 µL fractions were collected.

Two-dimensional gel electrophoresis and immunoblotting of hHR6 proteins

Nuclear extracts or purified Pol{eta} complexes were subjected to two-dimensional gel electrophoresis according to the Invitrogen instruction manual (ZOOM IPG Runner, NuPAGE, Invitrogen). Gels adjusted to pH 4.5–5.5 were used for first dimension electrophoresis and 4–12% Bis-Tris gels were used for second dimension electrophoresis. Proteins were blotted on to a PVDF membrane, and hHR6A and hHR6B were detected with an anti-Rad6 antibody (Boston Biochem).

Immunoprecipitation with the anti-Rev1 antibody

A portion (20 µL) of the affinity-purified fraction from nuclear extracts was diluted to 95 µL with HA-elution buffer (100 mM Tris-HCl [pH 6.8], 100 mM KCl, 0.2 mM EDTA, 0.1% Tween 20, 2 mM ß-mercaptoethanol, 0.2 mM PMSF) containing 20% glycerol. The diluted HA-eluted fraction was incubated with a 20 µL the bead suspension (50% slurry) of anti-hRev1 antibody-conjugated Protein G-Sepharose beads at 4 °C overnight. After centrifugation at 500 g for 3 min, the supernatant was collected as an unbound fraction. The beads were washed 3 times with wash buffer, and bound materials were eluted with 2.5% SDS-PAGE sample buffer. The samples were subjected to 8–14% SDS-PAGE and immunoblotted with the indicated antibodies.

Fractionation of UV-irradiated cells

HeLa/Pol{eta} cells were grown in tissue culture dishes, washed with phosphate-buffered saline (PBS), irradiated with UV at 15 J/m2, and incubated in fresh media for the periods indicated. To prepare nuclear extracts, cells grown on two 15 cm diameter dishes per sample were washed with PBS, collected by centrifugation at 200 g for 5 min at 4 °C. The cell pellets were suspended in six packed cell volumes (PCV) of hypotonic buffer (10 mM HEPES-NaOH [pH 7.9], 10 mM KCl, 1 mM EDTA, 1 mM EGTA, phosphatase inhibitor cocktail (Calbiochem), protease inhibitor cocktail (Complete, Roche)), 0.3 PCV of 10% NP40 was added, and the mixture was allowed to stand for 5 min on ice and then centrifuged at 5000 g for 10 min at 4 °C. The supernatant was collected as a soluble fraction. Nuclear extracts were prepared principally as described above. Briefly, the pellet was suspended in 1/2 PCV of low salt buffer (20 mM Tris-HCl [pH 7.3], 20 mM KCl, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 2 mM ß-mercaptoethanol and 0.2 mM PMSF), and then 1/2 PCV of high salt buffer (20 mM Tris-HCl [pH 7.3], 1.2 M KCl, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 2 mM ß-mercaptoethanol and 0.2 mM PMSF) was added with gentle mixing. After incubation on ice for 30 min, the sample was centrifuged at 20 000 r.p.m. for 30 min at 4 °C in a TLA 45 rotor (Beckmann). The nuclear pellet was stored at –80 °C except for Fig. 4D, in which unfrozen pellet was used. The supernatant was dialyzed against BC-100 (20 mM Tris-HCl [pH 7.3], 100 mM KCl, 20% glycerol, 0.2 mM EDTA, 2 mM ß-mercaptoethanol, 0.2 mM PMSF) at 4 °C for 2 h and then centrifuged at 20 000 r.p.m. for 20 min at 4 °C in a TLA 45 rotor. The supernatant was used as a nuclear extract fraction. The nuclear pellet was thawed and suspended in 1 PCV of micrococcal nuclease (MNase) buffer (20 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 0.3 M sucrose, 0.1% Triton X-100, 1 mM DTT, protease inhibitor cocktail [Complete, EDTA free, Roche]) as previously described (Groisman et al. 2003). MNase was added at 2.5 U/µL (Roche) and the mixture was incubated for 10 min at room temperature. After termination of the reaction by adding 5 mM EDTA, samples were centrifuged at 500 g for 5 min at 4 °C. The supernatant was collected as a MNase soluble fraction. The pellet was washed with MNase buffer and centrifuged, and the supernatant was collected as a wash fraction. The remaining pellet was suspended in MNase buffer and dissolved in LDS (Nu-PAGE) sample buffer. These fractions were loaded on 4–12% Bis-Tris Nu-PAGE gels (Invitrogen). Proteins were detected by Western analysis with indicated antibodies. Where indicated, MNase soluble fractions and wash fractions were combined and used for immuno-precipitation experiments with anti-FLAG M2 agarose affinity gel (Sigma).

Cell synchronization

HeLa/Pol{eta} cells were synchronized by the double thymidine block method as follows. HeLa/Pol{eta} cells were incubated in media containing 2.5 mM thymidine for 24 h, the excess media was removed, the cells were washed twice with PBS and incubated in fresh media without excess thymidine. After 10 h incubation in normal growth media, cells were incubated in 2.5 mM thymidine-containing media for 14 h. The synchronized cells were washed twice with PBS and then released by adding fresh growth media. For the 0 h sample, synchronized cells were harvested immediately after the replacement of thymidine-containing media with fresh media. After 2 h incubation in fresh media after release from the thymidine block, the cells were washed with PBS, irradiated with 15 J/m2 UV or mock irradiated, incubated for an additional 0.5 h, and then harvested. For HU treatment, HeLa/Pol{eta} cells were incubated in media containing 1 mM HU for 12 h, washed twice with PBS, incubated in fresh media without HU, and then harvested at the indicated time points.

FACS analysis

Asynchronous or synchronized HeLa/Pol{eta} cells (2 x 106) were harvested, washed twice with PBS and fixed in 70% ethanol in PBS at 4 °C overnight. Fixed cells were collected by centrifugation at 200 g for 5 min, washed twice with PBS and suspended in 1 mL PBS. Five hundred micrograms of RNase A was added to the cell suspensions and the mixtures were incubated for 30 min at 37 °C. Propidium iodide (20–50 µg) was added and the cell suspensions were subjected to FACS analyses (BectonDickinson, with CellQuest software).

Antibodies

Mouse monoclonal anti-Pol{eta} (B-7) and rabbit polyclonal anti-PCNA (FL-261) antibodies were purchased from Santa Cruz Biotechnology. Rat monoclonal anti-HA (3F10) antibody was purchased from Roche. Rabbit polyclonal anti-Rad6 (Ubch2) antibody was purchased from Boston Biochem. Anti-Rev1 antibody was prepared by immunizing a guinea pig with purified polypeptides consisting of amino acids (aa) 810–1251 of Rev1, as described elsewhere. Anti-Rad18 antibody was raised by immunizing rabbits with a Rad18 fragment (aa 383–495) as described (Tateishi et al. 2000).


   Acknowledgements
 
We would like to thank Yoshiaki Ohkuma, Masayuki Yokoi and other members of Hanaoka's laboratory at Osaka University for helpful discussions; Steven Gygi and colleagues at the Taplin Biological Mass Spectrometry Facility for mass spectrometric analyses. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Human Frontier Science Program, and by Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency. This work was also supported by the Bioarchitect Research and Chemical Biology Research Projects of RIKEN. M. S. Y. was supported by a fellowship from the Center of Excellence (COE) of the Japan Society for the Promotion of Science.


   Footnotes
 
Communicated by: Hiroyuki Araki

* Correspondence: E-mail: fhanaoka{at}fbs.osaka-u.ac.jp


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Top
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 Introduction
 Results
 Discussion
 Experimental procedures
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