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 e
Trang 1antigen (PCNA2) from Drosophila melanogaster
Tatsushi Ruike1, Ryo Takeuchi1, Kei-ichi Takata1,2, Masahiko Oshige1,3, Nobuyuki Kasai1,
Kaori Shimanouchi1, Yoshihiro Kanai1, Ryoichi Nakamura1, Fumio Sugawara1 and
Kengo Sakaguchi1
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 pigmentosumG (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 addiparticipa-tion, 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 SepSep-tember 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.
Trang 2and 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
(H2O2) treatments increased the level of DmPCNA2
associating with chromatin, suggesting that
DmPCNA2 may be another sliding clamp involved in
the repair of MMS- and H2O2-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 initiatransla-tion, 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 D2E2loop.
Trang 3Eukaryotic 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 D2E2 loop is
absent in DmPCNA2 [19] According to previous
stud-ies, PCNAs from several archaeans (A pernix,
P furiosusand S solfataricus) also lack the D2E2loop
[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
DmPCNA1was 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
DmPCNA2signal 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
DmPCNA2gene 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 heterotrimerihomotrimeri-zation 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 D2E2 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⁄ Pi (lanes 1–5 in Fig 4A), incubated at
4C 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 4C for
Trang 46 h in NaCl⁄ Pi containing 0.1% Tween-20, followed
by dialysis at 4C for 6 h in NaCl ⁄ Pi, 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
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, 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 anti-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.
Trang 5Flag-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.
Trang 6chromatin increases in cells treated with mutagens such
as MMS [25], H2O2 [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 H2O2treatment (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 H2O2 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 collecMMS-trea-ted 5 h after treatment, and S2 cells treated with H2O2, 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 H2O2treatments, 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 harvesindica-ted 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).
Trang 7not 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 DmPCNA2might 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 H2O2 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-, H2O2- 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
H2O2-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 H2O2-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)18primer 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.
Trang 8A B
D C
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 H2O2, 35 JÆm)2UV 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 m M ), 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.
Trang 9TGAG-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
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
GGGGTCATTCA-3¢; DmPCNA1 – forward, 5¢-ATGTTC
GAGGCACGCCT-3¢, and reverse, 5¢-TTATGTCTCGTT
TCCCGACA-3¢, and reverse, 5¢-ATCCCGATCCTGAC
TCTT-3¢ The PCR conditions were: DmPCNA2 – 94 C
for 5 min, 94C for 45 s, 55 C for 45 s, 72 C for 1 min,
24 cycles, 5 min extension at 72C; DmPCNA1) 94 C
for 5 min, 94C for 45 s, 55 C for 45 s, 72 C for 1 min,
21 cycles, 5 min extension at 72C; Act5c) 94 C for
5 min, 94C for 45 s, 55 C for 45 s, 72 C for 1 min 30 s,
17 cycles, 5 min extension at 72C 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
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 ngÆmL)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
over-expressed in Escherichia coli BL21 (DE3) (Novagen) and purified with His-Bind Resin according to the manufacturer’s
GST) protein was overexpressed in E coli BL21 (DE3) and purified with Glutathione Sepharose-4B (Amersham
DmPCNA1⁄ T7-(His)6protein and DmPCNA1⁄ GST protein were carried out as described above for DmPCNA2
Gel filtration column chromatography
DmPCNA1⁄ T7-(His)6 proteins were dialyzed against
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
Trang 10of 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⁄ Piand
incubated at 4C for 12 h, or mixed in NaCl ⁄ Picontaining
0.1% Tween-20 and incubated at 4C for 6 h The
mix-tures were then dialyzed in NaCl⁄ Piat 4C for 6 h GST
Sepharose-4B beads (Amersham Pharmacia Biotech) were
added to the samples, which were then incubated at 4C
for 1 h After being washed six times with 0.8 mL of
NaCl⁄ Pi, 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 25C 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
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· 107S2 cells were washed in NaCl⁄ Pi 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 4C 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 4C 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 4C 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⁄ Pi for
10 min at room temperature After several washes with NaCl⁄ Pi, 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 4C 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 H2O2 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⁄ Pi Aliquots of 1· 107
S2 cells were lysed in 500 lL of cytoskeleton buffer (CSK buffer: 10 mm Hepes, pH 7.4, 100 mm NaCl, 300 mm
2-mercapto-ethanol, 1 mm phenylmethanesulfonyl fluoride, 1 mm leu-peptin, 1 mm pepstatin A, 0.5% Triton X-100) at 4C 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
sulfate was added to a final concentration of 0.25 m The samples were incubated for 5 min at 4C 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 4C 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 For western blot analysis, equal cell equivalents from each fraction were subjected to SDS⁄ PAGE and probed with appropriate antibodies: mouse monoclonal anti-V5 serum, rabbit polyclonal anti-Flag serum (Sigma), mouse monoclonal anti-b-tubulin serum (Chemicon, Temecula, CA), or rabbit polyclonal anti-Histone H4 (Imgenex, San Diego, CA)