Deoxycytidyl Transferase Activity of the Human REV1 Protein Is Closely Associated with the Conserved Polymerase Domain*

Yuji Masuda, Mamoru Takahashi, Noriko Tsunekuni, Tomoyuki Minami, Masaharu Sumii, Kiyoshi Miyagawa, and Kenji KamiyaDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 zeta  (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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 lambda  phage vector, lambda 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 lambda RH2, was chosen for further analysis.

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 lambda 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.

For construction of full-length REV1S cDNA, the BamHI/XbaI fragment of REV1S cDNA from lambda 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 lambda RHT1 was inserted. The resulting plasmid, pREV1S, contained the fragment of the position 20-4227 of human REV1S cDNA (AB047646).

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 -80 °C.

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 beta -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 beta -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 beta -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, M NaCl, 10% glycerol, 10 mM beta -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.

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 -80 °C. The amount of DNA present in each band was quantified using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.).

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 -80 °C. The amounts of the binding fraction and free DNA were quantified using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 alpha -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.

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.


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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.

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.


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Fig. 3.   Northern blot analysis of REV1/REV1S mRNA in human tissues. The REV1 cDNA probe (upper) and beta -actin cDNA probe (lower) were hybridized.

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).


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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.

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 Delta N245 and Delta 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 Delta N245/Delta C885 protein, which has both truncations of N and C termini, sustained the activity. Delta C810 and Delta C729 proteins, which have further truncation of the C terminus, were inactive. The transferase activity of Delta 407 protein was dramatically decreased, but significant activity was still detected. However, the protein Delta N407/Delta C885, which has both truncations of 406 residues of the N terminus and 365 residues of the C terminus, was inactive.

                              
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Table I
List of the Rev1S-expressing plasmids

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 Delta N407/Delta 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 Delta N245 and Delta 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 Delta N245 was slightly higher than that of the wild type, and those of Delta C885 and Delta N245/Delta C885 proteins were slightly lower than the wild type level. The activity of the Delta 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 Delta 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.

                              
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Table II
Apparent molecular weight of the h6-REV1S protein and its derivatives by gel filtration chromatography
The Superdex 200 PC 3.2/30 column was calibrated with ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa), and a standard curve was produced. Apparent Mr was determined using the standard curve.

                              
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Table III
Summary of the deoxycytidyl transferase activities of the h6-REV1S and mutant proteins

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, Delta N245, Delta C885, and Delta N245/Delta C885 proteins were quite similar to that of the wild type REV1S protein. The affinities of Delta N407, Delta C810, and Delta N407/Delta C885 proteins were about one-third of that of the wild type, and the affinity of Delta 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%.

                              
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Table IV
DNA binding affinities of the h6-REV1S and mutant proteins


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Delta 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 Delta N407 protein was dramatically decreased, and that of the Delta 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 Delta C885 protein, which is 75 residues longer than the Delta 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 Delta 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.

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 Delta N407/Delta C885 protein (Table II). The apparent molecular weight of the Delta N407/Delta C885 protein is close to the calculated molecular weight (Table II), suggesting that only the Delta N407/Delta C885 protein is a monomer. (ii) The mobility of the Delta N407/Delta C885-DNA complex is much faster than that of others on gels (Fig. 7A). This supports the idea that only Delta N407/Delta C885 exists as a monomer and that the REV1S protein forms a dimer.

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 Delta 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.

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 Delta C885 with Delta N245 and Delta N245/Delta 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.

    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.

Dagger 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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RESULTS
DISCUSSION
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