From the Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595
Received for publication, August 23, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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The yeast two-hybrid screening method was used to
identify novel proteins that associate with human DNA polymerase DNA replication is essential not only for duplication of the
genome but also for maintenance of genomic integrity during DNA repair
(1, 2). Chromosomal DNA replication in eukaryotic cells requires three
distinct DNA polymerases- Yeast Two-hybrid Screening--
The Matchmaker system 2 and
human placental cDNA library (HL4025AH) were purchased from
Clontech (Palo Alto, CA). The procedures for
culture media and plates for yeast growth and selection were as
described in the manufacturer's protocols. Full-length cDNAs corresponding to the human pol Pairwise Yeast Two-hybrid Interactions--
Pairwise yeast
two-hybrid interactions were performed as described previously
(11).
PDIP38 Antibody--
A synthetic peptide corresponding to the
C-terminal 20 amino acid residues
(H2N-PPFSLESNKDEKTPPSGLHW-COOH) of the human PDIP38 sequence was used to generate rabbit polyclonal antibodies. These were
purified by affinity chromatography using the immobilized peptide
(SynPep, Dublin, CA).
Purification of pol Immunoprecipitation--
Fresh HeLa cell pellet was extracted
with high salt buffer (20 mM HEPES, pH 7.9, 25% glycerol,
420 mM NaCl, 1.5 mM MgCl2, 10 mM KCl, 20 mM EDTA, 0.2 mM PMSF, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 10 mM benzamidine)
for 20 min on ice, sonicated, and diluted with 2 volumes of wash buffer
(50 mM Tris-HCl, pH 7.8, 20 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40). The diluted extract was
precleared with protein A plus protein G beads and 5 µg of rabbit
preimmune serum at 4 °C for 30 min. After centrifugation, the
protein A and protein G beads were discarded. To the precleared HeLa
extract, 10 µg of PDIP38 rabbit polypeptide antibody or normal rabbit
serum, protein A and protein G beads were added and incubated for
2 h at 4 °C. After incubation, protein A plus G beads were washed 5 times with wash buffer. Bound proteins were extracted with
SDS-PAGE sample buffer by boiling and subjected to SDS-PAGE and
immunoblotted with PDIP38 rabbit polyclonal antibody, PCNA mouse
monoclonal antibody 74B1, or pol PCNA Overlay--
PCNA overlay was performed as described
previously (11).
GST Pull-down Assays--
The GST-PDIP38 fusion protein
expression vector was constructed by double digestion of pACT2-PDIP38
with EcoRI and XhoI and ligation of the isolated
fragment into the pGEX-5X-3 vector (Amersham Biosciences). The fusion
protein was expressed in Escherichia coli strain BL21(DE3,
pLysS) (Stratagene). The GST lysate or GST-PDIP38 fusion protein lysate
(240 µl) in GST binding buffer (50 mM Tris-HCl, pH 7.8, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 0.2 mM PMSF) was mixed with 200 µl of cell lysate containing
recombinant pol Non-denaturing PAGE--
Log phase 293 cells from five
75-cm2 flasks were washed with phosphate-buffered saline
and resuspended in l ml of buffer containing 10 mM HEPES,
pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM PMSF, 5 mM sodium bisulfite, 1 mM dithiothreitol, 5 µg/ml leupeptin, and 2.5 µg/ml
aprotinin. The cells were sonicated and centrifuged at 14,000 × g for 30 min at 4 °C. Aliquots of the supernatant (150 µl, 10 mg/ml protein) were run on a 5-15% gradient gel with a 3.5%
stacking gel at 4 °C as described previously (13). The proteins were
transferred to nitrocellulose membranes and immunoblotted. One of the
lanes was blotted with the p125 monoclonal antibody and another with
PDIP38 polyclonal antibody. The band that was immunoblotted by the p125
monoclonal antibody was excised and immersed in SDS-PAGE sample buffer
for 2 h at 37 °C. The extracted proteins were subjected to
SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted
with antibody against PDIP38.
Yeast Two-hybrid Screening--
A yeast two-hybrid screen of a
human placental cDNA library was performed (see "Experimental
Procedures") using human p125 and p50 as the baits. With pol
When p50 was used as the bait, 22 positives were obtained. These were
sorted into three groups by restriction enzyme digestion and
examination of the products on agarose gel electrophoresis. DNA
sequencing of the inserts was used to identify the positives. Two of
these were found to be novel genes of previously unknown function.
These will be referred to as polymerase
An important additional result was obtained in this experiment. The
interaction of PDIP38 with PCNA was tested and was shown to be positive
(Fig. 1). The finding that PDIP38 interacts with PCNA is highly
significant, because the latter is a crucial accessory protein that
functions as a molecular sliding clamp and endows pol Data Base Analysis of PDIP38 and PDIP46--
The PDIP46 isolate
from the two-hybrid screening was sequenced. A sequence of 1950 bp was
obtained. The sequence matched THC132984 and THC212521 in the TIGR
Tentative Human Consensus (THC) sequences and had over 100 hits in the
ESTdb. A search of the NRdb provided additional clones: a human genomic
sequence HS222E13 and a protein sequence named Isoform 1 (GenBankTM accession number CAB77058). The function of this
gene is unknown, and its interaction with pol
The entire insert of PDIP38 in the pACT2 vector was sequenced and found
to consist of 1967 bp. BLAST searches of the NRdb revealed that the
PDIP38 sequence matched the following three genomic sequences:
AC002094, from human chromosome 17; AC002324 from mouse Chromosome 11;
and L14429 from Caenorhabditis elegans cosmid ZK652.9. It
also matched over 100 EST sequences from the ESTdb. The human genomic
sequence AC002094 was found to have one error (Fig.
2A), in that it is missing a
cytosine at position 43699, which results in an incorrect open
reading frame (ORF). The corrected AC002094 sequence (Fig.
2A) was used for ORF prediction and allowed the construction
of an exon-intron map of the PDIP38 gene (Fig. 2B). The
predicted ORF matched the cDNA sequence derived from the clone
isolated from the yeast two-hybrid screen and was also consistent with
the sequences of a number of the EST clones found by BLAST searches.
The derived human PDIP38 cDNA (gi:15213478) encodes a protein of
368 a.a. (GenBankTM accession number AF179891) The
PDIP38 insert in the pACT2 vector contained the entire coding sequence
except for the first three N-terminal amino acids. The cognate mouse
PDIP38 ORF was derived from the mouse genomic clone (AC002324). The
predicted ORF also consists of 368 amino acid residues and has an
identity of 95% with human PDIP38 at the amino acid level. The
exon-intron map of the mouse PDIP38 gene is shown in Fig.
2B.
Analysis of the PDIP38 Amino Acid Sequence--
Data base searches
were performed with the BLASTp program using the National Center for
Biotechnology Information Blast2 service against the SWISS-PROT + TREMBL + TREMBL_new data bases. In addition to the mouse PDIP38
cDNA, sequences for the Drosophila and C. elegans proteins (accession numbers Q9VNC0 and Q95PW5) were found; these had 57 and 35% identity, respectively, to the human PDIP38 sequence. The C-terminal region of PDIP38 (residues 233-353) was found
to be related to the bacterial APAG proteins and to the F box A protein
(22). The APAG gene was found in E. coli (23) and has been
identified in a number of other bacteria. The E. coli APAG
gene encodes a protein of 125 a.a. whose function is unknown.
The human and mouse FBA proteins (371 a.a. and 367 a.a., respectively) contain the APAG domain at their C termini.
Alignments for the human, mouse, Drosophila, and C. elegans PDIP38 sequences with the mouse and human FBA proteins and
the APAG proteins from E. coli, Salmonella
typhimurium, and Rhizobium etli are shown in Fig.
3. The bacterial APAG proteins have
identities of 28-37% with the C-terminal region of PDIP38, whereas
the FBA proteins exhibit a 32% identity with PDIP38. A striking
feature of this alignment is a conserved region that conforms to the
motif GXGVVGXXPX(LI). This motif
contains a GXGXXG signature, a motif found in
NAD- and FAD-binding proteins (24, 25) that is involved in the binding
of the ADP moiety (26, 27). The region of similarity of PDIP38 proteins
encompasses most of the length of the APAG protein.
Northern Blot Analysis of PDIP38 Expression--
Northern blot
analysis revealed a single major transcript of ~2.0 kb of PDIP38 in
both HeLa and MCF7 total RNA extracts using a 323-bp probe (436-759
from the human PDIP38 cDNA sequence). The size of the transcript is
in good agreement with the sequence information. A minor transcript (1 kb) was also detected. At this point it cannot be ascertained if the
minor transcript (1 kb) represents a second transcript of the same
gene, the existence of a related gene, or a degradation product of
the 2-kb transcript (data not shown).
Demonstration of Protein-Protein Interactions between PDIP38 with
PCNA or p50 by Pull-down Assays--
The existence of protein-protein
interactions between human PDIP38 and PCNA and PDIP38 and p50 were
further investigated by GST pull-down experiments. The coding sequence
of PDIP38 was inserted into the pGEX-5X-3 vector, and the recombinant
GST-PDIP38 was expressed (see "Experimental Procedures"). The
GST-PDIP38 was used to demonstrate the ability to bind to PCNA and p50
by pull-down assays using E. coli lysates containing the
recombinant proteins (Fig. 4,
A and B). These experiments in which bacterially
expressed proteins were used indicate that PDIP38 directly interacts
with PCNA and also with the pol
Purified GST and GST-PDIP38 adsorbed onto glutathione-Sepharose beads
were then used in a variation of the pull-down assay to determine
whether pol
The interaction between human PCNA and PDIP38 that was identified by
the yeast two-hybrid pairwise assay (Fig. 1) was confirmed by the use
of PCNA overlay assays using either biotinylated or digoxigenin-labeled
PCNA. By this assay, PCNA binds to GST-PDIP38 (Fig.
5, B and C,
lanes 2). The specificity of the interaction was shown by a
negative reaction for GST alone (Fig. 5, B and C, lanes 1). Inspection of the PDIP38 sequence
shows the presence of three putative PCNA-binding motifs (19-21)
between residues 81-88, 151-158, and 193-200 (Table
I).
Demonstration of Interactions between PDIP38 and PCNA or pol PDIP38 Is Associated with pol Demonstration of the Association of PDIP38 with pol The motivation for these studies was to identify novel proteins
that interact with pol Obviously, protein-protein interactions revealed by the yeast
two-hybrid system do not necessarily indicate that the native (non-fusion) proteins are capable of interaction, nor do they provide
any evidence that such interactions take place in a cellular context.
In these studies, we have further characterized the interaction of
PDIP38 with the p50 subunit of pol The ability of PDIP38 to interact with p50 and also with PCNA was
demonstrated by coimmunoprecipitation and GST pull-down assays.
Pull-down experiments using bacterially expressed p50 and PCNA
established that the interactions were direct. These experiments were
important for the validation of the coimmunoprecipitation experiments
from cell extracts. The coimmunoprecipitation experiments revealed that
PDIP38 interacts with the pol Whereas the functions of PDIP38 are still unknown, our studies have
shown that it possesses one property that is of relevance to the
functions of pol In yeast, the third subunit of pol Although the functions of PDIP38 are at this point unknown, a protein
that binds to both p50 and PCNA could serve as a structural role to
strengthen the interaction of the pol Data base analysis showed that PDIP38 has a striking conservation
between the C-terminal 111 residues with the bacterial APAG proteins. A
similar conservation has been found for one other mammalian protein,
the F box A protein (22). We suggest this conserved region in PDIP38 be
called the APAG domain. The conservation of the APAG domain across a
wide evolutionary range argues that it is likely to have some
significant biological function. The functions of the APAG protein or
of the members of the family (Fig. 3) that includes PDIP38 are unknown.
The presence of the highly conserved GXGXXG motif
suggests the presence of a pyrophosphate-binding domain,
i.e. that they may be able to bind either pyrophosphate or
nucleotide triphosphates (27). Thus, exploration of the possible binding of these compounds to PDIP38 may provide clues to its function.
The FBA protein contains an F box at its N terminus (residues 9-58).
The latter motif provides the family of F box proteins with the ability
to bind to the core of the ubiquitin-protein ligase complex,
Skp1-cullin-F box protein (33). The F box proteins bind to specific
substrates, which are thereby targeted for intracellular degradation
via the proteosome. The presence of an APAG domain in the F box A
protein would suggest that it may be a protein interaction domain;
however, the target of the F box A protein is currently unknown.
The current work establishes that p50 interacts with PDIP38, and it is
noteworthy that p50 has also been reported to interact with a protein
termed p36 also known as PDIP1 (polymerase In summary, we have identified two novel proteins, PDIP38 and PDIP46,
that interact with the p50 subunit of pol (pol
). Two baits were used in this study. These were the large
(p125) and small (p50) subunits of the core pol
heterodimer. p50
was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen.
In this study, the interaction of PDIP38 with pol
was further
characterized. PDIP38 encodes a protein of 368 amino acids whose C
terminus is conserved with the bacterial APAG protein and with the F
box A protein. It was found that PDIP38 also interacts with
proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to
interact with both the p50 subunit of pol
and with PCNA was
confirmed by pull-down assays using glutathione
S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38
interaction was also demonstrated by PCNA overlay experiments. The
association of PDIP38 with pol
was shown to occur in calf thymus
tissue and mammalian cell extracts by GST-PDIP38 pull-down and
coimmunoprecipitation experiments. PDIP38 was associated with pol
isolated by immunoaffinity chromatography. The association of PDIP38
with pol
could also be demonstrated by native gel electrophoresis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
, and -
(1-6). pol1
is required for
replication of the leading strand and for completion of the lagging
strand synthesis at the replication fork (7). The action of pol
as
a processive enzyme requires its interaction with proliferating cell
nuclear antigen (PCNA), which functions as a molecular sliding clamp
(1). The core mammalian pol
enzyme consists of a tightly associated
heterodimer of 125- and 50-kDa subunits. pol
has been shown
recently (8-14) to consist of at least four subunits, consisting of
the core enzyme and two additional subunits in both yeast and mammalian
systems. In the yeast Schizosaccharomyces pombe, Cdc27 and
Cdm1 have been identified as the third and the fourth pol
subunits,
respectively, (8, 9). In Saccharomyces cerevisiae Pol32p has
been identified as the homologue of the S. pombe third
subunit (10). A human homologue of Cdc27, the KIAA0039 gene product
(11-13), and p12, a human homologue of Cdm1 (14), have recently been
identified and can be considered to be the third and fourth subunits of
human pol
. Our laboratory has been interested in the identification of additional protein components that are involved in the formation of
the pol
replication complex. In this study we report the identification of two novel proteins, PDIP38 and PDIP46, that interact
with the p50 subunit of pol
by the use of the yeast two-hybrid (15,
16) screening method. PDIP38 was shown to be a PCNA-binding protein,
and its interaction with pol
was established by additional experiments.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
catalytic subunit p125 and the small
subunit p50 were ligated in-frame with GAL4 DNA-binding domain (amino
acids 1-147) at the multiple cloning sites of the pAS2-1 vector.
Positive clones were confirmed by transfection of the plasmid DNA into
Y190 cells containing pAS2-1-p125 or pAS2-1-p50 for pairwise yeast
two-hybrid assays for protein-protein interactions. After confirmation
by pairwise yeast two-hybrid assays, the positive clones were
sequenced. The primer, 5'-AGA TTA CGC TAG CTT GGG TGG TC-3', was used
for automated sequencing. Blast searches were performed against the
NRdb and ESTdb in the National Center for Biotechnology Information.
from HeLa Cell Extracts by Immunoaffinity
Chromatography--
pol
was purified by immunoaffinity
chromatography on a column containing an immobilized antibody against
the p125 subunit of pol
(17). Frozen cells from 20 liters of mid
log phase HeLa cells (Cell Culture Center, Cellex Biosciences, Inc.)
were lysed by sonication in 140 ml of TGEE buffer (50 mM
Tris-HCl, pH 7.8, 10% glycerol, 1 mM EDTA, 0.5 mM EGTA) containing 10 mM benzamidine, 10 mM sodium bisulfite, 1 mM PMSF, 7 µg/ml
pepstatin A, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The lysate
was centrifuged for 10 min at 27,000 × g. The
supernatant was loaded onto a 20-ml pol
p125 immunoaffinity column.
The column was washed with 120 ml of TGEE buffer containing 100 mM NaCl and eluted with TGEE buffer containing 400 mM NaCl and 30% ethylene glycol. Fractions of 1.5 ml in
volume were collected. The fractions were assayed for pol
activity
with poly(dA)/oligo(dT) as the template and loaded onto a 5-15%
gradient SDS-polyacrylamide gel for Western blot analysis (14).
p125 monoclonal antibody 78F5.
p50 or PCNA. To this was added 100 µl of 50%
suspension of glutathione-Sepharose 4B beads. The suspension was
incubated with gentle rocking for 2 h at 4 °C. After collection
by centrifugation, the beads were washed five times with GST binding
buffer. Bound proteins were extracted with SDS-PAGE sample buffer by
boiling and were then subjected to SDS-PAGE and immunoblotted as
described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p125
as bait, all of the positive clones that were studied were identified
as the pol
p50 subunit. This is consistent with previous data on
the strong interaction between these two pol
subunits which still
coelute on glycerol gradient ultracentrifugation after treatment with
3.4 M urea (17).
interacting protein 38 (PDIP38) and polymerase
interacting protein 46 (PDIP46). Twelve
identical PDIP46 and six identical PDIP38 clones were isolated. The
third group, consisting of four isolates, was identified as p21Waf1, a known PCNA-binding protein (18-21).
Further tests of the interactions of p50 with p21Waf1,
PDIP38, and PDIP46 were performed by the use of a pairwise yeast two-hybrid analysis, followed by a liquid assay for
-galactosidase activity (11). The results confirmed the ability of p50 to interact with p21Waf1, PDIP38, and PDIP46 (Fig.
1).
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Fig. 1.
Demonstration of the interactions
between p50-PDIP38, PCNA-PDIP38, and p50-PDIP46 by pairwise yeast
two-hybrid assays. Y190 cells were transfected with pol p125,
pol
p50, or PCNA and grown in Trp dropout synthetic media. These
cells were transfected with PDIP38 or PDIP46 cDNA, selected for His
auxotrophy, and assayed for
-galactosidase activity (see
"Experimental Procedures").
with the
processivity required for chromosomal DNA replication (1, 7). Because
PDIP38 also interacts with the p50 subunit, it suggests that it could
interact both with pol
and with PCNA. Only very weak activities for
a p50-p50 interaction or a p50-PCNA interaction were found, and the
strength of the interactions was similar to those of negative controls
when only p50-pAS2-1 (the p50-GAL4 DNA-binding domain fusion construct)
or p50-ACT2 (the p50-GAL4 activation domain fusion construct) was
present in the yeast cells which grew in Trp
or
Leu
media, respectively.
is currently under investigation.
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Fig. 2.
Analysis of the human and mouse genomic
sequences of PDIP38. A, the 5' end of the PDIP38
transcript was aligned with the sequences available in the current
ESTdb (C06428, U46408, and AL079399) and NRdb (AC002094). The missing
cytosine at position 43699 of AC002094 is highlighted.
B, organization of the human and mouse PDIP38 genes. The
diagram shows the organization of the human PDIP38 gene, starting at
nucleotide 43620 of the AC002094 genomic clone and ending at nucleotide
53879. The organization of the mouse PDIP38 gene, starting at the
beginning of the open reading frame at nucleotide 33275 and ending at
nucleotide 23952 of the mouse AC002324 genomic clone, is shown
below that of the human gene.
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Fig. 3.
Alignment of PDIP38 with APAG and FBA
proteins. The C terminus of human PDIP38 (residues 243-353) was
aligned with PDIP38 sequences from mouse, Drosophila, and
C. elegans, together with the human and mouse FBA (F
box A or FBX3) proteins and the sequences of three bacterial APAG
proteins (E. coli, S. typhimurium, and R. etli). Selected identical residues are shown in
boldface, and residues that are conserved are
shaded, in order to show the relationships between the three
groups. The Clustal 1.74 program was used for the alignments.
GenBankTM accession numbers for the sequences are as
follows: human PDIP38, Q96JE4; mouse PDIP38, Q91VA6;
Drosophila PDIP38, Q9VNC0; C. elegans PDIP38,
Q95PW5; human FBA, mouse FBA Q9JIE4l; E. coli APAG, P05636;
S. typhimurium APAG (APAG SALTY) Q56017; R. etli
APAG (APAG_retli), Q9ZES3. Residues conserved across the alignment are
shown with asterisks on the bottom line.
p50 subunit, and the observed
interactions are not due to the presence of a bridging protein.
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Fig. 4.
Demonstration of the interaction of human
PDIP38 with PCNA, pol p50, and pol
125 by GST pull-down assays. A and
B, lanes 1 and 2, cell lysates of
E. coli cells expressing GST or GST-PDIP38 were mixed with
lysates of E. coli cells containing overexpressed PCNA
(A) or pol
p50 (B) and pulled down with
glutathione-Sepharose 4B beads (see "Experimental Procedures").
Western blots of the bound proteins were performed using monoclonal
antibody 74B1 against PCNA (A) or with monoclonal antibody
17D2 against pol
p50. Lane 3 shows the input recombinant
PCNA or recombinant pol
p50. C, lane 1, calf
thymus pol
purified to the DE52 column chromatography step (14);
lanes 2 and 3, the partially purified calf thymus
pol
preparation was mixed with E. coli lysates
containing GST and PDIP38-GST fusion proteins, respectively, and pulled
down with glutathione-Sepharose. Western blot analysis was performed
with monoclonal antibody 78F5 against pol
p125.
p125 could be bound from partially purified calf thymus
pol
preparations. The GST-PDIP38 pull-down assay was positive (Fig.
4C). This ability of GST-PDIP38 to bring down p125 is
presumably because of the tight association of p125 with p50, because
PDIP38 does not interact with p125, at least in the yeast two-hybrid
assay (data not shown), and PCNA is absent in the pol
preparation.
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Fig. 5.
Demonstration of the interaction of human
PDIP38 with PCNA by overlay assay. A, GST (lane
1) and GST-PDIP38 fusion protein (lane 2) were purified
by affinity chromatography on glutathione-Sepharose and stained with
Coomassie Blue after SDS-PAGE. B, overlay using
biotin-labeled PCNA. C, overlay using digoxigenin-labeled
PCNA.
Putative PCNA binding motifs in PDIP38
in
Cell Extracts by Coimmunoprecipitation--
The association of PDIP38
with p50, p125, and PCNA in HeLa cell extracts was demonstrated by
coimmunoprecipitation experiments. A polyclonal antibody to a PDIP38
peptide derived from the C terminus (see "Experimental Procedures")
was used. The PDIP38 antibody was able to immunoprecipitate PDIP38 from
HeLa lysates and also recognized a polypeptide of 38 kDa on SDS-PAGE by
Western blotting. (The PDIP38 polypeptide migrates on SDS-PAGE with an
apparent size of 38 kDa as determined by Western blotting, although its amino acid sequence indicates a calculated molecular mass of 42 kDa.
The reason for this difference is unknown.) The immunoprecipitates were
then Western blotted with antibodies against PCNA and p125 (Fig.
6). The results for the Western blot with
PCNA showed strong signals. This is consistent with evidence for a
direct interaction of PDIP38 and PCNA obtained by the overlay and GST
pull-down experiments (Figs. 4 and 5). p125 and p50 were also
immunoprecipitated. The most likely interpretation for the
coimmunoprecipitation of p125 with PDIP38 is that PDIP38 is bound to
the pol
heterodimer via p50. These results show that the
interaction of PDIP38 with p50 is not restricted to two proteins in
isolation but that the interaction can be demonstrated in cell extracts
with pol
.
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Fig. 6.
Demonstration of the interaction of human
PDIP38 with PCNA and pol 125 by
coimmunoprecipitation experiments. HeLa cell extracts were
immunoprecipitated (IP) with PDIP38 peptide polyclonal
antibody and protein A and protein G beads (see "Experimental
Procedures"). The bound proteins were analyzed by immunoblotting
(WB) with PDIP38 rabbit polyclonal antibody (lanes
1-3), PCNA monoclonal antibody 74B1 (lanes 4 and
5), or pol
p125 monoclonal antibody 78F5 (lanes
6 and 7), respectively. Lane 1, mock
immunoprecipitation with rabbit preimmune serum; lanes
2, 4, and 6, HeLa input; lanes
3, 5, and 7, immunoprecipitate with PDIP38
antibody.
Purified by Immunoaffinity
Chromatography--
pol
was purified from HeLa cells by a
modification of the immunoaffinity chromatography method used for the
isolation of pol
from calf thymus tissues (17) and HeLa cell
lysates. A HeLa cell lysate from 20 liters of cell culture was directly
chromatographed on a p125 immunoaffinity column without passage through
DE52 and phenyl-agarose steps, and a lower salt buffer (0.1 M NaCl) was used to wash the immunoaffinity column. The
column was eluted with TGEE buffer containing 0.4 M NaCl
and 30% ethylene glycol (see "Experimental Procedures"). Western
blotting for PDIP38 showed that it is present with all four subunits
(p125, p50, p68, and p12) in the peak fraction of pol
as judged by
pol
activity assay and Western blot analyses (Fig.
7).
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Fig. 7.
PDIP38 is present in pol isolated by immunoaffinity chromatography-purified pol
from HeLa cells. A cell lysate from 20 liters
of HeLa cell culture was fractionated on a pol
immunoaffinity
column (see "Experimental Procedures"). A, assay of the
fractions eluted from the column for pol
activity on a
poly(dA)·oligo(dT) template in the presence (solid
circles) and absence (open circles) of PCNA. The peak
fractions (numbers 43, 46, and 49) of
pol
activity were analyzed by Western blot for the presence of p125
and PDIP38 (B) and for the presence of p68, p50, and p12
(C).
by
Nondenaturing Gel Electrophoresis--
Cultured cell extracts (293 cells) were subjected to electrophoresis on native (i.e.
nondenaturing) polyacrylamide gradient gels and Western blotted for the
presence of the p125 subunit of pol
and PDIP38. This method has
been used to demonstrate a high molecular weight complex of pol
(13). The results are shown in Fig. 8.
The p125 antibody recognizes a single band that elutes close to
thyroglobulin (Mr 669,000). It is seen that the major band recognized by the PDIP38 antibody coincides with the same
band that is recognized by the p125 antibody (Fig. 8A). Two other smaller bands were also observed. The high molecular weight band
was excised from the gel and run in a second dimension on SDS-PAGE.
Western blotting with antibody against PDIP38 showed the presence of a
band corresponding to PDIP38 (Fig. 8B). Both p125 and p50
could also be shown to be present in the high molecular band by a
similar analysis (data not shown). These experiments show that PDIP38
is associated with the pol
complex in cell extracts.
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Fig. 8.
Association of PDIP38 with pol
during native gel electrophoresis. A,
nondenaturing gel electrophoresis of 293 cell extracts was performed as
described under "Experimental Procedures." The proteins were
transferred to nitrocellulose membranes which were then Western blotted
with a monoclonal antibody against the p125 subunit of pol
(left lane) or a rabbit polyclonal antibody against PDIP38
(right lane). The protein standards used as molecular weight
markers were thyroglobulin (669,000), ferritin (440,000), and catalase
(232,000). B, the band corresponding to that immunoblotted
by the antibody against the p125 subunit (A) was excised and
subjected to SDS-PAGE as described under "Experimental Procedures."
After electrophoresis, the proteins were transferred to a
nitrocellulose membrane and Western blotted with PDIP38 antibody
(right lane). The left lane is the 293 crude cell
extract.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
using the yeast two-hybrid system. pol
,
rigorously isolated from mammalian tissues, has been shown to consist
of a tightly associated heterodimer of the p125 and p50 subunits. Two
less tightly associated subunits, p68 and p12, have recently been
identified as homologues of the so-called third and fourth subunits in
S. pombe (11-14). In S. cerevisiae, evidence for
the presence of the third subunit has been obtained, but so far a
homologue of the S. pombe fourth subunit (10) has not been
identified. A yeast two-hybrid screen of a human cDNA library using
the p125 and p50 subunits was carried out. Our yeast two-hybrid screening using p50 as the bait revealed three positives. Two of these
were proteins of unknown function, and the third was identified as p21.
.
complex in cell extracts, in that we
could show the coimmunoprecipitation of p125 as well as p50. The
results indicate that PDIP38 is able to bind to p50 when the latter is
in association with p125. More significantly, an association of PDIP38
with pol
activity during immunoaffinity chromatography was
established. An association of PDIP38 with pol
was also
demonstrated by native gel electrophoresis of 293 cell lysates. These
studies strongly support the view that the interaction of PDIP38 with
the pol
complex is physiological, i.e. that this
interaction takes place in a cellular context and is likely to play
some physiological relevant function. The issues of whether it is an
accessory protein for pol
or could be considered to be a
subunit of pol
is currently under investigation.
, viz. the ability to bind to PCNA. PCNA has been shown to bind a number of proteins through a short peptide motif (18-21), exemplified by that present in
p21Waf1 which binds to the interdomain connector
loop of PCNA (18). Inspection of the PDIP38 sequence shows that there
are three putative PCNA-binding motifs. These are shown in alignment
(Table I) with those of examples of the known PCNA-binding proteins,
p21 (18-21, 28), DNA (cytosine-5)methyltransferase (29), the
pol
p68 subunit (11-14, 30), FEN1 (19), and DNA ligase I (32). The PCNA-binding motif is QXXZXXF(F/Y), where Z is
generally an aliphatic residue (20). The three candidate sequences
(PDIP38) all possess only a single aromatic residue, but there exists
at least one known PCNA-binding motif with a single aromatic residue,
that for DNA (cytosine-5)methyltransferase (29).
(10, 31) binds to both the
homologue of p50 and PCNA. p68, the mammalian third subunit of pol
,
binds to PCNA (11-13, 30) and interacts with the p50 subunit in the
yeast two-hybrid assay.2 In
this respect, the properties of PDIP38 parallel those of the third
subunit of pol
. This similarity raises a possibility that PDIP38
might be able to substitute p68 to form a variant pol
holoenzyme.
This is an intriguing consideration, bearing in mind that pol
can
be regarded as taking part in a number of cellular functions that
include (a) synthesis at the leading strand, (b) synthesis at the lagging strand, and (c) DNA repair and
recombination processes. Each of these processes may require a specific
and likely different assembly of proteins surrounding the pol
core heterodimer. This issue is also important in considerations of attempts
to isolate a pol
replication complex from tissues or cells, because
there may exist different multiprotein complexes involving pol
.
Further studies using the isolated PDIP38 protein are currently
underway to determine its ability to interact with the purified pol
complex and to assess its functional effects on pol
activity.
heterodimer with PCNA by
binding to pol
and PCNA, as already noted for p68 (14). This
possibility is consistent with studies of S. pombe Cdc27, where the three-subunit enzyme in which Cdc27 is absent was found to
bind much less strongly to PCNA than did the four-subunit pol
(31).
Given the trivalent nature of PCNA, there is also the possibility that
PDIP38 could serve as a bridging protein between two pol
-PCNA
complexes. This could be envisioned as a linking of two pol
-PCNA
assemblies via PDIP38 in which it is attached to one assembly via PCNA
and to the other via p50.
interacting protein 1)
and the Werner helicase (34, 35). This suggests that p50 may serve as a
nexus or scaffold for the interaction of other subunits or accessory
proteins with pol
.
. The interaction of
PDIP38 with p50 was extensively characterized, and although its
functions are currently unknown, these findings indicate that the
number of proteins that may be involved in the formation of the pol
enzyme complex may involve additional proteins besides the four known subunits.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. H. Xu for the digoxigenin- and biotin-labeled PCNA probes and Jing Su for performing the native gel electrophoresis.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM31973.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Valhalla, NY 10595. Tel.: 914-594-4070; Fax:
914-594-4058; E-mail: Marietta_Lee@nymc.edu.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M208694200
2 L. Liu and M. Y. W. T. Lee, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
pol, DNA polymerase;
PDIP, polymerase -interacting protein;
PCNA, proliferating cell
nuclear antigen;
ESTdb, expressed sequence tag data base;
NRdb, non-redundant nucleotide data base;
PMSF, phenylmethylsulfonyl
fluoride;
a.a., amino acid;
GST, glutathione S-transferase;
ORF, open reading frame.
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