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Originally published In Press as doi:10.1074/jbc.M602967200 on June 27, 2006

J. Biol. Chem., Vol. 281, Issue 34, 24314-24321, August 25, 2006
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Role of Single-stranded DNA in Targeting REV1 to Primer Termini*Formula

Yuji Masuda and Kenji Kamiya1

From the Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan

Received for publication, March 29, 2006 , and in revised form, June 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular functions of the REV1 gene have been conserved in evolution and appear important for maintaining genetic integrity through translesion DNA synthesis. This study documents a novel biochemical activity of human REV1 protein, due to higher affinity for single-stranded DNA (ssDNA) than the primer terminus. Preferential binding to long ssDNA regions of the template strand means that REV1 is targeted specifically to the included primer termini, a property not shared by other DNA polymerases, including human DNA polymerases {alpha}, beta, and {eta}. Furthermore, a mutant REV1 lacking N- and C-terminal domains, but catalytically active, lost this function, indicating that control is not due to the catalytic core. The novel activity of REV1 protein might imply a role for ssDNA in the regulation of translesion DNA synthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The majority of both spontaneous and DNA damage-induced mutations in eukaryotes results from replication processes in which REV1, REV3, and REV7 proteins play major roles. Studies of REV genes originated with the isolation of yeast rev mutants (1, 2), which exhibited a reduced frequency of mutations following treatment with a variety of DNA-damaging agents (3). Deoxycytidyltransferase activity of the REV1 protein and DNA polymerase activity of the REV3-REV7 complex for translesion DNA synthesis were discovered in the pioneering work of Lawrence and co-workers (4, 5). By using information obtained from yeast studies, homologues of the encoding genes were subsequently identified in mammals (6–13), and it is now well established that the pathway has been conserved in evolution from the yeast to humans.

REV1 is a member of the Y family of DNA polymerases, which also includes DNA polymerase (pol)2 IV and V in Escherichia coli, and DNA pol {eta}, {iota}, and {kappa} in eukaryotes (14, 15). These proteins are required for translesion DNA synthesis because many lesions block typical replicative DNA polymerases. However, the REV1 protein almost exclusively utilizes only dCTP, in contrast to the other members of the family, and preferentially inserts dCMP opposite template G and a variety of damaged bases and apurinic/apyrimidinic sites (4, 6, 7, 11, 16–18). Because of this preference, REV1 has been called a deoxycytidyltransferase (3, 4). This novel activity has been maintained throughout eukaryotic evolution, implying a contribution to survival (3). Recently, it was demonstrated that the specificity for dCMP is tightly regulated by formation of hydrogen bonds with an arginine residue in the protein, but not template G (19). Indeed, dCMP residues are known to be incorporated opposite apurinic/apyrimidinic sites in the majority of bypass events in wild type yeast cells but not the rev1{Delta} strain (20–23).

A second function of the REV1 gene product in the mutagenesis pathway has also been proposed, independent of its action as a deoxycytidyltransferase (24). Methyl methanesulfonate-induced mutagenesis has been shown to be normal in a site-directed mutant lacking deoxycytidyltransferase activity (25). Furthermore, although the REV1 protein does not allow bypass of thymine-thymine (6-4) photoproducts in vitro, the gene is required for bypass replication of this lesion in yeast cells (24, 26, 27). With respect to the second function, the BRCA1 C-terminal domain of REV1 has an essential function, mutation abolishing UV-induced mutagenesis, even if the protein retains normal levels of transferase activity in vitro (24, 27).

Evolutionary preservation of functions of the mammalian REV1 gene was first indicated by the finding that human cell lines expressing high levels of human REV1 antisense RNA exhibit a much reduced frequency of 6-thioguanine-resistant mutants induced by UV light (10). This feature was confirmed in another experimental system using a ribozyme that cleaves human REV1 mRNA (28) and with an RNA interference down-regulating mouse Rev1 function (29). Furthermore, mouse embryonic stem cells carrying a mutation lacking the BRCA1 C-terminal domain of the Rev1 gene also exhibit sensitivity to a wide range of DNA-damaging agents and a reduced level of UV light-induced mutations (30, 31). Recently, chicken {Delta}Rev1-DT40 cell lines were generated and found to exhibit slow growth and sensitivity to a wide range of DNA-damaging agents (32–34).

Although studies of the cellular functions of the REV1 gene in yeast to humans have shed light on its importance for maintaining genetic integrity, the biochemical basis is poorly understood. It is known that both mouse and human REV1 proteins interact with REV7 and other Y family polymerases through the same C-terminal region (35–38). In particular, the REV1-REV7 interaction is very stable and results in formation of a heterodimer in yeast and humans (39, 40). The C-terminal region of yeast Rev1 is also required for stimulation of yeast pol {zeta} (41), although the amino acid sequence is not conserved. From these observations, a noncatalytic role in translesion DNA synthesis has been proposed (35–41).

In this report, we document a novel biochemical property of human REV1. We show that REV1 has single-stranded DNA (ssDNA) binding activity with affinity much higher than that of the primer-template. After binding to ssDNA, REV1 can translocate and be targeted to primer termini. We consider that this property might reflect a particular biochemical function required for regulation of the repair pathway involved in lesion bypass replication.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides—The oligonucleotide sequences were as follows: H1, 5'-GACGCTGCCGAATTCTGGCTTGCTAGGACATCTTTGCCCACGTTGACCCG-3'; H2, 5'-CGGGTCAACGTGGGCAAAGATGTCCTAGCAATGTAATCGTCTATGACGTC-3'; H3, 5'-GACGTCATAGACGATTACATTGCTAGGACATGCTGTCTAGAGACTATCGC-3'; and H4, 5'-GCGATAGTCTCTAGACAGCATGTCCTAGCAAGCCAGAATTCGGCAGCGTC-3'. The primer-template, P5786T, was made by annealing the 5'-32P-labeled primer P5786 (5'-GTCTACAAGTTCAC-3') with the template 5786T (5'-ATTCTGAGCAGCCCGGATGGTGAACTTGTAGAC-3'). Others are shown in Fig. 3A.

Plasmids—Human POLB cDNA was amplified from HeLa cDNA by PCR and inserted into the NdeI-BamHI site of a pET20b(+) vector (Novagen) to yield plasmid pET-POLB. Human POLH cDNA was amplified by PCR from a plasmid carrying human POLH cDNA, kindly provided by Dr. F. Hanaoka, Osaka University, Osaka, Japan (42), and inserted into the NdeI-BamHI site of a pET15b vector (Novagen) to yield pET-h6-POLH. Human POLA cDNA was amplified by PCR from a plasmid carrying human POLA cDNA, kindly provided by Dr. M. Suzuki, Nagoya University, Nagoya, Japan (43), and inserted into the NdeI-XhoI site of a pET15b vector (Novagen) to yield pET-h6-POLA. Human POLA2 cDNA was amplified from HeLa cDNA by PCR and inserted into the NdeI-KpnI site of a pCDFK vector, which was made by deletion of an EcoNI-AflII fragment and replacement of the streptomycin resistance gene of pCDFDuetTM-1 (Novagen) with the kanamycin resistance gene from pSY343 (44), to yield pCDFK-POLA2. The nucleotide sequences were verified in all these plasmids.

Proteins—Intact REV1 was purified as described (17), along with mutants (6). Human pol beta was overproduced in BL21 (DE3) (45) harboring pET-POLB and similarly purified (46).

His-tagged human pol {eta} (h6-pol {eta}) was purified from overexpressing E. coli cells as follows. BL21 (DE3) (45) harboring pET-h6-POLH was grown in 500 ml of LB medium supplemented with ampicillin (250 mg/ml) at 15 °C with aeration until the culture reached an A600 value of 0.6. Isopropyl beta-D-thiogalactopyranoside was added to 0.2 mM, and incubation was continued for 10 h. The resultant cell paste was resuspended in 2 ml of buffer I (50 mM HEPES-NaOH, pH 7.5, 0.1 mM EDTA, 10 mM beta-mercaptoethanol) containing 1 M NaCl per 1 g of cells and frozen in liquid nitrogen. The cells were thawed in ice water and lysed after addition of phenylmethylsulfonyl fluoride to 0.1 mM by introduction of 100 mM spermidine and 4 mg/ml lysozyme in buffer I containing 1 M NaCl, 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 further incubated on ice for 30 min at 4 °C. Then the lysate was clarified by centrifugation at 85,000 x g for 30 min at 4 °C. Subsequent column chromatography was carried out at 4 °C using a fast protein liquid chromatography system (GE Healthcare). After adding imidazole to 50 mM, the lysate was applied at 0.1 ml/min to a 1-ml HiTrap chelating column (GE Healthcare), which had been treated with 0.1 M NiSO4 and then equilibrated with buffer A (50 mM HEPES-NaOH, pH 7.5, 10 mM beta-mercaptoethanol, 10% glycerol) containing 1 M NaCl and 50 mM imidazole. The column was washed with 10 ml of equilibration buffer, and then 10 ml of buffer A containing 1 M NaCl and 100 mM imidazole, and the h6-pol {eta} was eluted with 10 ml of a linear gradient of 100–300 mM imidazole in buffer A containing 1 M NaCl. Fractions containing the enzyme were pooled and concentrated and then loaded at 0.1 ml/min onto a Superdex 200 HR 10/30 column (GE Healthcare) equilibrated with buffer A containing 1 M NaCl. The h6-pol {eta} peak fractions were pooled, frozen in liquid nitrogen, and stored at –80 °C.

His-tagged human pol {alpha} p180 (h6-p180) was purified as a complex with p70 from overexpressing E. coli cells. BL21 (DE3) (45) harboring both pET-h6-POLA and pCDFK-POLA2 was grown in 10 liters of "terrific" broth (47) supplemented with ampicillin (250 mg/ml) at 15 °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 5 h. The resultant cell paste was resuspended in 2 ml of buffer I containing 0.5 M NaCl per 1 g of cells and frozen in liquid nitrogen. The cells were thawed in ice water and lysed after addition of phenylmethylsulfonyl fluoride to 0.1 mM by introduction of 100 mM spermidine and 4 mg/ml lysozyme in buffer I containing 0.5 M NaCl, 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 further incubated on ice for 30 min at 4 °C. Then the lysate was clarified by centrifugation at 85,000 x g for 30 min at 4 °C. Subsequent column chromatography was carried out at 4 °C using a fast protein liquid chromatography system. After adding imidazole to 50 mM, the lysate was applied at 0.5 ml/min to a 1-ml HiTrap chelating column, which had been treated with 0.1 M NiSO4 and then equilibrated with buffer A containing 0.5 M NaCl and 50 mM imidazole. The column was washed with 10 ml of equilibration buffer at 0.1 ml/min and then eluted with 10 ml of a linear gradient of 50–100 mM imidazole in buffer A containing 0.5 M NaCl. Fractions containing h6-p180-p70 were pooled, diluted with buffer A to 100 mM of NaCl, and applied at 0.1 ml/min to a 1-ml HiTrap Q HP column (GE Healthcare) equilibrated with buffer A containing 100 mM NaCl. The column was washed with 10 ml of equilibration buffer, and the h6-p180-p70 complex was eluted with 10 ml of a linear gradient of 100–500 mM NaCl in buffer A. Fractions containing h6-p180-p70 were pooled and loaded at 0.1 ml/min onto a Superose 6 HR 10/30 column (GE Healthcare) equilibrated with buffer A containing 0.5 M NaCl. The h6-p180-p70 peak fractions were pooled, frozen in liquid nitrogen, and stored at –80 °C. Protein concentrations were determined by protein assay using BSA (Bio-Rad) as the standard.


Figure 1
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  		FIGURE 1.
Inhibition of dCMP transferase activity of human REV1 protein by ssDNA. Inhibition of dCMP transferase activity of the REV1 protein (A–E) or its deletion derivatives (G) by various oligonucleotides is shown. Ten ng of REV1 or deletion derivatives (except for 5 ng of {Delta}5) and the primer-template, P5786T (100 nM), were incubated under standard reaction conditions in the presence of the indicated concentration of oligonucleotides. The double-stranded DNA represented as DS in B was made by annealing H3 and the complementary oligonucleotide. The reaction products were resolved on 20% polyacrylamide gels containing 8 M urea and autoradiographed at –80 °C (A, 0 and +1 represent positions of substrate and product, respectively), and the amounts of DNA present in each band were quantified (B–E and G). F, schematic representation of deletion mutants. The molar concentration of REV1 in the reactions was 2.8 nM and those of mutant REV1 were 3.1 nM {Delta}1, 3.8 nM {Delta}2, 3.9 nM {Delta}3, 4.2 nM {Delta}4, and 3.6 nM {Delta}5.

 
Primer Extension Assay—The primers were labeled using polynucleotide kinase (New England Biolabs) and [{gamma}-32P]ATP (GE Healthcare) and annealed to the respective templates. The standard reaction mixture (25 µl) contained 50 mM Tris-HCl buffer, pH 8.0, 2 mM MgCl2, 0.1 mg/ml BSA, 5 mM dithiothreitol, 0.1 mM dCTP, 100 nM primer-template, and 1 µl of protein sample diluted with buffer (50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 10 mM beta-mercaptoethanol, 10% glycerol, 0.1 mg/ml BSA) as indicated. After incubation at 30 °C for 10 min, 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.). The conditions for the primer extension assay for DNA polymerase shown in Fig. 4B were identical to those for the dCMP transferase assay.

DNA Polymerase Assay—DNA polymerase activities shown in Fig. 4A were measured by incorporation of [{alpha}-32P]dCMP using the unlabeled primer-template, P5786T, as a substrate. The reaction mixture (25 µl) contained 50 mM Tris-HCl buffer, pH 8.0, 2 mM MgCl2, 0.1 mg/ml BSA, 5 mM dithiothreitol, 0.1 mM each of dGTP, dATP, dTTP, and [{alpha}-32P]dCTP (GE Healthcare), 100 nM primer-template (P5786T), and 1 µl of protein sample diluted with buffer (50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 10 mM beta-mercaptoethanol, 10% glycerol, 0.1 mg/ml BSA) as indicated. Ten ng of REV1, 3 ng of pol {alpha}, 2 ng of pol beta, and 10 ng of pol {eta} were used in 25-µl reaction mixtures. After incubation at 30 °C for 10 min, reactions were terminated with 10 µl of 30 mM EDTA, and then 1-µl samples were spotted on DE81 paper (Whatman), which was washed three times with 0.5 M Na2HPO4. The amount of incorporated [{alpha}-32P]dCMP was determined as the radioactivity retained on the paper (48) and quantified using a Bio-Imaging Analyzer BAS2000 (Fuji Photo Film Co., Ltd.)

Electrophoretic Mobility Shift Assay—Poly[d(C-T)] oligonucleotides with various lengths were labeled using polynucleotide kinase (New England Biolabs) and [{gamma}-32P]ATP (GE Healthcare). Assays of DNA binding were performed with modification of a method described previously (6). Reaction mixtures (10 µl) contained 50 mM Tris-HCl buffer, pH 8.0, 2 mM MgCl2, 0.2 mg/ml BSA, 5 mM dithiothreitol, 0.1 mM dCTP, 50 pM oligonucleotide, and 1 µl of protein sample diluted with buffer (50 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 10 mM beta-mercaptoethanol, 10% glycerol, 0.1 mg/ml BSA) as indicated. Incubation was on ice for 20 min followed by loading on prerunning 4% polyacrylamide gels (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 for 2 h at 6°C. Following gel electrophoresis, the gels were dried and autoradiographed at –80 °C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
High Affinity Binding of REV1 to ssDNA—During biochemical characterization of the dCMP transferase reactions of human REV1 protein, we found a synthetic oligonucleotide, H1, to be a strong inhibitor of the transferase activity of the REV1 (Fig. 1A). In reactions containing 100 nM primer-template and 2.8 nM REV1, the transferase activity dropped to less than 10% in the presence of 10 nM of the H1 (Fig. 1, A and B). To ascertain whether the inhibitory effect was because of a specific nucleotide sequence, oligonucleotides of different sequences (H2–H4) were tested (Fig. 1B). The results suggested that the inhibitory effect was not because of a specific nucleotide sequence, although each oligonucleotide exhibited a different extent of inhibition. When the oligonucleotide was annealed with the complementary oligonucleotide and converted to double-stranded DNA, such an effect was much decreased (Fig. 1B). The remaining effect might be due to trace contamination of ssDNA (data not shown). To exclude the possibility that the effect is due to local secondary structures formed by the oligonucleotides, we tested 30- and 60-mer oligonucleotides composed of one or two nucleotides, which are guaranteed not to form secondary structures (Fig. 1, C and D). The results revealed general features for the inhibition. First, it was not because of secondary structures of the oligonucleotides. Second, the composition of the nucleotides affected the extent of inhibition, whereas polypurines, poly(dA) and poly[d(A-G)], showed no effect. Third, the extent of inhibition was stronger with 60- than 30-mer oligonucleotides with the same composition of nucleotides. Furthermore, we systematically addressed the effect of the length using poly[d(C-T)] as a model oligonucleotide (Fig. 1E) and found the extent of inhibition to correlate synergistically with the length. In a control experiment, we demonstrated that REV1 could not transfer dCMP to the 3' end of the d(C-T)30, 60-mer oligonucleotide, under those reaction conditions (supplemental Fig. 1), indicating that the inhibition is not because of random priming reactions with the oligonucleotide. In following experiments, we used poly[d(C-T)] oligonucleotides as model ssDNA substrates.


Figure 2
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  		FIGURE 2.
Analysis by electrophoretic mobility shift assay of ssDNA binding to the REV1 protein. A and B, electrophoretic mobility shift assays of ssDNA binding to REV1 (A) and its deletion derivatives (B). Poly[d(C-T)] consisting of the indicated repeats (A) or d(C-T)30 60-mer (B) were incubated with REV1 or deletion mutants at the indicated concentrations. Filled and open arrowheads indicate the positions of the DNA-REV1 complex and the free DNA, respectively. C, competition assay of REV1-ssDNA binding activity. In binding reactions with 0.9 nM of REV1 or deletion derivatives, the indicated concentrations of unlabeled primer-template, P5786T, were incubated as a competitor. D, quantified results of C.

 
The inhibitory effect might be due to high affinity binding of the REV1 to the ssDNA, and consequently, it should compete with the primer-template. To examine REV1 ssDNA binding activity, we performed gel mobility shift assays using poly[d(C-T)] oligonucleotides with various lengths as substrates and detected REV1-ssDNA complexes (Fig. 2A). When we tested the d(C-T)15, 30-mer oligonucleotide, as a substrate, no complexes could be detected. However, when the length was increased, complexes became visible and were stable. The apparent affinity of REV1 for the oligonucleotides proved to be relative to their lengths. On the 60-mer oligonucleotide, the apparent affinity was very high with less than 1 nM of estimated Kd. Most importantly, the degree of apparent affinity of REV1 for the oligonucleotides of various lengths showed good agreement with the degrees of their inhibitory effects on transferase activity (Fig. 1E).

Analysis of Truncated REV1 Proteins—Next, we examined the properties of truncated REV1 proteins ({Delta}1–{Delta}5) (Fig. 1F) with intact transferase activity (Fig. 3E, panel a). First, the inhibitory effects of 60-mer poly[d(C-T)] were examined (Fig. 1G). The effect of truncation of the C-terminal {Delta}3 and {Delta}4 was the same as observed with the full-length protein. On the other hand, truncation of the N-terminal {Delta}1 and {Delta}2 resulted in partial resistance to ssDNA. However, truncation of both N- and C-terminal {Delta}5 showed a much greater effect. The activity of the mutant protein {Delta}5 consisting of only the transferase domain was not inhibited by the oligonucleotide, indicating inhibition by ssDNA to be modulated by domains outside the transferase domain of REV1.

Then we tested ssDNA binding activity of the mutants (Fig. 2B). Surprisingly, binding of the 60-mer poly[d(C-T)] to all the truncated proteins was essentially identical to that to full-length REV1 (Fig. 2B). With the full-length REV1, the transferase activity could be inhibited by ssDNA binding. However, this was not the case with truncated protein {Delta}5. Therefore, we addressed the question of whether the primer-template is accessible to REV1-ssDNA complexes (Fig. 2, C and D). If REV1 could interact with a primer-template after forming a complex with ssDNA, the complex would be sensitive to an addition of a large amount of the primer-template. In the REV1-ssDNA binding reaction, a primer-template was therefore introduced as a competitor. The result clearly demonstrated that the {Delta}5-ssDNA complex was sensitive to addition of primer-template. The amount of the complex was decreased to 30% by addition of primer-template at 25 nM, indicating that the primer-template could access the catalytic site of {Delta}5 even after complex formation with ssDNA. In contrast, we could not detect any difference between full-length REV1 and {Delta}2, even though {Delta}2 exhibited decreased sensitivity to ssDNA (Fig. 1G) as compared with full-length REV1. We consider that the difference could be due to sensitivity of the assay systems to detect competition between primer-template and ssDNA, but the results from both experiments were essentially consistent. From the results, we conclude that ssDNA binding itself does not fill up the catalytic site of the transferase but rather prevents accession of primer-template, and this function is because of the presence of N- and C-terminal domains.


Figure 3
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  		FIGURE 3.
Template specificity of dCMP transferase reactions of REV1. A, nucleotide sequences of the primer-templates. B, inhibition of dCMP transferase activity of the REV1 protein by a primer-template containing long ssDNA. Ten ng of REV1 (2.8 nM) and the primer-template, P5786T (100 nM) as a substrate, were incubated under standard reaction conditions in the presence of the indicated concentration of 13/30- or 13/91-mer (A). Incorporation into the P5786T was measured and plotted in the graph. C, time course of dCMP transferase reactions using 13/30- and 13/91-mer (A) as substrates. REV1 (0.7 nM) and each primer-template (20 nM) were incubated for the indicated times. Incorporation into each 13-mer primer was measured. The errors in this assay were less than 5%. In the assays of B and C, the reaction products were resolved on 20% polyacrylamide gels containing 8 M urea, and amounts of DNA present in each band were quantified. D and E, competition assay of dCMP transferase activity using various primer-templates. The primer-templates shown in A (20 nM each) were used as substrates for the transferase assays (shown at the bottom of each panel) and were incubated with the indicated amount of REV1 (D) or deletion derivatives (E). The positions of 13- and 17-mer primer on the gels are shown by open and closed arrowheads, respectively. In the reactions with mixtures of two primer-templates, the respective primers were distinguished by differing size (panels d and e of D, and panels a and b of E). The reaction products migrating between 13- and 17-mer were derived from the 13-mer primer and those migrating larger than 17-mer were derived from the 17-mer primer. The molar concentrations of REV1 in the reactions were 0.7 nM (2.5 ng), 1.4 nM (5 ng), and 2.8 nM (10 ng), and those of mutant REV1 were 3.1 nM {Delta}1 (10 ng), 3.8 nM {Delta}2 (10 ng), 3.9 nM {Delta}3 (10 ng), 4.2 nM {Delta}4 (10 ng), and 1.8 nM {Delta}5 (2.5 ng).

 
Specific Utilization of a Primer-Template Containing Long ssDNA—For further investigation of this novel property of the REV1 protein, we addressed whether the catalytic site of the transferase reaction might be accessible to a primer terminus annealed with a template containing a long ssDNA region (cis effect of ssDNA). Because the apparent binding affinity of REV1 to ssDNA is much higher than to the primer terminus (6), if the template contained a long ssDNA region, it could be a target for REV1 binding and inhibit the transferase reaction. To test this possibility, we made two primer-templates: one a 30-mer template annealed with a 13-mer primer, and the other a 91-mer template in which a dCT repeat was attached to the 5' end of the 30-mer, and the same 13-mer was annealed (Fig. 3A). First, we examined whether the primer-template could inhibit REV1 in an ssDNA-dependent manner when it was introduced in the reaction in trans (Fig. 3B). The transferase activity of REV1 was monitored using a different primer-template, P5786T. The result demonstrated that the oligonucleotide, 13/91-mer, inhibited transferase activity when introduced in trans (Fig. 3B), the effect being much stronger than that of poly[d(C-T)] 60-mer (Fig. 1E). This agreed with the synergistic properties (Fig. 1E), considering that the single-stranded region of 13/91-mer is 78 bases. This inhibition was not because of the structure of primer-template, because it was not detected with 13/30-mer (Fig. 3B). Therefore, we concluded that the long ssDNA region in template acted as a target of REV1 binding and inhibited the activity.

We then labeled the primer termini of 13/30- and 13/91-mer with 32P and examined the extensions (the cis effect of ssDNA) (Fig. 3C). Interestingly, both primer-templates were utilized as good substrates. The time courses of the reactions revealed that the incorporation of dCMP increased to a greater extent than the amount equivalent to REV1 protein (18 fmol) in the reaction solution, indicating REV1 was turned over many times. Thus, the catalytic site of the REV1 protein was accessible in cis, meaning that even after binding to ssDNA, REV1 interacted with a primer terminus that is located on the long ssDNA template.

To further confirm this property of REV1, we designed an experiment in which two primer-templates, one contained a long ssDNA in the template and the other contained a shorter ssDNA in the template, were competed with each other in the same reaction for utilization as substrates for REV1. To distinguish the reaction products on a polyacrylamide gel, we made another oligonucleotide, 17/33-mer composed of a different sequence with a short template strand (Fig. 3A). Because the 17-mer primer is 4 bases longer than the 13-mer primer, the reaction products derived from 17-mer could not be overlapped by the products derived from the 13-mer when those were reacted together in a mixture. Those primer-templates were utilized to almost the same extent in the transferase reactions (Fig. 3D, panels a–c). When the 13/30- and 17/33-mer were reacted with REV1 in one tube, both were utilized to the same extent (Fig. 3D, panel d). However, when the 13/91- and 17/33-mer were reacted with REV1 in one tube, we observed no extension of the 17-mer (Fig. 3D, panel e), indicating that REV1 specifically utilized the primer-template followed by long ssDNA.

When the same experiment was carried out using truncated mutants (Fig. 3E), the specific activities of mutant proteins slightly differed possibly due to variation in stability under standard reaction conditions. In this assay, we compared the proteins with equivalent levels of activity, rather than amount of proteins themselves (Fig. 3E). When 13/30- and 17/33-mer were reacted with mutant REV1 proteins in one tube, both primers were extended (Fig. 3E, panel a). With the 13/91- and 17/33-mer and mutants, the specificity to 13/91-mer was abolished only with the mutant {Delta}5 (Fig. 3E, panel b). This result agreed with inhibition curves of the mutants with ssDNA (Fig. 1G) and the electrophoretic mobility shift assay (Fig. 2, C and D). Thus, REV1 is specifically targeted to the primer terminus, followed by the long ssDNA template, and the N- and C-terminal regions are required for the activity.

Effects of ssDNA on DNA Polymerase Activity of pol{alpha}, beta, and{eta}—Although REV1 is one member of the Y family of DNA polymerases (14), the N- and C-terminal regions are not conserved. To determine whether the novel activity is specific to REV1, we first tested the inhibitory effects of ssDNA on another Y family polymerase, human pol {eta} (42), and distinct family members, human pol {alpha} (B family) and beta (X family) (49, 50). Neither of the polymerases was inhibited by d(C-T)30, 60-mer, in contrast to REV1 (Fig. 4A). We also tested the specificity for primer-template following long ssDNA using combinations of 13/30- and 17/33-mer and 13/91- and 17/33-mer oligonucleotides (Fig. 4B). In this assay, the primer extension reactions were carried out with only dCTP as the dNTP source to prevent overlapping reaction products. Although pol {alpha} and {eta} showed a slight preference for 13/91-mer (Fig. 4B), the level was similar to that of {Delta}5 (Fig. 3E, panel b), suggesting that the novel property is REV1-specific.


Figure 4
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  		FIGURE 4.
The targeting mechanism is REV1-specific. A, effects of ssDNA on DNA polymerase activity of pol {alpha}, beta, and {eta}. Relative activities were measured by incorporation of [{alpha}-32P]dCMP in a reaction mixture containing dNTP into unlabeled primer-template, P5786T, in the presence of various concentrations of d(C-T)30 60-mer. B, competition assay on the primer extension activity of DNA polymerases using various primer-templates. The primer-templates (20 nM each) shown in Fig. 3A were used as substrates for the primer extension assay. The indicated amounts of DNA polymerases and a mixture of a set of primer-template shown at the bottom of each panel were incubated with only dCTP, because further primer extensions of 13-mer by incorporation of dNTP would result in overlapping of products from 17-mer and make the results confusing. The reaction products were resolved on 20% polyacrylamide gels containing 8 M urea, and the amounts of DNA present in each band were quantified. The molar concentrations of polymerases in the reactions were 0.2 nM pol {alpha} (1 ng), 0.5 nM (0.5 ng of pol beta) and 5 nM pol {eta} (10 ng).

 


Figure 5
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  		FIGURE 5.
Model for actions of REV1. A, sequestration of REV1 from primer-template by ssDNA. B, targeting REV1 to a primer terminus via ssDNA binding. The catalytic site of REV1 is shown as an open circle inside shaded oval. See text for details.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we provide evidence of a novel biochemical activity of human REV1 protein. First, we found that ssDNA inhibits the transferase activity of REV1, because of sequestration of the catalytic site by high affinity binding (Fig. 5A). The N- and C-terminal domains are required for the sequestration, because a mutant REV1 lacking both N- and C-terminal domains lost only the function for sequestration, and not ssDNA binding and transferase activity (Fig. 5A). Second, we demonstrated that sequestration by ssDNA is only effective in trans. When REV1 binds to the template strand annealing a primer, the primer terminus is accessible for the catalytic site (Fig. 5B). Therefore, the catalytic site is open in cis. Third, we showed that REV1 preferentially utilizes the primer-template following long ssDNA.

We speculate that REV1 translocates on ssDNA from the following considerations, according to models involved in protein translocation that have been well discussed with regard to repressor-DNA recognition (51). We found that the extent of inhibition by ssDNA correlated with the length (Fig. 1E). To explain the synergism observed, two models were considered. One is a "dissociation-reassociation model," in which a protein dissociated from a DNA molecule could reassociate within a closely spaced site of the same DNA molecule. Therefore, the protein would appear to be trapped in the DNA molecule. The frequency of both initial binding and re-association would be proportional to the length of DNA. Because the binding event is affected by two factors, the inhibition curve would become "synergistic." The other is a "sliding model," in which the dissociation rate would be inversely proportional to the length of the DNA, because the protein could translocate at any point within the molecule. Besides, the length of the DNA molecule is proportional to the frequency of initial binding of the protein. Therefore, the inhibition curve would again become synergistic. After the occasional dissociation or sliding out of the DNA molecule, however, the protein could reassociate to another molecule of DNA, and therefore would not appear to be trapped. Our biochemical data here support the latter model. The primer-template following long ssDNA did not inhibit transferase activity in cis, and we observed turnover of REV1 protein several times with respect to dCMP transferase enzyme activity. These results indicate that after binding to any site of the ssDNA region, REV1 can access the primer terminus and subsequently dissociate and re-bind to another molecule. Currently, we do not have direct evidence for sliding of REV1, but we cannot explain our biochemical data without consideration of such translocation.

Translesion DNA synthesis plays an important role in post-replication repair pathways. It has been postulated that after posing a replicative DNA polymerase at a damage base, it is replaced with specialized DNA polymerases for translesion DNA synthesis. However, the biochemical reactions have yet to be clarified. Genetic data from yeast to humans suggest that REV1 has a central function in organizing such a polymerase switch (3). Besides, in higher eukaryotes, it has been shown that mouse and human REV1 have potential for interaction with other translesion DNA polymerases (35–41). The novel property of REV1 may play a role in organization of translesion DNA synthesis (52).


   FOOTNOTES
 
* This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Y. M. and K. K.) and by the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima 734-8553, Japan. Tel.: 81-82-257-5842; Fax: 81-82-257-5844; E-mail: kkamiya{at}hiroshima-u.ac.jp.

2 The abbreviations used are: pol, DNA polymerase; ssDNA, single-stranded DNA; BSA, bovine serum albumin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Fumio Hanaoka (Osaka University, Osaka, Japan) and Dr. Motoshi Suzuki (Nagoya University, Nagoya, Japan) for providing the POLH cDNA and the POLA cDNA, respectively. We are grateful to Eriko Aoki for help with cDNA cloning, and Kumiko Mizuno, Masako Okii, Miki Yano, and Hatsue Wakayama for their laboratory assistance.



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