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INTRODUCTION |
In yeast Saccharomyces cerevisiae, rev
mutants, rev1, rev3, and rev7 were
initially isolated on the basis of reduced mutations after UV treatment
(1, 2) and classified into a subbranch of the RAD6 epistasis
group, with alteration in the error-prone postreplication repair
pathway (2-4). Yeast strains carrying rev mutations exhibit
a reduced frequency of mutations following treatments with a variety of
DNA damaging agents (5). The majority of mutations are induced through
replication processes, which directly copy the damaged DNA template
during DNA replication, a function of REV genes. Lesion
bypass DNA replication is one cellular system activated in response to
DNA damage that functions to prevent cell death caused by replication
arrest (5).
Rev1 protein, belonging to a family of translesion DNA polymerases (6,
7), contains a BRCA1 C terminus
(BRCT)1 domain (8) and
possesses deoxycytidyl transferase activity in template-directed
reactions (9). It is capable of extending a primer terminus by
insertion of dCMP opposite a variety of damaged bases and
apurinic/apyrimidinic (AP) sites (9-11). The in vivo role
of the REV1 gene in translesion replication of AP sites has been well studied using a yeast system (12, 13), and dCMP residues are
known to be incorporated opposite AP sites in the majority of bypass
events in the wild type but not the rev1
strain (13). Deoxycytidyl transferase activity of the Rev1 protein, first
observed for the yeast enzyme (9), has been conserved throughout
eucaryotic evolution (14-18), implying a contribution to survival (5).
However, the other role of the REV1 gene in the mutagenesis
pathway is not due to its action as a deoxycytidyl transferase.
Although the Rev1 protein does not allow bypass of T-T (6-4)
photoproducts in vitro, the gene is required for translesion DNA synthesis in vivo (13, 19). A mutagenesis-deficient BRCT domain mutant encodes a protein with normal deoxycytidyl transferase activity (13), while methyl methane sulfonate-induced mutagenesis has
been shown to be normal in a site-directed mutant lacking deoxycytidyl
transferase activity (10). The second function of the Rev1 protein may
be to incorporate large complexes with DNA polymerase
due to
protein-protein interactions (5, 10, 13).
REV3 encodes the catalytic subunit of DNA polymerase
,
which also contains Rev7 as a stabilizing and enhancing element for polymerase activity (20). DNA polymerase
has the ability to extend
terminally mismatched primers and distorted structures (10, 21-24). It
is also capable of bypass replication of T-T cyclobutane dimers (20).
These properties are likely to contribute to generation of mutations.
Yeast strains carrying rev3 or rev7 mutations
exhibit a similar phenotype on treatment with wide variety of
DNA-damaging agents, supporting the conclusion that both act in the
same process (5).
The damage-induced mutagenesis pathway is evolutionarily conserved from
yeast to humans. Preservation of the functions of human REV
genes is supported by the finding that human cell lines expressing high
levels of human REV1 or REV3 antisense RNA
exhibit a much reduced frequency of 6-thioguanine-resistant mutants
induced by UV (25, 26). The biochemical properties of the REV1 protein are also conserved from yeast to humans (14-16, 18). While the human
DNA polymerase
has yet to be purified, there is no direct evidence
for polymerase activity of human REV3 protein. Recently, Murakumo
and coworkers found that human REV7 interacts with human REV1 and REV3
proteins in a yeast two-hybrid study, as confirmed by
immunoprecipitation experiments (27, 28). With yeast proteins, a
Rev1-Rev7 interaction has not been observed (20). The responsible domain in the human REV1 protein is not found in the yeast Rev1 counterpart but highly conserved in the mouse Rev1 protein (17), suggesting a specificity for higher eucaryotes (28).
In the present paper we provide several lines of evidence that human
REV1 and REV7 form a stable heterodimer. In contrast to yeast Rev3, the
human REV7 subunit does not affect the transferase activity or
stability of the REV1 protein. A possible molecular role of the REV7
subunit may be to help assembly of the REV1 protein to a large complex
containing REV3 and/or other DNA polymerases in higher eucaryotes.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Human REV7 cDNA was
amplified from HeLa cDNA by PCR with primers
(5'-CATATGACCACGCTCACACGACAAGAC-3' and 5'-GGAGGCTGAGAAGTCGAGGTT-3') and
cloned into a pCR 2.1-TOPO vector (Invitrogen) to yield plasmid pREV7.
The nucleotide sequence was verified. Then a
NdeI-EcoRI fragment of the pREV7 was inserted
into the corresponding site of the pET20b(+) vector (Novagen) to yield
plasmid pET-REV7, which produces intact REV7 protein under control of
the T7 promoter. The REV1- and REV7- coexpressing
plasmid (pBAD-REV7-REV1S) was constructed by insertion of the
Klenow-treated XbaI-AvrII fragment of pET-REV7
into the Klenow-treated KpnI site of pBAD-I-REV1S (16). The
pBAD-REV7-REV1S produces intact REV1S and REV7 proteins, regulated by
the pBAD promoter of the arabinose operon. A plasmid producing a truncated his6-REV1S protein (245-847) was constructed by
insertion of a PCR-amplified fragment into the
NdeI-HindIII site of pET15b (Novagen).
Overproduction and Purification of Human REV7--
BL21 (DE3)
(29) harboring pET-REV7 was grown in 20 liters of LB medium
supplemented with ampicillin (250 µg/ml) at 15 °C with aeration
until the culture reached an A600 value of 0.6. Isopropyl
-D-thiogalactopyranoside was added to 0.2 mM, and the incubation was continued for 10 h. The
cells were harvested by centrifugation, resuspended in 2 ml of buffer I
(50 mM Tris-HCl, pH 7.5/0.1 mM EDTA/10
mM
-mercaptoethanol/10% sucrose) per 1 g of cells,
and frozen in liquid nitrogen. The frozen cells were thawed in ice
water and lysed after addition of phenylmethylsulfonyl fluoride to 0.1 mM by introduction of buffer I containing 100 mM spermidine, 1 M NaCl, and 4 mg/ml lysozyme
to 10 mM, 100 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 30 min. The cell lysate
was clarified by centrifugation at 75,000 × g for 30 min at 4 °C. Although the majority of the induced REV7 protein was
precipitated, a small amount was recovered in the soluble fraction.
Subsequent column chromatography was carried out at 4 °C using an
FPLC system (Amersham Biosciences). We found that the intact REV7
protein has affinity for a nickel-chelating column. After adding
imidazole to 10 mM to the crude lysate, it was applied at
0.5 ml/min to a 5-ml HiTrap chelating column (Amersham Biosciences), which had been treated with 0.1 M NiSO4 and
equilibrated with buffer A (50 mM Tris-HCl, pH 7.5/100
mM NaCl/10% glycerol/10 mM
-mercaptoethanol) containing 10 mM imidazole. The column
was washed with 50 ml of equilibration buffer at 0.5 ml/min and then the REV7 protein was eluted with 50 ml of a linear gradient of 10-100
mM imidazole in buffer A. Fractions containing REV7
protein, which were eluted at about 50 mM imidazole, were
pooled and dialyzed against buffer B (50 mM Tris-HCl, pH
7.5/1 mM EDTA/1 mM dithiothreitol/10% glycerol), then loaded at 0.1 ml/min onto a 5-ml HiTrap heparin column
(Amersham Biosciences) equilibrated with buffer B. Unbound material was
eluted in 1-column volume. The REV7 was eluted at around 6-column
volumes, and fractions containing REV7 were collected then loaded at
0.1 ml/min onto a MonoQ HR 5/5 column (Amersham Biosciences)
equilibrated with buffer B. The column was washed with 10 ml of buffer
B, and the REV7 protein was eluted with 10 ml of a linear gradient of
0-300 mM NaCl in buffer B. The REV7 eluted at the
beginning of the gradient, and then the peak fraction was loaded at 0.1 ml/min on a Superdex 200 HR 10/30 column (Amersham Biosciences)
equilibrated with buffer C (50 mM HEPES-NaOH, pH 7.5/10
mM
-mercaptoethanol/10% glycerol) containing 150 mM NaCl. The REV7 eluted as a single peak with an apparent
molecular mass of 27 kDa with a Stokes' radius of 25 Å. The peak
fractions of REV7 protein were pooled, frozen in liquid nitrogen, and
stored at
80 °C.
Overproduction and Purification of the Human REV1-REV7
Complex--
The bacterial strain, BL21 (DE3) (29), harboring
pBAD-REV7-REV1S was grown in 2 liters of SB medium (30) supplemented with ampicillin (250 µg/ml) with aeration until the culture reached an A600 value of 0.6. L-(+)-arabinose was added to 1%, and the incubation
was continued for 11 h. Then cells were harvested by centrifugation at 4 °C. The resultant cell paste was resuspended in
2 ml of buffer II (50 mM HEPES-NaOH, pH 7.5/500
mM NaCl/0.1 mM EDTA/10 mM
-mercaptoethanol) per 1 g of cells and frozen in liquid
nitrogen. The cells were thawed on ice and after addition of 100 mM phenylmethylsulfonyl fluoride to 0.1 mM were
lysed with 100 mM spermidine plus 4 mg/ml lysozyme (at 10 mM and 0.4 mg/ml, respectively) in buffer II.
Subsequent column chromatography was carried out at 4 °C using an
FPLC system (Amersham Biosciences). After adding imidazole to 10 mM, the lysate was applied at 0.1 ml/min to a 1-ml HiTrap chelating column (Amersham Biosciences), which had been treated with
0.1 M NiSO4, and then equilibrated with buffer
D (50 mM HEPES-NaOH, pH 7.5/500 mM NaCl/10
mM
-mercaptoethanol/10% glycerol) containing 10 mM imidazole. The column was washed with 10 ml of
equilibration buffer at 0.1 ml/min, and then the REV1 and REV7 proteins
were eluted with 10 ml of a linear gradient of 10-100 mM
imidazole in buffer D. Fractions containing both REV1 and REV7
proteins, which were eluted at about 50 mM imidazole, were
pooled and dialyzed against buffer C containing 400 mM NaCl
then loaded at 0.1 ml/min onto a 1-ml HiTrap heparin column (Amersham
Biosciences) equilibrated with the dialysis buffer. The column was
washed with 10 ml of equilibration buffer at 0.1 ml/min, and then the
REV1 and REV7 proteins were eluted with 10 ml of a linear gradient of
400-700 mM NaCl in buffer C. Fractions containing both
REV1 and REV7 proteins, which eluted at about 490 mM NaCl,
were loaded at 0.1 ml/min onto a Superdex 200 HR 10/30 column (Amersham
Biosciences) equilibrated with buffer C containing 500 mM
NaCl. The REV1 and REV7 proteins coeluted as a single peak with an
apparent molecular mass of 410 kDa with a Stokes' radius of 60.3 Å.
The REV-REV7 complex peak fractions were pooled, frozen in liquid
nitrogen, and stored at
80 °C.
Other Proteins--
Intact REV1 protein and deletion derivatives
of the his6-REV1 protein were purified as described previously (15,
16). Protein concentrations were determined by Bio-Rad protein assay using BSA (BIO-RAD) as the standard.
Reconstitution of the REV1-REV7 Complexes in Vitro--
REV1
protein (24 µg) and REV7 protein (3 µg) were mixed in 70 µl of
buffer C containing 500 mM NaCl. For truncated his6-REV1 derivatives,
N407 (4.2 µg) and
C885 (4.8 µg) proteins
(15) were mixed with REV7 (1.5 µg). After incubation on ice for
2 h, a 50-µl aliquot of the mixture was loaded on a Superdex 200 PC 3.2/30 (Amersham Biosciences) column and equilibrated with buffer C
containing 500 mM NaCl, and fractions (40 µl) were
collected using the SMART system (Amersham Biosciences).
Antibodies--
A rabbit polyclonal antiserum against REV7 was
kindly provided by Dr. Y. Murakumo (Nagoya University Graduate School
of Medicine, Nagoya, Japan) (27). To obtain polyclonal antibodies
against REV1, his6-REV1S-(245-847) was expressed in BL21 (DE3)
(29) at 15 °C, purified as described previously (15), and used to immunize a rabbit.
Western Blotting--
Protein samples were subjected to
SDS-PAGE, electrotransferred onto nitrocellulose filters (Bio-Rad), and
then probed with rabbit REV1 antiserum (1:1000 dilution) or rabbit REV7
antiserum (1:2000 dilution). The blots were visualized with the ECL
blotting system (Amersham Biosciences).
Sucrose Density Gradient Sedimentation--
Sucrose density
gradient sedimentation was performed as described (31). The purified
REV1 protein (15 µg), REV7 protein (7 µg), and REV1-REV7 complex
(12 µg) were sedimented through 2 ml of 10-40% sucrose gradient in
buffer C containing 500 mM or 150 mM NaCl by
centrifugation at 55,000 rpm for 20 h in a TLS 55 rotor (Beckman)
at 4 °C, and fractions (100 µl) were collected from the bottom of
the tube and analyzed by SDS-PAGE. Gel bands were stained with
Coomassie Brilliant Blue R-250 according to the method of Sambrook
et al. (32) and quantified using NIH image 1.60 software.
Sedimentation coefficients were determined relative to those of
standard proteins sedimented in parallel gradients.
Transferase Assays--
The oligonucleotide templates
5'-CTCGTCAGCATCTTCAXCATACAGTCAGTG-3'
(X = G; 30G, A; 30A, T; 30T, C; 30 °C,
tetrahydrofuran; 30F) and the primer 5'-CACTGACTGTATG-3' (P13) were
purchased (17). The latter was labeled using polynucleotide kinase (New
England Biolabs) and [
-32P]ATP (Amersham Biosciences)
and annealed to the template. The standard reaction mixture (25 µl)
contained 50 mM Tris-HCl buffer, pH 8.0, 25 mM
(NH4)2SO4, 2 mM
MgCl2, 0.1 mg/ml BSA, 5 mM dithiothreitol, 0.1 mM dNTP, 100 nM primer-template, and 1 µl of
protein sample diluted with buffer C containing 500 mM NaCl
and 0.1 mg/ml BSA as indicated. After incubation at 30 °C for the
indicated time, reactions were terminated with 10 µl of stop solution
(30 mM EDTA/94% formamide/0.05% bromphenol blue/0.05%
xylene cyanol), and products were resolved on 20% polyacrylamide gels
containing 8 M urea and autoradiographed at
80 °C. The
amount of DNA present in each band was quantified using a Bio-Imaging
Analyzer BAS2000 (Fuji Photo Film Co., Ltd.).
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RESULTS |
In Vitro Reconstitution of the Human REV1-REV7
Complex--
Recently, Murakumo and coworkers reported that the human
REV1 protein directly interacts with the human REV7 protein (28). To
determine whether the purified REV1 and REV7 proteins (Fig. 1A) form a stable complex,
they were mixed and incubated on ice, and then the mixture was
subjected to gel filtration chromatography in the presence of 500 mM NaCl (see "Experimental Procedures"). When the
individual proteins were loaded on the column, REV1 of 138 kDa eluted
at an apparent molecular mass of 380 kDa (Fig. 1B,
panel a), (16) and REV7 of 24 kDa eluted as a single peak at
an apparent molecular mass of 27 kDa (Fig. 1B, panel
b). When the mixture of two proteins were developed on gel
filtration chromatography, a limited but significant fraction of REV7
was coeluted with REV1 under these conditions in the presence of 500 mM NaCl (Fig. 1B, panel c),
suggesting that REV1 and REV7 form a salt-resistant complex.

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Fig. 1.
In vitro reconstitution of
REV1-REV7 complexes. A, 10% SDS-PAGE analysis of
purified REV1 (lane 1) and REV7 (lane 2)
proteins. Aliquots (0.5 µg) were electrophoresed and stained with
Coomassie Brilliant Blue R-250. Lane M, molecular mass
markers (Bio-Rad). B, gel filtration analysis of REV1
(panel a), REV7 (panel b), and a mixture of REV1
and REV7 proteins (panel C). Samples were loaded onto a
Superdex 200 PC 3.2/30 column and 40-µl fractions were collected,
analyzed by 10% SDS-PAGE, and stained with Coomassie Brilliant Blue
R-250. Ferritin (440 kDa), aldolase (158 kDa), and ovalbumin (43 kDa)
eluted at the positions indicated. C, gel filtration
analysis of deletion derivatives of the his6-REV1 protein and REV7.
Deletion mutants of his6-REV1 protein, N407 (a) and
C885 (b), were mixed with REV7 protein and loaded on a
Superdex 200 PC 3.2/30 column, and 40-µl fractions were collected,
subjected to 10% SDS-PAGE, and visualized by Western blotting probed
with anti REV1 (upper panels) or anti REV7 (lower
panels) antisera. Positions of the respective proteins on the
membrane are indicated by arrow heads.
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The binding domain in REV1 for REV7 has been mapped to the C terminus
(28). It was further examined using purified truncated his6-REV1
proteins.
N407 (lacking the BRCT domain of the N terminus) and
C885 (lacking the C terminus) (15) were mixed with purified REV7 and
loaded on the gel filtration column, and then the eluted fractions were
analyzed by Western blotting. Both
N407 and
C885 proteins eluted
at an apparent molecular mass of around 200 kDa on this gel filtration
chromatography (15). When the eluted fractions were analyzed by Western
blotting, signals of REV7 were detected in fractions containing
N407
protein (Fig. 1C, panel a), but not
C885 (Fig.
1C, panel b), in agreement with the previous report that the C terminus of REV1 contains the interaction domain with
REV7 (28).
Purification of the REV1-REV7 Complex from Overproducing
Escherichia coli Cells--
With in vitro reconstitution,
only a limited fraction of the proteins formed complexes. To purify a
large amount, we established a system coexpressing the REV1
and REV7 genes in E. coli. The plasmid, pBAD-REV7-REV1S expressing both genes from the
pBAD promoter as an operon, was employed with addition of
arabinose as an inducer. SDS-PAGE analysis of the total proteins of the
cells after induction revealed bands for REV1 and REV7 (Fig.
2A, lane 2) with
sizes in agreement with the predicted molecular masses of 138 and 24 kDa respectively. The mobilities of the proteins in the SDS-PAGE were
identical to those of individually purified REV1 and REV7 (Fig.
1A, data not shown).

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Fig. 2.
Purification of the REV1-REV7 complex.
A, overproduction of REV1 and REV7 proteins in E. coli cells. Total E. coli proteins
were analyzed by 10% SDS-PAGE before (lane 1) and after
induction (lane 2) and stained with Coomassie Brilliant Blue
R-250. Lane M, molecular mass markers (Bio-Rad).
B, the purification scheme. C, elution profile of
the REV1-REV7 complex from Superdex 200 gel filtration chromatography.
Fractions eluted from the gel filtration column were analyzed by 10%
SDS-PAGE and visualized by Coomassie Brilliant Blue R-250 (top
panel), Western blotting probed with anti REV1 (middle
panel), or anti REV7 antisera (bottom panel).
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Previously, we found that the intact REV1 protein has an affinity for a
nickel-chelating column, eluting at about 20 mM imidazole (16). When the crude lysate was loaded onto a nickel-chelating column,
the REV1 fractions were eluted at about 50 mM imidazole and
also contained REV7 as judged by Western blotting (data not shown).
This higher than expected affinity of the REV1 fraction for the
nickel-chelating column could be due to the actions of the REV7
protein, suggesting complex formation. Whereas REV7 alone did not bind
to heparin-Sepharose, as described under "Experimental Procedures," with the mixture a significant fraction of REV7 was retained in the column and coeluted with REV1 (data not shown). The
peak fraction from the heparin column was further loaded onto a gel
filtration column (Fig. 2C), and SDS-PAGE analysis revealed REV1 and REV7 to coelute (Fig. 2C, top panel).
Furthermore, the proteins were verified by Western blotting. The
membrane was probed with anti REV7 antiserum first (Fig. 2C,
bottom panel), stripped, and then probed with anti-REV1
antiserum (Fig. 2C, middle panel). The antibodies
specifically detected the respective proteins, and no cross-reaction
was observed (data not shown).
Physicochemical Properties of REV1, REV7, and the REV1-REV7
Complex--
In the gel filtration chromatography final step of the
purification, REV1, REV7, and the REV1-REV7 complex were eluted with apparent molecular masses of 380, 27, and 410 kDa, respectively. The
respective Stokes' radiuses were 59, 25, and 60.3 Å (Table I). To determine the molecular masses in
solution, they were analyzed by sucrose density gradient sedimentation.
They all sedimented as single peaks, and the REV1-REV7 complex
co-sedimented through the centrifugation (data not shown), suggesting
again a stable association. The determined sedimentation coefficients
are summarized in Table I. Employing the method described by Siegel and
Monty (33), we calculated the molecular masses of REV1, REV7, and the
REV1-REV7 complex to be 138, 13, and 141 kDa, respectively (Table I),
suggesting REV1 and REV7 to be monomers and the REV1-REV7 complex a
heterodimer in solution. Analysis of the stoichiometry of the REV1 and
REV7 proteins in the complex by scanning of a SDS-PAGE gel visualized
by Coomassie staining gave a REV1:REV7 ratio of 1:0.6, suggesting again
that the REV1-REV7 complex is a heterodimer.
Transferase Activity of the REV1-REV7 Complex--
The
deoxycytidyl transferase activity of the REV1-REV7 complex was analyzed
by primer extension assay using a template, with G as the nucleotide
immediately downstream from the annealed primer. When equivalent
amounts of REV1 and REV1-REV7 were subjected to the assay, the time
course of the deoxycytidyl transferase reaction was identical (Fig.
3A). To determine the
stability of the proteins in this reaction condition, incubation at
30 °C was performed for the indicated times and then residual
activity was examined. The half-lives (~20 min) of both were almost
the same (Fig. 3B). These results indicated that the REV7
protein did not affect the transferase activity or stability of the
REV1 protein.

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Fig. 3.
Transferase activity of the REV1-REV7
complex. A, time courses of deoxycytidyl transferase
reactions with REV1 and the REV1-REV7 complex. The REV1 protein
(open circles) and the REV1-REV7 complex (open
triangles) at 18 fmol were incubated with the primer-template
(P13-30G) and dCTP under standard reaction conditions for the
indicated times. The amount of DNA present in each band was quantified
using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.).
B, stability of the REV1 protein and the REV1-REV7 complex.
REV1 proteins (open circles) and REV1-REV7 complexes
(open triangles) were incubated in the standard buffer
containing dCTP without template at 30 °C for the indicated times,
and then the transferase reactions were started by addition of the
template, P13-30G, with further incubation for 1 min. The relative
residual activities were plotted. C, substrate specificity
of the transferase reactions. The REV1-REV7 complex (9.3 ng) and each
of primer-templates, P13-30G (panel a), P13-30A
(panel b), P13-30T (panel c), P13-30C
(panel d), and P13-30F (panel e) were incubated
with no dNTP ( ), a single dNTP (G, A,
T, C), or all four dNTPs (N) under
standard reaction conditions for 10 min. The reaction products were
resolved on 20% polyacrylamide gels containing 8 M urea
and autoradiographed at 80 °C.
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Then the substrate specificity of the transferase reactions was
analyzed using five different primer-templates in the presence of 100 µM dNTPs (Fig. 3C). In this experiment, the
respective primer-templates differed only at the template nucleotide
immediately downstream from the annealed primer (16). The REV1-REV7
complex inserts dCMP opposite template G, A, T, C, and an AP site and inserts dGMP and dTMP opposite template G (Fig. 3C). The
ability of the REV1-REV7 complex was essentially identical to that of REV1 alone (16). To confirm this, the kinetic parameters with the
REV1-REV7 complex were determined by steady-state gel kinetic assays
(Table II). The assays were all carried
out with a 5-min incubation because the time course of the reactions
was linear until 10 min (Fig. 3A). In the deoxycytidyl
transferase reaction, the template nucleotides slightly affected
kcat values, which were 3.7, 2.5, 1.6, 2.1, and
4.5 min
1 opposite templates G, A, T, C, and an AP site,
respectively (Table II). The template nucleotides strongly affected the
Km value for dCTP (Table II), the affinity of which
for template G was 53, 310, 380, and 28 times higher than for templates
A, T, C, and an AP site, respectively. These properties of the
REV1-REV7 complex were essentially identical to those of REV1 alone
(16).
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Table II
Kinetic parameters of deoxycytidyl transferase reactions of the
REV1-REV7 complex
Kinetic assays were performed for 5 min in 25-µl reaction solutions
using 57 fmol (9.3 ng) or 113 fmol (19 ng) of REV1-REV7 complex and 2.5 pmol of the primer-templates shown in Fig. 3C. dCTP
concentrations ranged from 1 to 2500 µM. The
Km for dCTP and kcat were
evaluated from the plot of the initial velocity versus the dCTP
concentration using a hyperbolic curve-fitting program. Data from two
to four independent experiments were plotted together, and the
correlation coefficients (R2) were more than 0.98.
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DISCUSSION |
Recently, Murakumo and coworkers found that human REV1 binds to
human REV7 at the C terminus (28), an interaction that has not been
reported in yeast, S. cerevisiae (20). Comparison of amino
acid sequences revealed the C-terminal region to be highly conserved in
the human and mouse REV1 proteins but not in the yeast counterpart (17,
28). This suggests that the interaction was acquired in higher
eucaryotes, although it is conceivable that it was lost in yeast during
evolution and might play an important role in translesion DNA synthesis
in mammalian cells (28). In this work, we provided several lines of
evidence that the human REV7 forms a stable heterodimer with REV1 protein.
Purified REV1 and REV7 were found to have a considerable potential to
form complexes in vitro even in the presence of 500 mM NaCl. Furthermore, both proteins co-purified after
several purification steps and co-sedimented through centrifugation
with constant stoichiometry. Indeed, it has been demonstrated that exogenously expressed REV1 protein co-immunoprecipitates with endogenous REV7 protein in HeLa cells (28).
The molecular masses of REV1, REV7, and the REV1-REV7 complex in
solution were calculated using Stokes' radius and sedimentation coefficients to be 138, 13, and 141 kDa, respectively. The value for
REV1 agrees with the theoretical molecular mass of 138 kDa, while that
of REV7 is smaller than the theoretical 25 kDa, suggesting the proteins
to be monomers in solution. The ratio of band densities of the REV1 and
REV7 proteins on a SDS-PAGE gel was assigned 1:0.6, suggesting a
stoichiometry of 1:1 (163 kDa) or 2:1 (301 kDa). The former value
agrees well with our result of 141 kDa, suggesting that the REV1-REV7
complex is a heterodimer in solution.
Previously, it was suggested that the REV7 forms a homodimer and
interacts through a common interface with REV1 (28). However, it is
very likely, considering the following, that purified REV7 exists as a
monomer in solution: (i) the obtained molecular mass of 13 kDa from our
experiments is much smaller than the theoretical molecular mass of 24 kDa; (ii) the purified REV7 has the potential to assemble with purified
REV1. If REV7 formed a stable homodimer, it could not interact with
free REV1 because the interaction interface would be hidden by the
homodimerization. Therefore, we suggest that REV7-REV7 interaction is
transient and dynamic or tightly regulated in vivo.
Surprisingly, our results demonstrated that the REV7 did not influence
the stability, substrate specificity, or kinetic parameters of the
transferase reactions of REV1 protein in contrast to the case with
yeast Rev3, which exhibits strongly enhanced DNA polymerase activity
and stability on assembly with Rev7 (20). It is possible that such
enhancement is yeast-specific or alternatively that in higher
eucaryotes one of the important functions of the human REV7 is to
assemble the REV1 protein to a large complex containing REV3 and/or
other DNA polymerases, and this function might be required for
translesion DNA replication.