Characterization of a second proliferating cell nuclear antigen (PCNA2) from Drosophila melanogaster Tatsushi Ruike 1 , Ryo Takeuchi 1 , Kei-ichi Takata 1,2 , Masahiko Oshige 1,3 , Nobuyuki Kasai 1 , Kaori Shimanouchi 1 , Yoshihiro Kanai 1 , Ryoichi Nakamura 1 , Fumio Sugawara 1 and Kengo Sakaguchi 1 1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Japan 2 University of Pittsburgh Cancer Institute, Hillman Cancer Center, PA, USA 3 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, IN, USA In eukaryotes, proliferating cell nuclear antigen (PCNA), a trimeric and ring-shaped protein, is involved in various nuclear events. During chromosomal DNA replication, PCNA is loaded onto DNA by the repli- cation factor C complex and acts as a DNA sliding clamp for DNA polymerase d [1] and DNA polymerase e [2]. PCNA also participates in the DNA repair machinery with multiple binding partners such as Xero- derma pigmentosum G (XP-G), apurinic ⁄ apyrimidinic (AP) endonucleases, DNA glycosylases, and translesion DNA synthesis polymerases [3]. Other studies have demonstrated the interaction of PCNA with proteins that contribute to cell cycle regulation [4], DNA methy- lation [5] and chromatin remodeling [6]. In Drosophila melanogaster, DmPCNA1 is encoded by the gene mus209 [7]. Most mus209 mutants show nonconditional lethality. However, some mus209 mutant alleles show a temperature-sensitive phenotype, and are hypersensitive to methyl methanesulfonate (MMS) and ionizing radiation, reflecting the participa- tion of DmPCNA1 in DNA repair [8]. In addition, DmPCNA1 gene expression is controlled by the DNA replication-related element (DRE) ⁄ DRE-binding factor (DREF), or DRE ⁄ DREF system [9], which is respon- sible for activating the promoters of the 180 and 73 kDa subunits of DNA polymerase a and cyclin A, among others. Therefore, like other eukaryotic PCNAs, DmPCNA1 is also closely linked to chromo- somal DNA replication and cell cycle progression. Recently, two or three types of PCNA have been identified in archaeans such as Aeropyrum pernix, Pyrococcus furiosus and Sulfolobus solfataricus [10–12] Keywords DNA repair; Drosophila melanogaster; proliferating cell nuclear antigen; sliding clamp Correspondence K. Sakaguchi, Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba-ken 278, Japan Fax: +81 471 23 9767 Tel: +81 471 24 1501 E-mail: kengo@rs.noda.tus.ac.jp (Received 09 August 2006, revised 14 Sep- tember 2006, accepted 18 September 2006) doi:10.1111/j.1742-4658.2006.05504.x The eukaryotic DNA polymerase processivity factor, proliferating cell nuc- lear antigen, is an essential component in the DNA replication and repair machinery. In Drosophila melanogaster, we cloned a second PCNA cDNA that differs from that encoded by the gene mus209 (for convenience called DmPCNA1 in this article). The second PCNA cDNA (DmPCNA2) enco- ded a 255 amino acid protein with 51.7% identity to DmPCNA1, and was ubiquitously expressed during Drosophila development. DmPCNA2 was localized in nuclei as a homotrimeric complex and associated with Drosophila DNA polymerase d and e in vivo. Treatment of cells with methyl methanesulfonate or hydrogen peroxide increased the amount of both DmPCNA2 and DmPCNA1 associating with chromatin, whereas exposure to UV light increased the level of association of only DmPCNA1. Our observations suggest that DmPCNA2 may function as an independent sliding clamp of DmPCNA1 when DNA repair occurs. Abbreviations GST, glutathione-S-transferase; MMS, methyl methanesulfonate; PCNA, proliferating cell nuclear antigen; S2 cells, Drosophila Schneider 2 cells. 5062 FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS and in higher plants such as carrots [13]. In contrast, it is known that several sequences homologous to PCNA are present in mammalian genomes, although they are reportedly pseudogenes [14,15]. In the present study, we identified and characterized a second Drosophila PCNA (DmPCNA2). DmPCNA2 was present as a 29 kDa protein product and localized in nuclei as a homotrimer in a similar fashion to DmPCNA1. Both MMS and hydrogen peroxide (H 2 O 2 ) treatments increased the level of DmPCNA2 associating with chromatin, suggesting that DmPCNA2 may be another sliding clamp involved in the repair of MMS- and H 2 O 2 -induced DNA lesions. Results Molecular cloning of Drosophila PCNA2 The studies on PCNA in archaeans and higher plants suggest that some organisms may have several PCNA proteins [10–13]. A search of the Drosophila genome sequence database [16] identified a gene, listed as CG10262 in FlyBase, that has homology to DmPCNA1, the original Drosophila PCNA encoded by mus209 [7]. According to the genome sequence data- base, this PCNA-like gene is located at 37F2 on the long arm of chromosome 2 and is composed of two exons and one intron. In comparison, the DmPCNA1 gene is located at 56F11 on the short arm of chromo- some 2 and is composed of two exons and one intron. We cloned the cDNA of this PCNA-like gene, design- ated as DmPCNA2 in this study, by RT-PCR ampli- fication and determined the 5¢- and 3¢-termini of the gene by 5¢- and 3¢-RNA ligase mediated rapid amplifi- cation of cDNA elements (RLM-RACE) analysis. The cDNA of the DmPCNA2 gene was 1019 bp in length, and had a Drosophila consensus sequence for transla- tion initiation, 5¢-(C ⁄ A)AA(A ⁄ C)ATG, and a putative poly(A) addition signal sequence, 5¢-AATAAA [17,18]. It encoded a predicted product of 255 amino acids with a molecular mass of 28.5 kDa, and showed 51.7% sequence identity with DmPCNA1 . Addition- ally, it showed 44.7% sequence identity to the human PCNA and 40.7% to Schizosaccharomyces pombe PCNA, whereas DmPCNA1 shows 70.7% and 49.6% identity, respectively. The nucleotide sequence data of DmPCNA2 cDNA have been submitted to the DDBJ ⁄ EMBL ⁄ GenBank nucleotide sequence data- bases (accession number: AB195794). We carried out a multiple sequence alignment of DmPCNA2 and DmPCNA1 to identify conserved structural domains in the two proteins (Fig. 1). Fig. 1. Amino acid sequence alignment of Drosophila melanogaster proliferating cell nuclear antigen 2 (DmPCNA2) and DmPCNA1. Identical and similar amino acid residues are boxed in black and gray, respectively. The interdomain connecting loop and the C-terminal tail, known to be important for interaction of PCNA-binding proteins, are indicated. The DmPCNA2 polypeptide lacked amino acid residues from positions 190 to 194 of DmPCNA1, which corresponds to part of the D 2 E 2 loop. T. Ruike et al. Second PCNA in Drosophila melanogaster FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS 5063 Eukaryotic PCNA proteins have an interdomain connecting loop that interacts with proteins such as DNA polymerase d, flag endonuclease 1 (FEN-1), and XP-G, and also a C-terminal tail that interacts with DNA polymerase e and replication factor C [3]. These regions are conserved in both DmPCNA2 and DmPCNA1, but a small region of the D 2 E 2 loop is absent in DmPCNA2 [19]. According to previous stud- ies, PCNAs from several archaeans (A. pernix, P. furiosus and S. solfataricus) also lack the D 2 E 2 loop [10–12]. The biophysical role of this loop is still unknown. Analysis of DmPCNA2 expression during Drosophila development In Drosophila, DmPCNA1 is highly transcribed in pro- liferating tissues [20], and its transcription is controlled by the transcription factors DREF and early region 2 transcription factor (E2F) [9,21]. These transcription factors regulate the expression of DNA replication- and cell proliferation-related genes. To investigate the biolo- gical role of DmPCNA2, we first performed northern hybridization experiments on a range of Drosophila developmental stages. A high level of expression of DmPCNA1 was detected in embryos at 0–2 and 8–12 h of development; moderate expression was present in unfertilized eggs, 4–8 and 12–16 h embryos, and adult females, and in Kc cells (Fig. 2A). Similar results were found in a previous study [20]. In contrast to the expres- sion pattern of DmPCNA1, it was difficult to detect a DmPCNA2 signal at any developmental stage (Fig. 2A). We therefore carried out an RT-PCR screen for DmPCNA2 expression in the different stages of Dro- sophila development. Expression levels were compared in the linear range of RT-PCR amplification. As shown in Fig. 2B, a DmPCNA2 cDNA-specific band was vis- ible ubiquitously throughout Drosophila development. To determine whether the putative DRE and E2F sites found in DmPCNA1 were present in the 5¢-upstream region of DmPCNA2, we isolated genomic DNA from adult Drosophila and cloned the 5¢-upstream region (approximately 1000 bp) of the DmPCNA2 gene. We searched this 1000 bp nucleotide sequence using genetyx-mac v. 9 processing software but did not find either a DRE site (5¢-TATCGATA-3¢) or an E2F site (5¢-TATCCCGC-3¢) in the 5¢-upstream region of the DmPCNA2 gene. Next, we sought to detect endogenous DmPCNAs by antibodies raised against peptides unique to either DmPCNA2 or DmPCNA1. Using specific antibodies for DmPCNA2 or DmPCNA1, protein bands of approximately 29 or 35 kDa, respectively, were observed in western blots of Drosophila Schneider 2 (S2) cells (Fig. 2C). In contrast, no significant staining was detectable with preimmune rabbit IgG. Further- more, using anti-Flag serum, protein bands of approxi- mately 30 or 36 kDa, respectively, were also observed in western blots of S2 cells that have stable expression of Flag-tagged DmPCNA2 or Flag-tagged DmPCNA1 (Fig. 2C). Analysis of trimer formation by DmPCNA2 and DmPCNA1 As PCNA forms a ring-shaped homotrimer with a central cavity [19], we analyzed the homotrimeri- zation of DmPCNA2 and its heterotrimerization with DmPCNA1. We first produced recombinant proteins and purified these to near homogeneity. The purified DmPCNA2 ⁄ T7-(His) 6 eluted as a single peak with a calculated molecular mass of 100 kDa in a Sephacryl S-300 gel filtration column, as also did DmPCNA1 ⁄ T7-(His) 6 (Fig. 3A). This result suggests that DmPCNA2 is able to form a homotrimer. We therefore simulated the three-dimensional structures of DmPCNA2 and DmPCNA1, using the data from human PCNA (Fig. 3B). The possible structures of the DmPCNA2 homotrimer resemble that of the DmPCNA1 homotrimer, except for the small region of the D 2 E 2 loop. This loop in DmPCNA2 was shorter than that in DmPCNA1. The simulation of the possible structure of the DmPCNA2 homotrimer suggests that the sizes of the homotrimers of DmPCNA2 and DmPCNA1 are sim- ilar. We therefore investigated whether DmPCNA2 could form a heterotrimer complex with DmPCNA1. We performed a glutathione-S-transferase (GST) pull-down experiment using DmPCNA2 ⁄ GST or DmPCNA1 ⁄ GST and DmPCNA2 ⁄ T7-(His) 6 or DmPCNA1 ⁄ T7-(His) 6 . The indicated GST fusion pro- teins and T7-(His) 6 -tagged proteins of DmPCNAs were mixed in NaCl ⁄ P i (lanes 1–5 in Fig. 4A), incubated at 4 °C for 12 h, and precipitated with GST Sepharose- 4B beads. Under these conditions, however, we were unable even to find an interaction of DmPCNA1 with itself (lane 3 in Fig. 4A). Unsurprisingly, therefore, we could not detect an interaction between DmPCNA2 and DmPCNA1 (lanes 4 and 5 in Fig. 4A). It is poss- ible that both DmPCNA2 and DmPCNA1 might already have been present as homotrimers prior to mixing and that they could not exchange monomers under the experimental conditions used. Therefore, we sought to reconstitute the trimeric forms of DmPCNAs. When the proteins were forced to disso- ciate to the monomeric state by incubation at 4 °C for Second PCNA in Drosophila melanogaster T. Ruike et al. 5064 FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS 6 h in NaCl ⁄ P i containing 0.1% Tween-20, followed by dialysis at 4 °C for 6 h in NaCl ⁄ P i , DmPCNA2 could form a heterotrimeric complex with DmPCNA1 in vitro (lanes 9 and 10 in Fig. 4A). Next, we used an immunoprecipitation assay to determine whether DmPCNA2 and DmPCNA1 could form a heterotrimer in vivo. As we could not detect endogenous DmPCNA2 in the crude extracts from S2 cells unless SuperSignal West Femto Maximum was used as the chemiluminescence reagent (Fig. 2C), we considered that further analyses of endogenous DmPCNA2 with anti-DmPCNA serum were impracti- cal. Therefore, using the Drosophila Expression Sys- tem, we carried out an immunoprecipitation using S2 cells that stably expressed V5-tagged DmPCNA1 and Flag-tagged DmPCNA1, V5-tagged DmPCNA2 and AB C Fig. 2. Expression of Drosophila melanogaster PCNA2 (DmPCNA2) during Drosophila development. (A) Northern hybridization analysis. 3¢-UTRs of DmPCNA2 and DmPCNA1 cDNAs (33.6% nucleotide sequence homology) were used as specific probes. RP-49 mRNA served as a loading control. (B) RT-PCR analysis of DmPCNA2. Expression of Act5C was used as an internal control; expression of DmPCNA1 was also analyzed to ensure that RT-PCR reflected the results of the northern hybridization. The cycle numbers used are indicated. NC is the neg- ative control. (C) Western blotting analysis of endogenous DmPCNAs. Crude extracts from Drosophila Schneider 2 (S2) cells were separated by 12.5% SDS ⁄ PAGE and blotted with preimmune rabbit IgG (left panel) serum, anti-DmPCNA2 serum (second panel from the left), or anti- DmPCNA1 serum (middle panel). The 29 kDa protein band of DmPCNA2 and the 35 kDa protein band of DmPCNA1 are indicated by arrows. Crude extracts from S2 cells expressing Flag-tagged DmPCNA2 (second panel from the right) or Flag-tagged DmPCNA1 (right panel) were separated by 12.5% SDS ⁄ PAGE and blotted with anti-Flag serum. The 30 kDa protein band of Flag-tagged DmPCNA2 and the 36 kDa pro- tein band of Flag-tagged DmPCNA1 are indicated by arrows. The sizes of the molecular mass markers are indicated on the left. T. Ruike et al. Second PCNA in Drosophila melanogaster FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS 5065 Flag-tagged DmPCNA2, or V5-tagged DmPCNA1 and Flag-tagged DmPCNA2. We detected interactions between DmPCNA1 molecules and between DmPCNA2 molecules with V5 and Flag tags, but found no evidence of an interaction between DmPCNA2 and DmPCNA1 (Fig. 4B). These data sug- gest that DmPCNA2 can only form a homotrimer in vivo, and that the heterotrimerization in vitro may be an artificial event. Association of DmPCNA2 with Drosophila DNA polymerases d and e PCNA was originally identified as a DNA sliding clamp for DNA polymerases [22]. In humans, PCNA associates with the p120 catalytic subunit of DNA polymerase d through interaction with the p66 third subunit [23]. In Schizosaccharomyces pombe, PCNA interacts with the Pol2p catalytic subunit of DNA polymerase e [24]. We therefore tested whether DmPCNA2 could associate with the catalytic subunits of Drosophila DNA polymerase d and DNA poly- merase e. We carried out an immunoprecipitation assay using crude extract from S2 cells that had stable expression of V5-tagged DmPCNA2. As shown in Fig. 5, both DNA polymerase d and DNA polymerase e are precipitated with anti-V5 serum, indicating that DmPCNA2 can associate with DNA polymerase d and DNA polymerase e in vivo. Properties of binding of DmPCNA2 and DmPCNA1 to chromatin damaged by various mutagens As described earlier, PCNA is involved in DNA repair [3]. In humans, the amount of PCNA binding to A B Fig. 3. Homotrimer formation of Drosophila melanogaster proliferating cell nuclear anti- gen 2 (DmPCNA2). (A) Gel filtration chroma- tography analysis. Two hundred micrograms of purified DmPCNA2 ⁄ T7-(His) 6 or DmPCNA1 ⁄ T7-(His) 6 was loaded onto a Sephacryl S300 gel filtration column. The cir- cle indicates the position of the maximum peak at which DmPCNA2 (left panel) or DmPCNA1 (right panel) was found. Molecu- lar mass standards (open squares) used were ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa) and ribonuclease A (13.7 kDa). (B) Building of a model of a ring-shaped, three-dimensional structure of DmPCNA2 (left panel) and DmPCNA1 (right panel): upper panel, back view; lower panel, side view. In the dia- grams for DmPCNA1, purple balls represent amino acid residues from 190 to 194 that are present in DmPCNA1 but absent from DmPCNA2. Second PCNA in Drosophila melanogaster T. Ruike et al. 5066 FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS chromatin increases in cells treated with mutagens such as MMS [25], H 2 O 2 [26] and UV light [27]. We there- fore examined the association of DmPCNA2 and DmPCNA1 with chromatin following treatment with DNA-damaging agents. First, we visualized the sub- cellular localization of V5-tagged DmPCNA2 and Flag-tagged DmPCNA1 in S2 cells using immunofluo- rescence microscopy. We found that both DmPCNA2 and DmPCNA1 were localized in the nucleus (Fig. 6A). Next, we prepared fractions from the cyto- plasm, the whole chromatin, a high-salt wash and the core nuclear matrix from S2 cells. As controls for the fractionation procedure, we used western blotting with antibodies against b-tubulin (a nonchromatin-bound protein) and histone H4 (a chromatin-bound protein). DmPCNA2 and DmPCNA1 were present in the cyto- plasmic and the whole chromatin fractions (Fig. 6B). Following exposure to DNA-damaging agents, both DmPCNA2 and DmPCNA1 showed increased levels of association with chromatin, with a time-dependent relationship (Fig. 6C). The level of DmPCNA2 in the whole chromatin fraction reached a maximum at 5–8 h after MMS treatment and at 3 h after H 2 O 2 treatment (Fig. 6C). In contrast, the amount of DmPCNA1 in this fraction continued to increase up to 8 h after MMS treatment and 5 h after H 2 O 2 treatment (Fig. 6C). UV light treatment increased the level of DmPCNA1 associating with chromatin but not of DmPCNA2. Mitomycin C did not alter the levels of either DmPCNA2 or DmPCNA1 associating with chromatin. We also investigated the binding of DmPCNA2 to chromatin after treatment with various doses of DNA-damaging agents (Fig. 6D). MMS-trea- ted S2 cells were collected 5 h after treatment, and S2 cells treated with H 2 O 2 , UV light or mitomycin C were harvested at 3 h. The amounts of DmPCNA2 in the whole chromatin fractions increased in a dose-depend- ent fashion after MMS and H 2 O 2 treatments, but were A B Fig. 4. Interaction of Drosophila melano- gaster proliferating cell nuclear antigen 2 (DmPCNA2) and DmPCNA1. (A) In vitro interaction of DmPCNA2 and DmPCNA1. Lanes 1–5: the indicated proteins were mixed in NaCl ⁄ P i at 4 °C for 12 h. Lanes 6–10: the indicated proteins were mixed in NaCl ⁄ P i containing 0.1% Tween-20 at 4 °C for 6 h, and this was followed by dialysis in NaCl ⁄ P i at 4 °C for 6 h. The proteins bound to GST Sepharose-4B beads were analyzed by western blotting with anti-T7 or anti-GST serum. (B) In vivo interaction between DmPCNA2 and DmPCNA1. Drosophila Schneider 2 (S2) cells expressing the indica- ted DmPCNAs were harvested and lysed. The lysates were immunoprecipitated (IP) with anti-V5 serum. The washed immuno- precipitates were separated by 12.5% SDS ⁄ PAGE and blotted for either Flag or V5 (left panel). The lysates were immunoprecip- itated with anti-Flag serum and blotted sequentially for V5 or Flag (right panel). T. Ruike et al. Second PCNA in Drosophila melanogaster FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS 5067 not influenced by UV light or mitomycin C treatments (Fig. 6D). Discussion In this study, we identified a second PCNA cDNA from Drosophila melanogaster. This PCNA, which we call here DmPCNA2, had two conserved regions, an interdomain connecting loop and a C-terminal tail. DmPCNA2 formed homotrimers and associated with DNA polymerase d and DNA polymerase e in vivo.In addition, DmPCNA2, as well as DmPCNA1, was present in the whole chromatin fraction of cellular proteins. Taken together, these results suggest that DmPCNA2 can act as a DNA sliding clamp for these DNA polymerases. Yamaguchi and colleagues reported that the expres- sion of DmPCNA1 is controlled by the transcription factors DREF and E2F, which are abundant in tissues such as the ovary and in unfertilized eggs and early embryos [9,21]. Thus, DmPCNA1 mRNA is highly expressed in proliferating tissues and decreases rapidly during development [20]. In contrast to DmPCNA1, there was no evidence for putative binding sites for DREF and E2F in the 5¢-upstream region of DmPCNA2. Moreover, DmPCNA2 was constantly expressed even in pupae in which few cells are pro- liferating. These data suggest that expression of DmPCNA2 might not be related to cell proliferation. We found different patterns of binding to chromatin between DmPCNA2 and DmPCNA1 in S2 cells trea- ted with DNA-damaging agents. MMS and H 2 O 2 induced a more rapid association of DmPCNA2 with chromatin than of DmPCNA1. UV light induced the association of DmPCNA1 with chromatin, but not of DmPCNA2. These results suggest that each DmPCNA functions independently when DNA is damaged. It has been reported that PCNA cannot load itself onto DNA in vitro and requires a clamp loader protein to achieve this association [28,29]. Therefore, the patterns of association of DmPCNA2 and DmPCNA1 with chromatin might reflect differential loading onto dam- aged DNA by clamp loaders. DmPCNA1 probably functions in the repair of MMS-, H 2 O 2 - and UV light- induced lesions in a similar manner to other eukaryotic PCNAs. In eukaryotes, base excision repair is known to be the major pathway for repair of MMS- and H 2 O 2 -induced DNA lesions and is often initiated by several DNA glycosylases [30]. In S. pombe, PCNA and Rad9 ⁄ Rad1 ⁄ Hus1 differentially participate in base excision repair through interaction with the DNA gly- cosylase MutY homolog [31]. Although the precise function of DmPCNA2 remains unclear, one hypothe- sis is that DmPCNA2 might participate in the base excision repair pathway through interaction with some of the Drosophila DNA glycosylases. Another possibil- ity is that DmPCNA2 might simply support DmPCNA1 in the repair of MMS- and H 2 O 2 -induced DNA damage. Our next task in the near future will be to elucidate how DmPCNA2 functions in the DNA repair system. The analysis of flies with mutation of DmPCNA2 will help us to understand its biophysiologic roles as well as enable identification of the DmPCNA2 binding partners. Experimental procedures Cloning of DmPCNA2 Total RNA from Kc cells was reverse transcribed using the SuperScript First-Strand Synthesis System (Invitrogen, Car- lsbad, CA) with an oligo-(dT) 12)18 primer. Amplification of the DmPCNA2 cDNA was performed using ExTaq thermo- stable DNA polymerase (TaKaRa, Ohtsu, Japan) and the following primers: forward, 5¢-ATGCTCGAGGCGCGTT Fig. 5. Association of Drosophila melanogaster proliferating cell nuclear antigen 2 (DmPCNA2) with DNA polymerase d (Dmpol d) and Dmpol e. Drosophila Schneider 2 (S2) cells expressing V5-tagged DmPCNA2 were harvested and lysed. The lysates were immunoprecipitated (IP) with anti-V5 serum. The washed immuno- precipitates were separated by 5% SDS ⁄ PAGE and analyzed by western blotting with anti-Dmpol d, anti-Dmpol e or anti-V5 serum. Second PCNA in Drosophila melanogaster T. Ruike et al. 5068 FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS AB DC Fig. 6. Chromatin-binding patterns of Drosophila melanogaster proliferating cell nuclear antigen 2 (DmPCNA2) and DmPCNA1 in response to DNA-damaging agents. (A) Immunofluorescent analysis of the localization of V5-tagged DmPCNA2 and Flag-tagged DmPCNA1. DmPCNA2 is shown in red, DmPCNA1 in green, and DNA in blue after DAPI staining. Bar represents 5 lm. (B) Fractionation of DmPCNA2 and DmPCNA1. Drosophila Schneider 2 (S2) cells expressing V5-tagged DmPCNA2 and Flag-tagged DmPCNA1 were extracted to obtain cyto- plasmic, whole chromatin, high-salt-wash and core nuclear matrix fractions. The fractions were analyzed by western blotting with the indica- ted antibodies. (C) Chromatin binding of DmPCNA2 and DmPCNA1 in response to DNA-damaging agents [0.02% methyl methanesulfonate (MMS), 1.5 m M H 2 O 2 ,35JÆm )2 UV light and 0.02% mitomycin C (MMC)]. S2 cells were collected at the indicated post-treatment intervals. (D) Chromatin binding of DmPCNA2 after various doses of DNA-damaging agents. S2 cells were treated with MMS (concentration range 0.01–0.1%), H 2 O 2 (concentration range 0.5–2.5 mM), UV light (dose range 15–70 JÆm )2 ) or MMC (concentration range 0.01–0.1%). The chro- matin fractions were analyzed by western blotting with anti-V5 serum. T. Ruike et al. Second PCNA in Drosophila melanogaster FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS 5069 TGAG-3¢; and reverse, 5¢-CTAGAAATCGGGGTCATT CA-3¢. The amplified cDNA was cloned into the pGEM-T vector (Promega, Madison, WI). To identify the 5¢- and 3¢-termini of the gene, 5¢- and 3¢-RLM-RACE was per- formed in accordance with the manufacturer’s recom- mended protocol (FirstChoice RLM-RACE kit; Ambion, Austin, TX). Northern hybridization and RT-PCR analysis Total RNAs were extracted using Trizol (Invitrogen) from unfertilized Drosophila eggs, embryos, larvae, adult flies and from Kc cells. Northern hybridization was carried out as described previously [32]. The 3¢-UTR of DmPCNA2 cDNA (nucleotides 863–1019) or that of DmPCNA1 (nucle- otides 873–997) was used as the specific probe. Full-length ribosomal protein 49 (Rp-49) cDNA was used as a control. For RT-PCR analysis, total RNAs (tissue and cell sources described above) were treated with DNase I (TaKaRa) to remove traces of genomic DNA contamin- ation, and purified with phenol ⁄ chloroform. First-strand cDNA was synthesized from 1 lg of total RNA using the SuperScript First-Strand Synthesis System (Invitrogen) with random hexamers, and then amplified using the following primers: DmPCNA2 ) forward, 5¢-ATGCTCGA GGCGCGTTTGAG-3¢, and reverse, 5¢-CTAGAAATC GGGGTCATTCA-3¢; DmPCNA1 – forward, 5¢-ATGTTC GAGGCACGCCT-3¢, and reverse, 5¢-TTATGTCTCGTT GT CCTCGA-3¢; Act5c ) forward, 5¢-TGTGGATACTCC TCCCGACA-3¢, and reverse, 5¢-ATCCCGATCCTGAC TCTT-3¢. The PCR conditions were: DmPCNA2 – 94 °C for 5 min, 94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min, 24 cycles, 5 min extension at 72 °C; DmPCNA1 ) 94 °C for 5 min, 94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min, 21 cycles, 5 min extension at 72 °C; Act5c ) 94 °C for 5 min, 94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min 30 s, 17 cycles, 5 min extension at 72 °C. PCR products were visualized by staining with SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR) after agarose gel electrophoresis. Generation of antibodies to DmPCNA2 and anti-DmPCNA1 A keyhole limpet haemocyanin (KLH)-conjugated syn- thetic peptide with an extra cysteine on the N-terminus (CKKDYTCFIQLPSS, amino acids 129–142 of DmPCNA2) or (CKLAQTGSVDKEEEA, amino acids 181–194 of DmPCNA1) was used for inoculation into rab- bits (Bio Matrix Research, Kashiwa, Japan). For detection of endogenous DmPCNA2, anti-DmPCNA2 serum or pre- immune rabbit IgG diluted to 0.5 lgÆmL )1 served as pri- mary antibodies. Horseradish peroxidase-conjugated goat anti-(rabbit IgG) (Vector Laboratories, Burlingame, CA) diluted to 2 ng Æ mL )1 served as the secondary antibody. Chemiluminescence was detected with SuperSignal West Femto Maximum (Pierce, Rockford, IL). For detection of endogenous DmPCNA1, anti-DmPCNA1 serum diluted to 1 lgÆmL )1 and horseradish peroxidase-conjugated goat anti-(rabbit IgG) diluted to 50 ngÆmL )1 served as primary and secondary antibodies, respectively. Chemiluminescence was detected with enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Animals were fed water and standard rabbit food and maintained on a 12 h light/dark cycle. Polyclonal antiserum to the peptide was raised in rabbits by subcutaneous injec- tion of 0.15 mg of the peptide emulsified in Freund’s com- plete adjuvant. Two weeks after the primary injection, boosts of 0.3 mg of the peptide in Freund’s incomplete adju- vant were injected every 2 weeks. The rabbits were bled one week after the final boost under anesthesia. The rabbits were treated in accordance with procedures approved by the Ani- mal Ethics Committee of the Science University of Tokyo. Purification of recombinant DmPCNA2 or DmPCNA1 proteins The DmPCNA2 coding region was cloned into pET21a (Novagen, Darmstadt, Germany) or pGEX-6P-1 vectors (Amersham Pharmacia Biotech). T7-(His) 6 -tagged DmPCNA2 [DmPCNA2 ⁄ T7-(His) 6 ] protein was over- expressed in Escherichia coli BL21 (DE3) (Novagen) and purified with His-Bind Resin according to the manufacturer’s protocol (Novagen). GST fusion DmPCNA2 (DmPCNA2 ⁄ GST) protein was overexpressed in E. coli BL21 (DE3) and purified with Glutathione Sepharose-4B (Amersham Pharmacia Biotech). Production and purification of DmPCNA1 ⁄ T7-(His) 6 protein and DmPCNA1 ⁄ GST protein were carried out as described above for DmPCNA2. Gel filtration column chromatography Samples of purified DmPCNA2 ⁄ T7-(His) 6 and DmPCNA1 ⁄ T7-(His) 6 proteins were dialyzed against TEMG buffer (50 mm Tris ⁄ HCl, pH 7.9, 1 mm EDTA, pH 8.0, 5 mm 2-mercaptoethanol, 10% glycerol) containing 0.2 m NaCl. A 200 lg sample of each protein was sepa- rately loaded onto a gel filtration column (Sephacryl S-300 gel column; Amersham Pharmacia Biotech) equilibrated with the same buffer. The molecular mass was estimated from a calibration curve using ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa) and ribonuclease A (13.7 kDa). Three-dimensional structure model building The predicted structure of the human PCNA protein was used to set the parameters for constructing models Second PCNA in Drosophila melanogaster T. Ruike et al. 5070 FEBS Journal 273 (2006) 5062–5073 ª 2006 The Authors Journal compilation ª 2006 FEBS of DmPCNA2 and DmPCNA1. We used the swiss-model program [33–35] to generate three-dimensional models of the DmPCNA2 and DmPCNA1 proteins. GST pull-down assay Equal amounts of purified GST fusion proteins and puri- fied T7-(His) 6 -tagged proteins were mixed in NaCl ⁄ P i and incubated at 4 °C for 12 h, or mixed in NaCl ⁄ P i containing 0.1% Tween-20 and incubated at 4 °C for 6 h. The mix- tures were then dialyzed in NaCl ⁄ P i at 4 °C for 6 h. GST Sepharose-4B beads (Amersham Pharmacia Biotech) were added to the samples, which were then incubated at 4 °C for 1 h. After being washed six times with 0.8 mL of NaCl ⁄ P i , the bound proteins were eluted with TEMG buf- fer containing 10 mm reduced glutathione and analyzed by western blotting with mouse monoclonal antibody T7 (Novagen) and rabbit polyclonal anti-GST serum. Cell culture, plasmid construction, and transfection S2 cells were cultured in Schneider’s Drosophila Medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum at 25 °C. The expression vector for V5-tagged DmPCNA2 was constructed by cloning the DmPCNA2 coding region into pAc5.1 ⁄ V5-His C (Invitrogen). Flag- tagged DmPCNA2 was constructed by cloning the N-ter- minally Flag-tagged DmPCNA2 coding region into pAc5.1 ⁄ V5-His C from which the V5-His tag had been removed. Expression vectors for V5-tagged DmPCNA1 and Flag-tagged DmPCNA1 were constructed as described above for DmPCNA2. All transfections and establishment of the stable cell lines were performed in accordance with the manufacturer’s protocols (Invitrogen). Immunoprecipitation experiments Aliquots of 1 · 10 7 S2 cells were washed in NaCl ⁄ P i and suspended in TEMG buffer containing 0.15 m NaCl, 0.01% NP-40, and the protease inhibitors phenylmethanesulfonyl fluoride (1 mm), leupeptin (1 mm) and pepstatin A (1 mm). After sonication, the lysates were rocked at 4 °C for 30 min, and then centrifuged at 10 000 g for 10 min (MX- 201; TOMY; TMA-29 rotor). The supernatants were pre- cleared by treatment with protein G Sepharose beads (Amersham Pharmacia Biotech) at 4 °C for 1 h. Cleared lysates were immunoprecipitated with protein G Sepharose beads and a mouse monoclonal V5 antibody (Invitrogen) or anti-Flag serum (Sigma, St Louis, MO) at 4 °C for 2 h. Immunoprecipitates were washed three times with the same buffer, solubilized in SDS ⁄ PAGE sample buffer, and ana- lyzed by western blotting. For generation of antibodies to DNA polymerase d, the purified recombinant DNA polymerase d fragment (amino acid residues 104–445) was used for inoculation into rabbits. The generation of anti- DNA polymerase e was described in a previous report [36]. Immunofluorescence analysis S2 cells were placed on poly-(l-lysine)-coated coverslips and fixed with 4% paraformaldehyde in NaCl⁄ P i for 10 min at room temperature. After several washes with NaCl ⁄ P i , the cells were treated with methanol for permeabi- lization. The samples were incubated with primary antibod- ies, mouse monoclonal anti-V5 serum and rabbit polyclonal anti-Flag serum, at 4 °C overnight, and then treated for 1 h with the secondary antibodies Alexa546 anti-(mouse IgG) and Alexa488 anti-(rabbit IgG) (Molecular Probes). They were also counterstained with 4¢,6-diamidine-2-phenylindole (DAPI). The preparations were observed under a fluore- scence microscope and the data were collected using a CCD camera (Nikon, Chiyoda, Japan). Fractionation of cellular proteins S2 cells were exposed to MMS or mitomycin C for 1 h or to H 2 O 2 for 15 min. The cells were then washed once and incubated prior to sampling. UV-irradiated S2 cells were incubated in the dark in order to distinguish the effects of UV irradiation from those of the photoreacti- vating mechanism. After incubation, S2 cells were washed three times with ice-cold NaCl ⁄ P i . Aliquots of 1 · 10 7 S2 cells were lysed in 500 lL of cytoskeleton buffer (CSK buffer: 10 mm Hepes, pH 7.4, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl 2 ,1mm EGTA, 5 mm 2-mercapto- ethanol, 1 mm phenylmethanesulfonyl fluoride, 1 mm leu- peptin, 1 mm pepstatin A, 0.5% Triton X-100) at 4 °C for 5 min and centrifuged at 3000 g for 5 min (MX-201; TOMY; TMA-29 rotor). The soluble cytoplasmic fraction was removed, and the pellet was washed once with 500 lL of CSK buffer. The pellet was then resuspended in 200 lL of CSK buffer containing 100 U of RNase-free DNase I (TaKaRa). After 30 min at 37 °C, ammonium sulfate was added to a final concentration of 0.25 m. The samples were incubated for 5 min at 4 °C and centrifuged as above. The soluble chromatin fraction was removed, and the pellet was extracted in CSK buffer with 2 m NaCl for 5 min at 4 °C. After another centrifugation, the 2 m NaCl wash was removed, and the nuclear matrix pel- let was resuspended in 50 lL of SDS ⁄ PAGE sample buf- fer. 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