From the Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
Received for publication, September 5, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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The REV1 protein is a member of the growing
family of translesion DNA polymerases. A cDNA of the human
REV1 gene that we had originally isolated encoded 1250 amino acids residues, which was one amino acid shorter than previously
reported ones. The shorter form of REV1 was named REV1S. All
individuals examined expressed equivalent amounts of REV1S
and REV1 mRNA, suggesting that the REV1S
mRNA is a splicing variant. We show that the REV1S protein also
possesses deoxycytidyl transferase activity that inserts a dCMP
opposite a DNA template apurinic/apyrimidinic site. Deletion and
point mutation analysis of the REV1S protein revealed that the domain
required for deoxycytidyl transferase and DNA binding activities of the
REV1S protein are located in a conserved domain of translesion DNA
polymerases. This result indicates that the structure of the catalytic
site of the deoxycytidyl transferase closely resembles that of the
translesion DNA polymerases. Therefore, the molecular mechanism of the
dCMP transfer reaction of the REV1S protein and maybe also the REV1
protein might be the same as that of the dNTP transfer reaction of the
translesion DNA polymerases.
In yeast Saccharomyces cerevisiae, almost all induced
mutations arise during translesion replication, a process that promotes elongation past sites of unrepaired lesions (1). The mutagenesis pathway (rev) mutants were initially isolated by their
reduced mutations after UV treatment (2). The REV1,
REV3, and REV7 genes are required for the major
pathway for translesion replication in yeast (3-6). The
REV1 gene encodes a deoxycytidyl transferase that
incorporates dCMP opposite apurinic/apyrimidinic
(AP)1 sites in the template
(7). The REV3 and REV7 genes encode a translesion
DNA polymerase, pol The REV1 gene is a member of a growing family including
umuC (15) and dinB/dinP (16) of
Escherichia coli; RAD30 (17) of S. cerevisiae; and XPV/RAD30A (18, 19),
RAD30B (20), and DINB1 (21, 22) of humans. Recent
studies have shown that those genes encode translesion DNA polymerases
(18, 23-29). However, the REV1 protein does not possess such
polymerase activity although it contains the conserved domain in
translesion polymerases.
In this work, we made various mutants of the human REV1S protein.
Biochemical analysis of those mutant proteins showed that the domain
required for deoxycytidyl transferase activity and DNA binding is
located in a conserved domain of translesion DNA polymerases.
Isolation of Human REV1 cDNA
We found a partial sequence of a candidate of the human
REV1 cDNA in a data base (accession number AJ131720).
Based on the sequence, a portion of the cDNA fragment of the human
REV1 was amplified by RT-PCR from human breast cancer
using primers 5'-AATCTAGCTGGAGCTGTTGA-3' and
5'-GTAAAACCACCTGGACATTG-3', and it was used as a probe for
screening a human testis cDNA library (CLONTECH). In this library, the cDNA was
inserted into a To obtain the promoter-proximal side of the REV1 cDNA,
another portion of the cDNA fragment was amplified by RT-PCR from
human breast cancer using primers 5'-TTCTGTTTTGTCAAGGGCTG-3' and
5'-TTGGTCTCTGCAAGGATTTC-3', and it was used as a probe for screening
the same library. One positive clone, named For construction of full-length REV1S cDNA, the
BamHI/XbaI fragment of REV1S cDNA
from Analysis of the REV1 and REV1S mRNA
Total mRNA from human mononuclear cells was used as a source
of RT-PCR (30, 31). First-strand cDNA synthesis was carried out
with reverse transcriptase (RAV-2) (TaKaRa) from an oligo(dT) (12-18)
primer (Life Technologies, Inc.). To amplify a portion of the human
REV1/REV1S cDNA fragment, including the different region, the PCR primers 5'-GGGTCCGCAAGGCCGGAGAA-3' and
5'-GCCTGGCCTCATAACTACAA-3' were used. After heating at 95 °C for 2 min, 35 cycles of amplification were performed according the following
conditions: 1 min of denaturation at 95 °C, 1 min of annealing at
58 °C, and 2 min of extension at 72 °C with TaKaRa
ExTaqTM polymerase (TaKaRa). The amplified 1618-base
pair cDNA fragment was directly sequenced from the primer
5'-GCGCAGAATCTGGATTCTCCCACA-3' using an ABI 373 DNA sequencing system
(Applied Biosystems). To quantify the ratio of the mRNA species,
the amplified 1648-base pair cDNA was diluted and reamplified with
PyrobestTM DNA polymerase (TaKaRa) by PCR using
the primers 5'-GCGCAGAATCTGGATTCTCCCACA-3' and
5'-32P-CCCAGCTGGAGTGGCAGTATTACC-3'. The PCR products were
separated on a 5% polyacrylamide gel containing 8 M urea.
The radioactivity of each band of 96 and 93 bases, respectively,
derived from REV1 and REV1S cDNA was measured
using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.).
Northern Blot Analysis of REV1 mRNA
The BglII/XbaI fragment from the human
REV1S cDNA was used as a probe. Human multiple-tissue
Northern filters (MTN1 and -2, CLONTECH) were
hybridized with the probe in ExpressHybTM Hybridization
Solution (CLONTECH) at 65 °C and washed with
0.1 × SSC and 0.1% SDS at 65 °C. The hybridized human
REV1/REV1S mRNA was visualized by autoradiography at
Construction of Mutant REV1S Genes
To introduce site-directed mutations, we used a PCR-based
method. DNA fragments of the limited portions were amplified by RCR and
cloned. After confirming the nucleotide sequence, the small fragments
were assembled with other fragments of the wild-type cDNA. All of
the expression constructs are listed in Table I. Proteins were tagged
with six histidines at their N termini.
Construction of pET-C729--
The 5'-portion of the
REV1S gene was amplified by PCR with primers
(5'-GAAGCTCCCATATGAGGCGA-3' and 5'-TGCTTGGACCTGGCAGAGGA-3') to
introduce an NdeI site at the first ATG (position 173 of the REV1S cDNA). The PCR fragment was cloned into the pCR
2.1-TOPO vector (Invitrogen), and the resulting plasmid was named
pRHFNde2. The NdeI/BamHI fragment from pRHFNde2
was inserted into the corresponding site of the pET15b vector
(Novagen). The resulting plasmid, pET-C153 (Table I), was digested with
BamHI and HindIII, and then the BamHI/HindIII fragment of pREV1S was inserted.
The resulting plasmid, pET-C729, produces a truncated protein
containing extra amino acid residues, YR, at the C terminus (Table
I).
Construction of pBAD-REV1S--
An
XbaI/HindIII fragment that includes a
ribosome-binding site and the 5'-portion of the REV1S gene
of pET-C729 was inserted into the corresponding site of pBAD22A. The
resulting plasmid, pBADRN, was digested with HindIII, and
then the HindIII fragment of the 3'-portion of the
REV1S gene of pREV1S was inserted. The resulting plasmid,
pBAD-REV1S, produces a full-length REV1S protein (Table I). The
expression of the h6-REV1S gene is induced by arabinose (32).
Construction of pET-N407--
To introduce an NdeI
site at position 1391 of the REV1S cDNA, PCR was carried
out using primers (5'-ACACAGGACATATGTCAGTA-3' and
5'-GATGGATCCGGTCTAGATGCTTTG-3'). The PCR fragment was cloned into the
pCR 2.1-TOPO vector. The resulting plasmid, pRHNXB6, was digested with
XbaI and partially digested with BglII, and then
the BglII/XbaI fragment from pRH2IN was inserted.
The resulting plasmid, pRHNX, was digested with ApaI and
XbaI, and then the ApaI/XbaI fragment
of pRH2IN was inserted. The resulting plasmid was named pRHNXA. The
NdeI/BamHI fragment of pRHNXA was inserted into
the corresponding site of pET15b. The resulting plasmid, pET-N407,
produces a truncated protein starting from the 407th methionine (Table
I).
Construction of pBAD-REV1SA--
To introduce the D569A/E570A
mutation, PCR was carried out using primers (5'-ACACAGGACATATGTCAGTA-3'
and 5'-TACCAGCGCTGCAGCACAAC-3'). The PCR fragment was cloned into the
pCR 2.1-TOPO vector, and the resulting plasmid was named pTUNE6. The
SphI/Eco47III fragment of pREV1S was replaced
with the corresponding fragment of pTUNE6. The resulting plasmid,
pREV1SA, was identical to pREV1S except for the D569A/E570A mutation.
The BamHI/HindIII fragment of pREV1SA was
inserted into the corresponding site of pET-C153. The resulting plasmid
was named pET-C729A (Table I). An XbaI/HindIII
fragment that includes a ribosome-binding site and the 5'-portion of
the REV1SA gene of pET-C729A was inserted into the
corresponding site of pBAD22A. The resulting plasmid, pBADRNA, was
digested with HindIII, and then the HindIII
fragment of the 3'-portion of the REV1S gene of pREV1S was
inserted. The resulting plasmid, pBAD-REV1SA, produces a full-length
mutant protein, D569A/E570A (Table I).
Construction of pET-C885--
pET-C729 was digested with
HindIII, and then the HindIII fragment of pREV1S
was inserted. The resulting plasmid, pET-REV1S (Table I), was digested
with SmaI and partially digested with XbaI. The
3'-end of this DNA was filled in with Klenow fragment (TaKaRa) and
self-ligated. The resulting plasmid, pET-C885, produces a truncated
protein containing extra amino acid residues, GCRNSISSLSMISCQT, at the
C terminus (Table I).
Construction of pET-N245--
To introduce an NdeI
site at position 905 of the REV1S cDNA, PCR was carried
out using primers (5'-TTGGTGCATATGGTCAACAG-3' and
5'-GATGGATCCGGTCTAGATGCTTTG-3'). The PCR fragment was cloned into the
pCR 2.1-TOPO vector, and the resulting plasmid was named pTOMO3. The
NdeI/BamHI fragment of pTOMO3 was inserted into
the corresponding site of pET15b. The resulting plasmid was named pET-N245/C479 (Table I). The pET-REV1S was digested with
BglII to remove two small BglII fragments, which
were of the 5'-portion of the REV1S gene, and then the
BglII fragment of pET-N245/C479 was inserted. The resulting
plasmid, pET-N245, produces a truncated protein starting from the 245th
methionine (Table I).
Construction of pET-N245/C885 and pET-N407/C885--
A
3'-portion of the REV1S gene, the HindIII
fragment, of each of pET-N245 and pET-N407 was replaced with the
HindIII fragment of pET-C885. The resulting plasmids,
pET-N245/C885 and pET-N407/C885, respectively, produce truncated
proteins starting from the 245th and 407th methionine to the 885th
arginine containing extra amino acid residues, GCRNSISSLSMISCQT, at the
C termini (Table I).
Construction of pET-C810--
To replace the 811th serine codon
with a stop codon and introduce a HindIII site at position
2615 of the REV1S cDNA, PCR was carried out using
primers (5'-TGATGAAGCGCTGGTAGACA-3' and
5'-AAGCTTCATATCTCATATATTTAG-3'). The PCR fragment was cloned into the
pCR 2.1-TOPO vector. The resulting plasmid was named p2620H-7. The
HindIII fragment of p2620H-7 was inserted into the
HindIII site of pET-C729. The resulting plasmid produces a
truncated protein at codon 810 (Table I).
Purification of the REV1S Protein and Its Derivatives
Five hundred ml of BL21 (DE3) (33) harboring pET-C885, pET-C810,
pET-C729, pET-N245/C885, and pET-N407/C885 was grown in LB broth
supplemented with ampicillin (100 µg/ml) at 37 °C with aeration
until the culture reached an A600 value of 0.6. isopropyl Deoxycytidyl Transferase Assays
Deoxycytidyl transferase assays were performed essentially as
described by Nelson et al. (7) and Lin et al.
(10). For the DNA substrate, a 5'-end 32P-labeled
oligonucleotide primer 5'-CACTGACTGTATG-3' annealed to an
oligonucleotide template, 5'-CTCGTCAGCATCTTCAUCATACAGTCAGTG-3', was
used (18). The substrate was treated with 0.1 units of E. coli uracil-DNA glycosylase (New England Biolabs) at 30 °C for 30 min in a reaction mixture just before adding enzyme. The reaction mixture (25 µl) contained 25 mM potassium phosphate
buffer, pH 7.4, 5 mM MgCl2, 0.1 mg/ml bovine
serum albumin, 10% glycerol, 5 mM dithiothreitol, 0.1 mM dCTP, 100 nM substrate, and 1 µl of protein sample. The protein samples had been diluted with buffer A
containing 0.1 mg/ml bovine serum albumin as indicated. After incubation at 30 °C for 30 min, reactions were terminated with 10 µl of stop solution (30 mM EDTA, 94% formamide, 0.05%
bromphenol blue, 0.05% xylene cyanole). The reaction products were
resolved on 20% polyacrylamide gel containing 8 M urea and
autoradiographed at DNA Binding Assays by EMSA
The following binding method was adapted from the protocols of
Masuda et al. (34, 35). As a substrate for binding
measurements, the primed template for the deoxycytidyl transferase
assay was used, but it was not treated with uracil-DNA glycosylase.
Reaction mixtures (10 µl) contained 25 mM potassium
phosphate buffer, pH 7.4, 0.2 mg/ml bovine serum albumin, 5 mM dithiothreitol, 0.5 nM substrate, and 1 µl
of protein sample. The mixtures were incubated on ice for 15 min and
loaded on a prerunning 4% polyacrylamide gel (79:1,
acrylamide/bisacrylamide). The electrophoresis buffer contained 6 mM Tris-HCl (pH 7.5), 5 mM sodium acetate, and
0.1 mM EDTA, and the gels were subjected to a constant
voltage of 8 V/cm applied for 2 h at 6 °C. Following gel
electrophoresis, the gels were dried and autoradiographed at
Cloning of the Human REV1S cDNA--
By searching a data base,
we found a human cDNA that encoded a homologue of the yeast REV1
protein. The cDNA has been isolated by yeast two-hybrid screening
as a protein interacting with
The human REV1 cDNA encodes an expected protein of 1250 residues, which is different from the recently reported REV1 protein of
1251 residues (Fig. 1). The former
protein lacks an alanine residue at position 479 (Fig. 1). We denote
here the shorter form of REV1 as REV1S. It was possible that the
REV1S mRNA was a minority in the cell. To clarify this
point, a portion of the REV1/REV1S cDNA fragments
including position 479 was amplified by RT-PCR from mononuclear cells
of 10 individuals and directly sequenced. Fig.
2A shows a typical result. The
sequencing signals from all individuals are duplicated, since CTG and
the intensity of both signals is a ratio of about 1:1, indicating that
both forms exist in all individuals at similar amounts. DNA fragments
including the region were amplified using a 5'-end
32P-labeled primer as one of the PCR primers and separated
on a 5% polyacrylamide gel containing 8 M urea (Fig.
2B). A short band, 93-base product, was derived from the
REV1S cDNA, and a band 3 bases longer was derived from
REV1 cDNA. The average ratio of the shorter and longer
bands, REV1/REV1S, from 10 individuals was 1.25. We
concluded that REV1S mRNA is one of the major forms.
Expression of the REV1 Gene in Human Tissues--
Expression of
the human REV1 gene in various human tissues was examined by
Northern blot analysis (Fig. 3). The
human REV1/REV1S mRNA was detected as a 4.4-kilobase
transcript in all tissues examined, indicating that the REV1
gene is ubiquitously expressed. Expression of the REV1 gene
was relatively high in the testis and ovary and relatively low in the
thymus and small intestine.
Deoxycytidyl Transferase Activity of the REV1S and Mutant
Proteins--
The human REV1 protein possesses a deoxycytidyl
transferase activity (10). First, we examined the deoxycytidyl
transferase activity of the REV1S protein. The REV1S protein was tagged
with six histidine residues at its N terminus and expressed in E. coli cells. The tagged REV1S protein (h6-REV1S) was purified by
affinity chromatography on a nickel chelating column. The purified
protein was analyzed by SDS-polyacrylamide gel electrophoresis (Fig.
4, lane 1). Full-length
h6-REV1S protein with Mr 140,000 and lower bands
were detected. The lower bands appeared specifically when the REV1S
protein was induced (Fig. 4, compare lane 1 with lanes 3-9), indicating that the bands were degraded products of the h6-REV1S protein. This was confirmed by Western blot analysis (data not
shown). Using a primed 30-mer DNA template containing an AP site, we
assayed the transferase activity of the purified h6-REV1S. Transferase
activity that extended the 32P-labeled primer by 1 nucleotide opposite the AP site was detected (Fig.
5B). This evidence proves that
the REV1S protein, as well as the REV1 protein, possesses deoxycytidyl
transferase activity (10).
Next, we made a series of deletion mutants with six histidine residues
at their N termini (Table I, Figs. 1 and
5A) and purified the proteins (Fig. 4, lanes
3-9). As shown in Fig. 5B, the
Johnson et al. (37) introduced a mutation in the highly
conserved SIDE sequence in yeast Rad30, in which the aspartate and glutamate residues had each been changed to alanine, and they found
that the mutant Rad30 protein lacks DNA polymerase activity. We also
changed the corresponding residues to alanines (D569A/E570A) (Table I,
Figs. 1 and 5A). The protein was purified like the wild-type
h6-REV1S protein (Fig. 4, lane 2). In striking contrast to
the transferase activity of the wild-type h6-REV1S protein, the
D569A/E570A protein showed no transferase activity (Fig.
5B).
The proteins were further analyzed by gel filtration chromatography
(Fig. 6). Interestingly, the apparent
molecular weight of each protein was much larger than the calculated
molecular weight except for the DNA Binding Activity of REV1S and Mutant Proteins--
The DNA
binding activities of the peak fractions of gel filtration
chromatography were analyzed by EMSA. To detect a protein-DNA complex
on the gel, the primed template for the deoxycytidyl transferase assay
was used as a substrate for EMSA. However, because an AP site is
unstable in gels (38), the primed template was not treated with
uracil-DNA glycosylase. Therefore, the template nucleotide immediately
downstream from the annealed primer was uracil, not an AP site. The
uracil template is a good substrate as well as the AP site for
deoxycytidyl transfer
reaction2 (10). Using this
substrate, we successfully detected a REV1S-DNA complex by EMSA (Fig.
7A). The amount of the binding
fraction increased in proportion to the increase in protein
concentration (Fig. 7B), although the complex was unstable
in the gel, as indicated by the smear of DNA migrating in the gel
between the protein-DNA complex and the free DNA (Fig. 7A).
Similarly, all of the mutant proteins formed protein-DNA complexes
(Fig. 7A). The mobility of each complex appeared to be
inversely proportional to the molecular weight of each protein. The
affinities of D569A/E570A, The REV1 gene has been isolated and identified as one
of the genes responsible for damage-induced mutagenesis in yeast. In humans, the pathway of damage-induced mutagenesis is largely unknown. Mutagenesis is a key event in carcinogenesis and genetic diseases. To
elucidate the pathway of mutagenesis in humans, we cloned and characterized the human orthologue of the REV1 gene.
A human REV1 cDNA that we isolated encoded a one-amino
acid shorter protein than recently reported clones (Fig. 1) (10, 11).
This shorter form of the REV1 protein was named REV1S. Gibbs et
al. (11) also reported a shorter form of the REV1
mRNA and suggested that this mRNA resulted from slippage at the
3' splice site of an intron rather than a polymorphism. We examined the
distribution of REV1S mRNA in a human population. Direct
sequencing of RT-PCR-amplified fragments from normal human mononuclear
cells of 10 individuals showed that all individuals expressed both of the REV1 and REV1S mRNA (Fig. 2). The ratio
of REV1 and REV1S mRNA was about 1:1 (Fig.
2). These results support the proposal of Gibbs et al. (11).
The alternative possibility that humans have two copies of the gene is
unlikely for the following reasons: (i) the position of the alanine
codon is an exon-intron junction (11); (ii) the human REV1
gene was mapped to only one locus (10); (iii) the nucleotide sequence
of REV1 cDNA reported by Gibbs et al. (11) is
identical to that of REV1S cDNA except for the insertion
of the alanine codon; and (iv) only one species of DNA fragment
containing introns was amplified by genomic PCR (data not shown).
Lin et al. (10) showed that expression of the
REV1 gene was ubiquitous in human tissues using the RT-PCR
method. The results of Northern blot analysis confirmed their
observation (Fig. 3). Moreover, we found that expression of the gene
was relatively high in the testis and ovary and relatively low in the
thymus and small intestine (Fig. 3).
It has recently been shown that the human REV1 gene encodes
a deoxycytidyl transferase (10). Although the REV1 protein is a member
of a family of translesion DNA polymerases, the REV1 protein does not
possess any DNA polymerase activity (7, 10). To elucidate the molecular
mechanism for the deoxycytidyl transfer reaction, we made several
deletion and point mutants of the REV1S cDNA and
purified recombinant proteins as histidine-tagged forms (Fig. 4). We
showed that the REV1S protein also possesses deoxycytidyl transferase
activity. The value of specific activity, 320 pmol of dCMP
transfer/µg of protein in our reaction conditions (Table III), is
higher than that previously reported for the REV1 protein (10).
However, we believe that our results from mutation analysis of the
REV1S protein are applicable to the case of the REV1 protein. We are
currently preparing REV1 protein to compare its activity with that of REV1S.
The domain conserved in translesion DNA polymerase is located in the
central region of the REV1 protein (motifs II to VII) (Fig. 1). The
BRCT domain and motif I are specific to REV1 proteins (Fig. 1). We
found that the It is very likely, considering the following observations, that the
REV1 protein exists as a dimer in solution. (i) The apparent molecular
weight of each protein is roughly twice the calculated molecular
weight, except for the EMSA results indicated that the DNA binding domain was located in the
conserved polymerase domain (motifs II-VII). We found that the
affinities of the inactive proteins were slightly decreased. Since the
lack of transferase activity might be a result of the decreased
affinity to DNA, the transferase activity of One of the characteristic features of the structure of the REV1 protein
is the BRCT domain. The BRCT domain is a characteristic motif of DNA
repair and cell cycle check point proteins (39). A mutation was found
in the BRCT domain of the yeast rev1-1 mutant, which lacks damage-induced mutability (Fig. 1) (15). However, the
Rev1-1 protein retains a significant fraction of its deoxycytidyl transferase activity (40). These findings suggest the existence of a
regulatory mechanism of deoxycytidyl transferase activity of the REV1
protein by the BRCT domain in vivo. Our results showed the
removal of the BRCT domain instead slightly increased the activity
(Table III, compare h6-REV1S and
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(8, 9), which works together with the REV1
protein for translesion replication (7). Recently, the human
orthologues of REV1 (10, 11), REV3 (12, 13), and
REV7 (14) have been identified. It has been shown that the
human REV1 protein possesses a deoxycytidyl transferase activity (10)
and that the human REV1 and REV3 genes are
required for mutagenesis induced by UV light in humans (11,
12).
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phage vector,
DR2
(CLONTECH). Four positive clones were obtained from 5 × 105 plaques. In all of the clones in these
phages, the promoter-proximal side of the REV1 cDNA was
truncated at an XbaI site, because XbaI had used
to construct the cDNA library. One clone, named
RH2, was chosen
for further analysis.
RHT1, was obtained. This
phage contained cDNA from the 5'-untranslated region to the
XbaI site. Since the assembled cDNA encoded 1250 amino
acid residues, 1 amino acid shorter than previously reported ones, we
gave the name REV1S cDNA to the shorter form of the
REV1 cDNA. The DDBJ/EMBL/GenBankTM accession
number of the nucleotide sequence of the human REV1S cDNA is AB047646.
RH2 was inserted into the corresponding site of pBluescript II
SK (
). The resulting plasmid, pRH2IN, was digested with
SacII and XbaI, and the
SacII/XbaI fragment of REV1S cDNA
from
RHT1 was inserted. The resulting plasmid, pREV1S, contained the
fragment of the position 20-4227 of human REV1S cDNA
(AB047646).
80 °C.
-D-thiogalactopyranoside was added to 0.2 mM, and the incubation was continued for 1 h. BL21
(DE3) harboring pET-N245 and pET-N407 was grown at 15 °C, and
incubation was continued for 10 h after the addition of isopropyl
-D-thiogalactopyranoside at an
A600 value of 0.6. For the full-length
h6-REV1S induction, BL21 (DE3) harboring pBAD-REV1S and
pBAD-REV1SA was grown in SB medium (32) at 15 °C, and incubation was
continued for 10 h after the addition of
L-(+)-arabinose to 1% at an A600
value of 0.6. The cells were harvested by centrifugation, resuspended
in 3 ml of buffer I (50 mM HEPES-NaOH, pH 7.5, 1 M NaCl, 0.1 mM EDTA, 10 mM
-mercaptoethanol) per 1.5 g of cells, and frozen in liquid
nitrogen. The cells were thawed on ice, added to 100 mM phenylmethylsulfonyl fluoride to 1 mM, and
lysed by adding 100 mM spermidine/lysozyme (4 mg/ml) in
buffer I to 10 mM and 0.4 mg/ml, respectively. The cells
were incubated on ice for 30 min, heated in a 37 °C water bath for 2 min, and then incubated on ice for 20 min. The cell lysate was clarified by centrifugation at 35,000 × g for 30 min at
4 °C. The following column chromatography was carried out at 4 °C
using a SMART system (Amersham Pharmacia Biotech). Two ml of the lysate was applied at 0.1 ml/min to a 1-ml HiTrap chelating column (Amersham Pharmacia Biotech), which had been flashed with 2 ml of 0.1 M NiSO4 and equilibrated with buffer A (50 mM HEPES-NaOH, pH 7.5, 1 M NaCl, 10% glycerol,
10 mM
-mercaptoethanol) containing 10 mM
imidazole. The column was washed with 10 ml of equilibration buffer and
then washed with 12 ml of buffer A containing 100 mM imidazole. The h6-REV1S and its derivatives were eluted with buffer A
containing 300 mM imidazole. For further purification, 50 µl of the peak fraction was applied at 0.01 ml/min to a Superdex 200 PC 3.2/30 column (Amersham Pharmacia Biotech) equilibrated with buffer
A, and 40-µl fractions were collected. Protein concentrations were
determined by the Bio-Rad protein assay using bovine serum albumin
(Bio-Rad) as the standard.
80 °C. The amount of DNA present in each band
was quantified using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film
Co., Ltd.).
80 °C. The amounts of the binding fraction and free DNA were
quantified using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co.,
Ltd.).
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ABSTRACT
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DISCUSSION
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-integrin (36). The gene was a good
candidate for the human REV1 gene. Because the cDNA was
partial, we screened a human testis cDNA library to obtain a
full-length cDNA. A clone isolated (accession number AB047646)
looks like a truncated form, because an open reading frame started from
the first ATG codon out-of-frame to the main open reading frame.
Therefore, we screened a human liver cDNA library, and 5'-rapid
amplification of cDNA ends was performed using total RNA from human
breast cancer. However, we were not able to obtain any longer cDNA.
This suggests that the cDNA is full-length. Lin et al.
(10) and Gibbs et al. (11) also presented the same
conclusion in recent reports.
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Fig. 1.
Amino acid sequence of the REV1S
protein. The open triangle indicates the
position of the missing alanine residue in the REV1S protein.
The underlined sequences show conserved regions
in REV1 proteins of different species. The boxes show highly
conserved sequences in translesion DNA polymerases. Vertical
bars represent positions of truncation in the REV1S
derivatives that were studied in this work. The closed
triangle indicates the position of mutation of yeast
rev1 1 mutant (15).
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Fig. 2.
Analysis of REV1 and
REV1S mRNA. A, direct sequencing
of RT-PCR-amplified fragments of REV1 cDNA. The expected
nucleotide sequences of REV1 and REV1S are
shown. The boxed sequence of the REV1S
cDNA is duplicated in the boxed sequence of
the REV1 cDNA. B, quantitation of
REV1 and REV1S mRNA of 10 individuals. RT-PCR
amplified fragments of REV1 cDNA were separated on 5%
polyacrylamide gel containing 8 M urea. The positions of
the 93- and 96-base fragments, which are derived from REV1S
and REV1 cDNA, respectively, are indicated on the
left. The band intensities were measured using a Bio-Imaging
Analyzer BAS2000. Ratios of REV1 to REV1S are
shown at the bottom.
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Fig. 3.
Northern blot analysis of
REV1/REV1S mRNA in human tissues. The
REV1 cDNA probe (upper) and -actin
cDNA probe (lower) were hybridized.
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Fig. 4.
SDS-polyacrylamide gel electrophoresis
analysis of purified h6-REV1S and its derivatives. Each
lane contained 0.5 µg of protein. Electrophoresis was
performed using an 8% polyacrylamide gel, followed by Coomassie
Brilliant Blue R-250 staining according to Sambrook et al.
(41).
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Fig. 5.
Deoxycytidyl transferase activity of the
h6-REV1S and mutant proteins. A, schematic
representation of mutant proteins. The conserved motifs are shown as
boxes with numbers, corresponding to Fig. 1.
B, titration of the proteins. Deoxycytidyl transferase
activity of each protein was examined. The amount of each protein is
increased in order by 4, 20, and 100 ng from left to
right. , no protein.
N245 and
C885
proteins as well as the full-length h6-REV1S protein were active,
indicating that 244- and 365-amino acid residues of the N and C
termini, respectively, are dispensable for the transferase activity.
Consistently, the
N245/
C885 protein, which has both truncations
of N and C termini, sustained the activity.
C810 and
C729
proteins, which have further truncation of the C terminus, were
inactive. The transferase activity of
407 protein was dramatically
decreased, but significant activity was still detected. However, the
protein
N407/
C885, which has both truncations of 406 residues of
the N terminus and 365 residues of the C terminus, was inactive.
List of the Rev1S-expressing plasmids
N407/
C885 protein (Table
II). The peak of each protein (Fig.
6A) corresponded to the peak of the activity (Fig.
6B), indicating that the activity is intrinsic to each
protein. This result confirmed the former conclusion. In
N245 and
N407, two peaks of activity were detected (Fig. 6B). The
second peaks of activity were co-eluted with C terminus-truncated
polypeptides (data not shown). The elution profile of the D569A/E570A
protein is identical to that of the wild-type protein (Fig.
5A and Table II), suggesting that the amino acid
replacements do not affect the global structure of the REV1S protein.
This chromatography successfully separated the degraded fractions.
Using the fractions of the leading edges of the chromatography, the
specific activities of mutant proteins were quantitatively compared
(Table III). The relative specific
activity of
N245 was slightly higher than that of the wild type, and
those of
C885 and
N245/
C885 proteins were slightly lower than
the wild type level. The activity of the
N407 protein was about
0.1% of the wild type level (Table III).
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Fig. 6.
Analysis by gel filtration
chromatography. Fractions from the Superdex 200 PC 3.2/30 column
chromatography were analyzed. A, each fraction was separated
on an 8% SDS-PAGE and stained with Coomassie Brilliant Blue R-250
according Sambrook et al. (41). Ferritin (440 kDa), aldolase
(158 kDa), albumin (67 kDa), and ovalbumin (43 kDa) were eluted at the
indicated positions. Fr, fraction number. B,
fractions were diluted to optimal concentration, and deoxycytidyl
transferase activity was assayed. The reaction products were separated
on a 20% polyacrylamide gel containing 8 M urea. The band
intensities of substrates and products were measured using a BAS2000
Bio-Imaging Analyzer, and the fraction of product was calculated as the
ratio to total DNA (2.5 pmol) in the reaction mixture. The scale for
the N407 polypeptide is shown on the right. One unit of
deoxycytidyl transferase was defined as the amount of activity that
incorporates 1 pmol of dCMP at 30 °C for 30 min.
Apparent molecular weight of the h6-REV1S protein and its derivatives
by gel filtration chromatography
Summary of the deoxycytidyl transferase activities of the h6-REV1S
and mutant proteins
N245,
C885, and
N245/
C885
proteins were quite similar to that of the wild type REV1S protein. The
affinities of
N407,
C810, and
N407/
C885 proteins were about
one-third of that of the wild type, and the affinity of
C729 protein
was about one-fifth of that of the wild type (Fig. 7B and
Table IV).
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Fig. 7.
DNA binding activity of the h6-REV1S and
mutant proteins. A, titration of proteins. Reaction
mixtures, each containing 0.5 nM of labeled DNA and the
indicated concentration of protein, were incubated and analyzed as
described under "Experimental Procedures." The protein
concentrations were calculated as monomers. The arrowheads
indicate the positions of origins of the electrophoresis. F,
free DNA; C, protein-DNA complexes. B,
quantitation of EMSA results. The intensities of free DNA and total
shifted fractions were measured using a Bio-Imaging Analyzer BAS2000,
and the fraction of DNA shifted was calculated as the amount of total
shifted fractions divided by total DNA and normalized to 100%.
DNA binding affinities of the h6-REV1S and mutant proteins
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N245 protein possessed transferase activity at the
wild-type level (Fig. 5). This finding indicated that the BRCT domain
is dispensable for transferase activity in vitro. The
activity of the
N407 protein was dramatically decreased, and that of
the
C810 protein was under the detection limit, although the protein
contains the total polymerase domain (motifs II-VII) (Figs. 1 and 5).
On the other hand, the
C885 protein, which is 75 residues longer
than the
C810 protein, retains deoxycytidyl transferase activity at
the wild-type level (Fig. 5). This result suggests that the region
consisting of 75 residues of the C terminus of the
C885 protein is
required for the activity. In this region, we found a motif (motif
VIII) that is well conserved among the REV1 proteins of different
species (Fig. 1). Johnson et al. (37) reported that the
replacement of DE residues in motif III of the Rad30 protein abolished
the polymerase activity of the Rad30 protein of yeast. These residues
also play a critical role in the polymerase activity of other
polymerases (23, 25, 27, 28). Motif III of the Rad30 protein
corresponds to motif IV of the REV1 protein (Fig. 1). In the REV1S
protein, we made a mutant, D569A/E570A, that has mutations in motif IV,
and we found that D569A/E570A completely lacks deoxycytidyl transferase
activity (Fig. 5). It is possible that the global structure of
D569A/E570A is not disrupted by the amino acid replacements, because
the elution profile from gel filtration chromatography (Fig. 6) and the
DNA binding property of D569A/E570A (Fig. 7) are identical to those of
the wild-type protein. Therefore, we believe that the ED residues are
essential for catalysis of the deoxycytidyl transferase reaction of the REV1/REV1S protein but not DNA binding. These results indicated that
the structure of the catalytic site of the deoxycytidyl transferase closely resembles that of the translesion DNA polymerases. Therefore, the molecular mechanism of the dCMP transfer reaction of the REV1/REV1S protein might be the same as that of the dNTP transfer reaction of the
translesion DNA polymerases.
N407/
C885 protein (Table II). The apparent
molecular weight of the
N407/
C885 protein is close to the
calculated molecular weight (Table II), suggesting that only the
N407/
C885 protein is a monomer. (ii) The mobility of the
N407/
C885-DNA complex is much faster than that of others on gels
(Fig. 7A). This supports the idea that only
N407/
C885 exists as a monomer and that the REV1S protein forms a dimer.
N407 was measured at
increased concentrations of substrate DNA. However, even the highest
substrate concentration (1 µM) did not improve the
decreased transferase activity. This substrate concentration is 70 times higher than the apparent binding constant of the protein. Therefore, we believe that the loss of deoxycytidyl transferase activity for the mutant proteins did not directly result from a
decrease in the affinity to substrate DNA. Rather, the stability of the
protein-DNA complex of the inactive proteins seems to decrease. The
result of EMSA showed that the size of the h6-REV1-DNA complex seemed
to gradually increase depending on the protein concentration (Fig. 7).
This observation suggests that the REV1 proteins might multimerize on
the DNA. The multimerization might play an important role in the
transferase activity. The molecular nature of the REV1-DNA complex is
currently being investigated.
C885 with
N245 and
N245/
C885). This might suggest that the BRCT domain negatively regulates the transferase activity in vivo and that the
in vivo role of the BRTC domain of the REV1 protein is
conserved from yeast to humans.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Saburo Fukuda, Dr. Hiroaki Yasumoto, and Dr. Jun Teishima for generating figures. We thank Kumiko Mizuno, Hatsue Wakayama, and Emi Yagi for laboratory assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.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.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB047646.
To whom correspondence should be addressed. Tel.: 81-82-257-5842;
Fax: 81-82-257-5844; E-mail: kkamiya@hiroshima-u.ac.jp.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M008082200
2 Y. Masuda, M. Sumii, and K. Kamiya, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: AP, apurinic/apyrimidinic; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR.
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