Mutagenic and Nonmutagenic Bypass of DNA Lesions by Drosophila DNA Polymerases dpoleta and dpoliota *

Tomoko IshikawaDagger , Norio UematsuDagger , Toshimi Mizukoshi§, Shigenori Iwai§, Hiroshi Iwasaki, Chikahide Masutani||, Fumio Hanaoka||, Ryu Ueda**, Haruo OhmoriDagger Dagger , and Takeshi TodoDagger §§

From the Dagger  Radiation Biology Center, Kyoto University, Kyoto 606-8501, § Biomolecular Engineering Research Institute, Suita, Osaka 565-0874,  Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, || Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565-0871, ** Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, and Dagger Dagger  Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan

Received for publication, October 27, 2000, and in revised form, January 10, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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cDNA sequences were identified and isolated that encode Drosophila homologues of human Rad30A and Rad30B called drad30A and drad30B. Here we show that the C-terminal-truncated forms of the drad30A and drad30B gene products, designated dpoleta Delta C and dpoliota Delta C, respectively, exhibit DNA polymerase activity. dpoleta Delta C and dpoliota Delta C efficiently bypass a cis-syn-cyclobutane thymine-thymine (TT) dimer in a mostly error-free manner. dpoleta Delta C shows limited ability to bypass a 6-4-photoproduct ((6-4)PP) at thymine-thymine (TT-(6-4)PP) or at thymine-cytosine (TC-(6-4)PP) in an error-prone manner. dpoliota Delta C scarcely bypasses these lesions. Thus, the fidelity of translesion synthesis depends on the identity of the lesion and on the polymerase. The human XPV gene product, hpoleta , bypasses cis-syn-cyclobutane thymine-thymine dimer efficiently in a mostly error-free manner but does not bypass TT-(6-4)PP, whereas Escherichia coli DNA polymerase V (UmuD'2C complex) bypasses both lesions, especially TT-(6-4)PP, in an error-prone manner (Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018). Both dpoleta Delta C and DNA polymerase V preferentially incorporate GA opposite TT-(6-4)PP. The chemical structure of the lesions and the similarity in the nucleotides incorporated suggest that structural information in the altered bases contribute to nucleotide selection during incorporation opposite these lesions by these polymerases.

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ABSTRACT
INTRODUCTION
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DNA is frequently damaged by environmental and endogenous genotoxic agents. Although various mechanisms exist to ensure that the majority of DNA damage is recognized and repaired and the integrity of the DNA is faithfully restored, some DNA lesions escape repair and persist in the cell. Unrepaired DNA lesions can block the progress of the replication machinery. To help resolve this problem, cells have specialized polymerases that carry out translesion DNA synthesis (TLS)1, which is a mechanism that permits nucleotides to be incorporated opposite lesions. After TLS bypasses the DNA damage, replication can continue with the normal replication machinery downstream of the site of the damage.

Recently, a new family of DNA polymerases was identified called the UmuC/DinB/Rev1/Rad30 superfamily (1-3). Several of these polymerases participate in TLS (4-11), and members of this protein family exhibit lower fidelity and processivity than replicative DNA polymerases (10, 12-15). The lower fidelity enables these polymerases to carry out TLS and the lower processivity facilitates dissociation of these enzymes after bypass of a lesion; this is important because it allows the normal replication machinery, with high fidelity and processivity, to preferentially carry out DNA synthesis distal to the lesion.

However, TLS is mutagenic when the base inserted opposite the DNA lesion is different than the base normally inserted opposite an undamaged base at that site. The chemical structure of the lesion is an important determinant for mutagenic or nonmutagenic bypass during TLS. Irradiation of DNA with UV light produces a variety of photoproducts that can cause mutations (16). cis-syn-cyclobutane pyrimidine dimer (CPD) and (6-4)-photoproducts ((6-4)PP) are the two major classes of UV-induced DNA photoproducts. In DNA irradiated with UV, CPD is the most abundant lesion, and it is ~3-5-fold more abundant than (6-4)PP. However, the mutagenic properties of these two lesions are different, and the relative contributions of the two lesions to the mutagenesis are not simply proportional to their relative abundance. The mutagenic specificities of CPD and (6-4)PP have been studied using well characterized site-specific UV photoproducts in DNA substrates. The mutation frequency obtained either with a site-specific TT-CPD or a TC-CPD is quite low (17-18), and in comparison, (6-4)PP is more mutagenic. The (6-4)PP at the 5'-TT site was highly mutagenic, and most of the mutations induced by this lesion are T to C transitions at the 3'-T (19). However, this type of mutation is not very common, because the (6-4)PP at the TT site is relatively rare. On the other hand, the TC-(6-4)PP is less mutagenic than the TT-(6-4)PP but more mutagenic than the TT-CPD. Of the mutations at this site, 80% were C to T transitions at the 3' base (20). Since TC to TT is frequently observed in DNA exposed to UV and (6-4)PP forms most frequently in the TC sequence, the TC-(6-4)PP could be a candidate for a strongly premutagenic lesion.

Human cells have three TLS polymerases that belong to the UmuC/DinB/Rev1/Rad30 superfamily: poleta (encoded by the XPV/RAD30A gene (21, 22)), poliota (encoded by RAD30B (10, 23)), and polkappa (encoded by DINB1 (24)). In addition, the human protein hRev1 is a homologue of yeast REV1 (25). Poleta , poliota , and polkappa bypass UV-induced damage with different efficiencies. Poleta bypasses TT-CPD efficiently and inserts AA opposite the lesion; however, it adds only one base opposite the 3'-T of TT-(6-4)PP (5). Poliota bypasses TT-CPD and TT-(6-4)PP with low efficiency, adding one or a few bases opposite these lesions (26, 27). Polkappa stops DNA synthesis and is blocked one base before TT-CPD and TT-(6-4)PP (9). Thus, in human cells, none of these polymerases bypass (6-4)PP. In contrast, Escherichia coli DNA polymerase V (UmuD'2C (28)) efficiently bypasses both lesions, inserting AA or GA opposite TT-CPD or TT-(6-4)PP, respectively (8). Thus, the chemical structure and the properties of each TLS polymerase determine which base is inserted and the likelihood of a mutation at a specific lesion.

The goal of this work was to identify and characterize the Drosophila homologue(s) of the UmuC/DinB/Rev1/Rad30 superfamily. Two cDNAs were isolated that encode Drosophila Rad30A and Rad30B, called drad30A and drad30B. Truncated forms of dRAD30A and dRAD30B proteins, designated dpoleta Delta C and dpoliota Delta C, respectively, have been purified, and their properties were studied, including the mechanism of UV-induced mutagenesis and the products of lesion bypass in reactions with these enzymes. The bypass templates used in this study include TC-(6-4)PP, TT-CPD, or TT-(6-4)PP; a template with TC-(6-4)PP was studied because of the biological relevance of this lesion. The results indicate that both polymerases bypass the TT-CPD in an error-free manner. Furthermore, dpoleta Delta C bypasses TT-(6-4)PP and TC-(6-4)PP in a highly error-prone manner.

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Isolation of Drosophila rad30A and rad30B cDNAs-- To identify the genes encoding the UmuC/DinB/Rev1/Rad30 superfamily proteins in Drosophila, we searched the data base of expressed sequence tag (GenBankTM dbEST) for Drosophila cDNAs that share homology with the proteins of this family. Several Drosophila cDNA sequences that share significant homology with the proteins of the superfamily were identified. Three such clones, SD05329, LD09220, and GH11153, were obtained from Research Genetics (Huntsville, AL), and the entire insert of each clone was sequenced with an automated DNA sequence analyzer (Applied Biosystems PRISM310). Translation of the large continuous open reading frame from each cDNA revealed that each translated protein shares significant similarity to Rad30A, Rad30B, and Rev1 proteins, respectively. Thus, the clones were designated as drad30A, drad30B, and drev1, respectively. The cDNAs did not contain in-frame stop codons. Thus, the 5' ends of each clone were amplified by 5'-rapid amplification of cDNA ends. The sequence determination of the amplified 5' ends revealed the presence of the in-frame stop codon. Screening of the dbEST indicated that Drosophila seems to lack a true DinB ortholog, which was confirmed by searching the recently completed sequence of the Drosophila genome. The drad30A and drad30B cDNAs each hybridized to Drosophila polytene chromosomes at a single site corresponding to bands 3L-79BC and 3R-84EF, respectively.

Overproduction of the dRAD30A and dRAD30B Proteins-- The expression vector used in these studies was pGEX-4T (Amersham Pharmacia Biotech). To overexpress the Drosophila dRAD30A and dRAD30B proteins, each gene was cloned in-frame with the glutathione S-transferase gene. A chimeric GST-drad30A gene was constructed by recloning the 3-kilobase pair EcoRI/XhoI DNA fragment from the EST clone SD05329 in EcoRI/XhoI-digested pGEX-4T-1, thus generating plasmid pGEX-dRad30A. The C-terminal deletion mutant of the drad30A gene was constructed by recloning the 1640-base pair EcoRI/Eco52I DNA fragment, which contains the N-terminal 545 residues, in EcoRI/NotI-digested pGEX-4T-1, thus generating pGEX-dRad30ADelta C. The chimeric GST-drad30B gene was constructed as follows. The EcoRI restriction site was generated just upstream of the start codon by polymerase chain reaction using the primer 30B5'Eco, 5'- CGGAATTCATGGACTTCGCTAGCGTAC-3', and the 3-kilobase pair EcoRI/XhoI DNA fragment from the EST clone LD09220 was then cloned in-frame with GST in the EcoRI/XhoI-digested pGEX4T-1, thus generating plasmid pGEX-dRad30B. The C-terminal deletion mutant of the GST-rad30B gene was constructed by first synthesizing the oligomer 30Bdel3'Xho, 5'- CCGGCTCGAGTGGGACTTGTGAGACTC-3'. Two polymerase chain reaction primers, 30B5'Eco and 30Bdel3'Xho, were used to amplify a 1.36-kilobase pair DNA fragment that contained the N-terminal 448 residues of the drad30B gene. The polymerase chain reaction products were digested with EcoRI and XhoI, and the resultant 1.35-kilobase pair fragment was cloned in the EcoRI/XhoI-digested pGEX-4T-1, thus generating pGEX-dRad30BDelta C.

Mutant forms of the GST-rad30A and GST-rad30B genes were constructed by utilizing the Mutant-Super Express mutagenesis kit (Takara, Japan). The adjacent residues, Asp126-Glu127 in drad30A and D115-E116 in drad30B, were changed to alanine using primers R30A-AA 5'-GCTTCCGTGGCAGCCGCGTACT -3' and R30B-AA 5'-CTAGGCTTCGCTGCAAACTTTA-3', respectively. The 400-base pair SphI fragment from pKF30A and the 800-base pair EcoRI/BglII fragment from pKF30B were sequenced to verify that the region only contained the desired mutation, and then each fragment was subcloned into the similarly digested pGEXrad30ADelta C and pGEXrad30BDelta C to create pGEXrad30A-DE126AA and pGEXrad30B-DE115AA, respectively.

E. coli JM109 harboring pGEX-dRad30ADelta C, pGEX-dRad30BDelta C, pGEX-dRad30A-DE126AA, or pGEX-drad30B-DE115AA was grown in 1 liter of LB medium containing 150 mg/liter ampicillin at 25 °C until an A600 of 0.9-1.0 was reached. Isopropyl beta -D-thiogalactopyranoside (0.1 mM) was added to induce expression of the chimeric gene. After incubation at 25 °C for 12 h, the cells were harvested by centrifugation and were stored at -80 °C.

Purification of the dRAD30A and dRAD30B Protein-- To purify the GST-tagged dRAD30A or dRAD30B, E. coli cells were resuspended in phosphate-buffered saline and disrupted using a sonicator before centrifugation at 10,000 × g. 10 mM Mg-ATP and 5 mg/ml casein were added to the extract, which was then incubated at room temperature for 20 min. The extract was then passed over a 5-ml glutathione-Sepharose 4B column (Amersham Pharmacia Biotech) and washed with 10 column volumes of wash buffer (1 M NaCl and 50 mM Tris-HCl (pH 8.0)). The GST-tagged protein was eluted from the GST column in a stepwise fashion by a wash with five column volumes of elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM glutathione). The GST fusion protein in each eluate was monitored by the GST activity according to the manufacturer's protocol (Amersham Pharmacia Biotech), and the eluates containing GST activity were pooled. The pooled eluates were concentrated by Centriplus 50 ultrafiltration (Amicon) to an approximate final volume of 1.0 ml. The concentrated eluate was applied to a HiTrap Q column (Amersham Pharmacia Biotech) equilibrated with buffer A (50 mM Tris (pH 8.0)). The column was subsequently washed with 10 column volumes of buffer A, and the GST fusion protein was eluted with a 0-1 M linear gradient of NaCl in buffer A. The GST activity in each fraction was measured, and the positive fractions were pooled. The pooled fractions were dialyzed against buffer A and loaded on a HiTrap heparin column (Amersham Pharmacia Biotech) equilibrated with buffer A. The column was washed with buffer A, and the protein was eluted by a linear gradient of 0-0.5 M NaCl in buffer A. Glycerol was added to the GST-containing fraction (10% v/v) before storage at -80 °C.

The eluate from the glutathione-Sepharose 4B column contained a contaminating DNA polymerase activity derived from E. coli because the eluate from the column of the cell extract from E. coli harboring the GST vector had a weak DNA polymerase activity. To remove the contaminating DNA polymerase activity, we tested two kinds of antibodies, one specific for E. coli pol I and another specific for pol II. The addition of the antibody for pol I to the eluate, but not the antibody for pol II, eliminated the DNA polymerase activity of the eluate from E. coli expressing GST, indicative that the contaminating DNA polymerase is E. coli pol I. The pol I antibody also eliminated the DNA polymerase activity in the eluate of the cell extract from E. coli harboring the mutant plasmids, pGEXrad30A-DE126AA and pGEXrad30B-DE115AA. Thus, the pol I antibody was added to the purified protein in all experiments in this paper. However, after the addition of pol I antibody, a slight 3'-exonuclease activity still remained.

DNA Polymerase Assays-- The 30-mer oligomer (non-damaged TT) with the sequence 5'-CTCGTCAGCATCTTCATCATACAGTCAGTG-3' was used as the nondamaged template. The same 30-mer oligomers containing the CPD (29) and the (6-4)PP (30) at the underlined sites were synthesized as described. The 30-mer oligomers, in which the underlined TT sequence of the "nondamaged TT" oligomers was changed to TC, were also used as nondamaged templates (designated nondamaged TC), and the 30-mer oligomers containing (6-4)PP at the TC site were synthesized as described (31). Various lengths (16-18-mer) of oligomers with sequences complementary to the 30-mer template were synthesized and used as primers. Each primer was labeled at the 5' end using T4 polynucleotide kinase and [gamma -32P]ATP and was annealed with the template at a molar ratio of 1:1. Standard reactions (10 µl) contained 40 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 100 µM each of the four dNTPs, 10 mM dithiothreitol, 250 µg/ml bovine serum albumin, 60 mM KCl, 2.5% glycerol, 40 nM primer-template, and the indicated amount of enzyme. After incubation at 37 °C for 15 min, the reactions were terminated by the addition of 10 µl of formamide followed by boiling. The products were subjected to 20% polyacrylamide, 7 M urea gel electrophoresis followed by autoradiography.

Detection of Correctly Replicated Products Opposite Lesions-- Two kinds of oligomers were used: 49-mers bearing the centrally located (6-4)PP or CPD at the TT site (32) and 30-mers bearing the centrally located (6-4)PP at the TC site (31). The substrate sequence is as follows (the introduced pyrimidine dimer is underlined): 49-mer, 5'-AGCTACCATGCCTGCACGAATTAAGCAATTCGTAATCATGGTCATAGCT-3', and 30-mer, CCTGGTTAGTACTTCGATTTAGGTGACACT. The photoproduct in the 49-base pair duplex DNA is at an MseI site (MseI cleaves between the two thymines in the TTAA sequence), and that in the 30-base pair duplex DNA is at a TaqI site (TaqI cleaves between the two pyrimidines in the TCGA sequence). The assay measures the susceptibility of the bypass product to MseI cleavage. A 20-mer oligomer (5'-ACCATGATTACGAATTGCTT-3') and a 15-mer oligomer (5'-AGTGTCACCTAAATC-3') with sequences complementary to the 49- and 30-mer damage-containing strands, respectively, were synthesized and used as primers. After labeling with [gamma -32P]ATP, the primer was annealed with the template and was used as the substrate for DNA synthesis. DNA synthesis was carried out as described in the DNA polymerase assay. After DNA synthesis, the replication products were denatured by incubation at 95 °C for 5 min and then annealed with a 10-fold excess of the 49-mer nondamaged oligonucleotide (400 nM). The annealed duplex DNA was digested with one unit of MseI or TaqI for 90 min at 37 or 65 °C, respectively. The digested DNA was denatured at 95 °C for 5 min in the presence of 50% formamide and was then separated on a 10% polyacrylamide sequencing gel. The MseI or TaqI-susceptible and -resistant fractions were quantified by scanning the gels with a Fuji Bioimage analyzer (BAS-2000, Fuji Film, Japan), and the ratio was calculated as follows. The amounts of the undigested bypass products (Counts-bp) were obtained by measuring the radioactivity of the areas corresponding to the 24-42-base fragments or the 19-30-base fragments in each lane for the template containing no damage, TT-CPD and TT-(6-4)PP, or TC-(6-4)PP, respectively. The radioactivity of the same area in the control sample, which did not contain enzyme, was used as the background for bypass products (Counts-bp-bg). The amounts of the bypass products susceptible to each enzyme were determined by measuring the radioactivity of the 19-base or 14-base fragments (Count-dig). The radioactivity of the same area of the undigested reaction was measured and used as the background of the digested products (Counts-dig-bg). The % restriction enzyme-sensitive bypass products in Table I was calculated as (Count-dig) - (Counts-dig-bg)/(Counts-bp) - (Counts-bp-bg) × 100. 

                              
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Table I
Ratio of bypass products with normal sequences
Values are the average of counts of 19- or 14-base fragments after digestion with MseI or TaqI, respectively and are normalized to those of the undigested bypass products (see "Experimental Procedures"). Data are the average of two independent experiments. Values in parentheses are normalized to data for the No damage control of each enzyme. ND, not determined.


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Isolation of Drosophila drad30A and drad30B cDNAs-- Two Drosophila cDNA clones were identified in the GenBankTM dbEST data base, which had significant homology to human Rad30A and Rad30B protein, and designated drad30A and drad30B. DNA sequence analysis of the full-length cDNAs revealed that drad30A encodes a protein of 885 amino acids, with 31% identity to the human XPV/RAD30A protein (hpoleta ), and drad30B encodes a protein of 737 amino acids, with 30% identity to the human RAD30B protein (hpoliota ). Previous studies show that the proteins in the UmuC/DinB/Rev1/Rad30 superfamily are most highly conserved in their N-terminal portion, which includes five discrete conserved regions (1, 33). As shown in Fig. 1, A and B, these regions (I-V) are conserved in the N-terminal portion of all the RAD30-like proteins including the Drosophila proteins encoded by drad30A and drad30B.


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Fig. 1.   Deduced amino acid sequences of the dRAD30A and dRAD30B proteins. A, schematic representation of the conserved regions in the dRAD30A and dRAD30B proteins. The conserved regions I-V are indicated. Schematic representations of the C-terminal deletion mutants are also shown, and the sites of the conserved DE sequences where the mutations were introduced are indicated by arrows. aa, amino acids. B, alignment of the region III amino acid sequences of the Rad30A proteins from Homo sapiens (XPV), Arabidopsis thaliana (AtRad30), Caenorhabditis elegans (CeRad30), S. cerevisiae (ScRad30), Schizosaccharomyces pombe (SpRad30), and Drosophila melanogaster (DRad30A), and the Rad30B proteins from H. sapiens (hRad30B) and D. melanogaster (DRad30B). The alignment was performed using the MegAlign program (DNA star Inc., Madison, WI). The exact locations of the amino acids in each protein are indicated at the left-hand side of the figure. Residues identical to dRAD30A or dRAD30B are indicated in black. The conserved DE sequence within which mutations are produced is marked with asterisks below the sequence. C, possible phylogenetic tree of the DinB/UmuC/Rev1/Rad30 family proteins. The accession numbers in the Swiss-Prot and GenBankTM for the sequences and the species names are as follows EcUMUC (E. coli, P04152), AtRev1 (A. thaliana, T19K24.14 in AC02342), CeRev1 (C. elegans, ZK675.2 in Z46812), ScREV1 (S. cerevisiae, P12689), SpREV1 (S. pombe, SPBC1347.01c in AC035548), HsRev1 (H. sapiens, AF151538), MmREV1 (Mus musculus, AF179302), DmRev1 (D. melanogaster, AB049435), EcDINB (E. coli, Q47155), HsDINB1 (H. sapiens, AA576919), MmDINB1 (M. musculus, AB027563), SpDINBh (S. pombe, SPCC553.07c), CeDINBh (C. elegans, F22B7.6), AtRAD30 h (A. thaliana, T19K24.15 in AC02342), CeRAD30h (C. elegans, F53A3.2 in AF025460), HsRAD30A (XPV) (H. sapiens, AB024313 and AF158185), MmRAD30A (M. musculus, AB027128), DmRAD30A (D. melanogaster, AB049433), ScRAD30 (S. cerevisiae, S69702), SpRAD30 (S. pombe, SPBC16A3.11 in AL021748), HsRAD30B (H. sapiens, AF140501), MmRAD30B (M. musculus, AF151691), DmRAD30B (D. melanogaster, AB049434).

The amino acid sequences were aligned for all the members of the UmuC/DinB/Rev1/Rad30 protein family, and the alignment was used to generate a phylogenetic tree (Fig. 1C). The tree reveals several distinct subgroups of proteins; the validity of these subgroups is convincingly supported by the bootstrap test. Since the Rad30B subfamily is equally distant from the RAD30A and DinB subfamilies, the superfamily can be classified into five subgroups; UmuC, DinB, Rev1, Rad30A, and Rad30B. The Drosophila genes drad30A and drad30B belong to the Rad30A and Rad30B subfamilies, respectively. A Drosophila homologue of human Rev1 was also identified, which belongs to the Rev1 subfamily as shown in Fig. 1C; however, no Drosophila homologue of the human DINB1 protein (hpolkappa ) was identified in the cDNA libraries that were searched. In addition, the sequence of the Drosophila genome (34) does not include a homologue of polkappa .

Overproduction and Purification of dRAD30A and dRAD30B Proteins-- The proteins encoded by drad30A and drad30B (dRAD30A and dRAD30B, respectively) were purified as recombinant fusion proteins with a GST tag at the N terminus. Initially, the entire coding sequences of the drad30A and drad30B genes were fused with the GST gene; however, these constructs produced a very low yield of the desired recombinant proteins in E. coli. However, when a mutation was introduced in each gene that creates a stop codon in the C-terminal region, the resulting C-terminal-truncated proteins were expressed at a higher level in E. coli. These truncated forms of the drad30A and drad30B gene products are similar in size to the truncated forms of hpoleta and hpolkappa proteins and include all of the N-terminal conserved regions of the UmuC/DinB/Rev1/Rad30 superfamily; thus, they were expected to be active polymerases despite the missing C-terminal region (5, 9, 14, 21). The truncated forms of dRAD30A (dRAD30ADelta C) and dRAD30B(dRAD30BDelta C) include the N-terminal 545 residues and 445 residues of each polypeptide, respectively. They were purified by GST-Sepharose affinity chromatography (see "Experimental Procedures") and characterized enzymatically as described below (Fig. 2).


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Fig. 2.   Detection of DNA polymerase activity. A, an SDS-polyacrylamide gel electrophoresis of the purified dRAD30ADelta C and dRAD30BDelta C protein is shown. Lane 1, molecular weight marker. 0.5 µg each of GST-dRAD30ADelta C (lane 2), GST-dRAD30A-DE126AA (lane 3), GST-dRAD30BDelta C (lane 4), and GST-dRAD30B-DE115AA (lane 5) were applied. The dRAD30ADelta C and dRAD30BDelta C proteins are indicated by the arrow. B and C, DNA polymerase activities of dRAD30ADelta C (B) and dRAD30BDelta C (C). Three or five different amounts of dRAD30ADelta C, dRAD30A-DE126AA, dRAD30BDelta C, and dRAD30B-DE115AA (10, 100, and 500 fmol in lanes 2-4, respectively, and 0.1, 1, 10, 100, and 500 fmol in lanes 5-9) were added in the reaction mixture. Control reactions with no enzyme are shown in lane 1.

DNA Polymerase Activities of dRAD30ADelta C and dRAD30BDelta C-- The dRAD30ADelta C and dRAD30BDelta C proteins were assayed for DNA polymerase activity using a primer extension assay with a 5'-end-labeled 16-mer primer annealed to a 30-mer template. As shown in Figs. 2, B and C, both dRAD30ADelta C (Fig. 2B, lanes 5-9) and dRAD30BDelta C (Fig. 2C, lanes 5-9) synthesize DNA in a template-dependent fashion, and the size of the replication products gradually increases with increasing enzyme concentration in the reaction. The polymerase activity of dRAD30ADelta C is 10 times higher than that of dRAD30BDelta C.

If the observed DNA polymerase activity is intrinsic to the purified protein, then site-directed mutagenesis of amino acids in the enzyme active site should create a mutant deficient in DNA polymerase activity. This prediction was tested by carrying out site-directed mutagenesis on adjacent Asp and Glu residues within motif III that are believed to be critical for the catalytic activity of the UmuC/DinB/Rad30 family proteins (4, 9, 10, 22, 28, 35, 36). The highly conserved Asp and Glu residues are present at positions 126 and 127 and at positions 115 and 116 in dRAD30A and dRAD30B, respectively (Fig. 1B). In the mutants generated for this study, these residues were changed to alanine. The mutant proteins displayed the same chromatographic properties as the wild type protein, but they lacked DNA polymerase activity (Figs. 2, B and C, lanes 2-4). Therefore, the observed DNA polymerase activity is intrinsic to the dRAD30A and dRAD30B proteins. We therefore propose that dRAD30A be renamed dpoleta and dRAD30B be renamed dpoliota . In the following text, dpoleta Delta C will be used to refer to the truncated protein dRAD30ADelta C, and dpoliota Delta C will be used to refer to dRAD30BDelta C.

The nucleotide selectivity of dpoleta Delta C and dpoliota Delta C was analyzed in DNA synthesis reactions using a nondamaged DNA template in the presence of only one deoxyribonucleotide (i.e. dA, dG, dC, or T). Incorporation of the correct complementary nucleotides and, to a less extent incorrect nucleotides opposite each template, occurred (data not shown), suggesting that both dpoleta Delta C and dpol iota Delta C have relatively low fidelity.

Translesion Synthesis by dpoleta Delta C and dpoliota Delta C-- To determine whether dpoleta Delta C and dpoliota Delta C bypass DNA lesions, polymerase assays were carried out using templates with TT-CPD or TT-(6-4)PP lesions. These templates were described in previous studies of hpoleta (5 7), hpoliota (26), and hpolkappa (9). In addition, a template with TC-(6-4)PP in the same sequence context as the above two templates was constructed and used in this study. A 16-mer primer was annealed to the template-containing lesion, which terminates just before the lesion site (Fig. 3). dpoleta Delta C and dpoliota Delta C efficiently bypassed TT-CPD (Fig. 3A), and the efficiencies of DNA synthesis on the lesion-containing template were almost the same as on the nondamaged template. dpoleta Delta C showed some ability to bypass TT-(6-4)PP (Fig. 3B, lanes 2-4) and TC-(6-4)PP (Fig. 3C, lanes 2-4) but in most cases added one or two bases opposite the lesions before arresting DNA synthesis. dpoliota Delta C added one base opposite the 3'-T of TT-(6-4)PP (Fig. 3B, lanes 6-8) or the 3'-C of TC-(6-4)PP (Fig. 3C, lanes 6-8); in addition, it bypassed TC-(6-4)PP at a very low level when the enzyme was present at a high concentration in the reaction (Fig. 3C, lane 8). As reported previously (7), hpoleta did not bypass TT-(6-4)PP, arresting DNA synthesis after the addition of one base opposite the 3'-T of the lesion. Therefore, the ability to bypass TT- and TC-(6-4)PP is a remarkable feature of dpoleta Delta C, which distinguishes it from other RAD30-like polymerases.


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Fig. 3.   Translesion synthesis by dpoleta Delta C and dpoliota Delta C. Increasing amounts of dpoleta Delta C (10, 100, and 500 fmol in lanes 2-4, respectively) and dpoliota Delta C (30, 300, and 1500 fmol in lanes 6-8, respectively) were incubated with the 5'-32P-labeled primer-template indicated above the panel for 15 min at 37 °C in the standard reaction mixture. The products were subjected to polyacrylamide gel electrophoresis, and the autoradiograms of the gel are shown. The DNA lesions on the templates are TT-CPD (A), TT-(6-4)PP (B), and TC-(6-4)PP (C). Control reactions with no enzyme are shown in lanes 1 and 5.

Nucleotide Selectivity of dpoleta Delta C and dpoliota Delta C during Incorporation Opposite Lesions-- Polymerization reactions by dpoleta Delta C and dpoliota Delta C were performed with the lesion-containing template-primer substrates described above in the presence of a single deoxynucleotide. The reaction products were analyzed and are shown in Fig. 4. On a template with TT-CPD, dpoleta Delta C and dpoliota Delta C preferentially incorporated dAMP and arrested synthesis after adding four dAMPs opposite TT-CPD and the following CT residues (Fig. 4A, CPD). dpoleta Delta C and dpoliota Delta C preferentially incorporated dAMP opposite the 3'T of TT-(6-4)PP, incorporating dGMP at a significant level (Fig. 4A, TT-(6-4)PP). Both enzymes incorporated dGMP, dAMP, and dTMP opposite the 3'-C of TC-(6-4)PP (Fig. 4B, TC-(6-4)PP). Thus, the selectivity of the correct nucleotide on the template containing (6-4)PP was lower than on the template containing CPD.


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Fig. 4.   Selectivity of dpoleta Delta C and dpoliota Delta C nucleotide incorporation opposite the lesions. dpoleta Delta C (20 fmol; upper panels) and dpoliota Delta C (100 fmol; lower panels) were incubated with a 30-mer template containing either a TT-(6-4)PP (A) or a TC-(6-4)PP (B) annealed to 5'-32P-labeled 16-mer primer with one of the indicated dNTPs. The sequence of the template and the primer are the same as that used in Fig. 3, and the partial sequences of the 3' ends of each primer and template are shown above each panel. No damage (TT) or (TC) are undamaged controls of TT-CPD and TT-(6-4)PP or TC-(6-4)PP, respectively.

It should be noted that on the template containing no damage, both dpoleta Delta C and dpoliota Delta C incorporated not only A but also G opposite T (Fig. 4A, no damage). Misincorporation of G opposite T is a common feature of the poleta and poliota family, and especially, hpoliota incorporates G opposite T more efficiently than A opposite T (10, 13, 27, 37). However, dpoliota Delta C misincorporates G opposite T less often than it correctly incorporates A opposite T.

Influence of Base-pairing at the Lesion on Elongation Past the Lesion by dpoleta Delta C and dpoliota Delta C-- The results shown in Fig. 4 indicate that dpoleta Delta C and dpoliota Delta C can insert a variety of bases opposite the 3' base of TT-CPD, TT-(6-4)PP, or TC-(6-4)PP. However, it is possible that the misincorporated base may not be extended as efficiency as the correctly incorporated base is, as shown in the case of hpoleta for the bypass of various lesions (7). To explore this question, primers were synthesized in which each of the four bases would be correctly or incorrectly paired with the 3' or 5' base of the above three lesions. First, the ability of dpoleta Delta C to elongate such primers annealed to TT-CPD was tested, and the results are shown in Fig. 5. dpoleta Delta C elongated DNA chains more efficiently from the correctly base-paired primers, the 17-mer with a 3'-terminal A opposite 3'-T of TT-CPD (Fig. 5B) and the 18-mer with AA opposite the lesion (Fig. 5D). Similar results were obtained with dpoliota Delta C (data not shown).


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Fig. 5.   Ability of dpoleta Delta C to elongate DNA chains past TT-CPD. Sets of 5'-32P-labeled 17-mer oligomers (N17-mer, A and B) or 18-mer (AN18mer, C and D) primers, which contain different sequences at their 3' ends (indicated by N, where N is A, C, G or T, as seen beneath each panel), were annealed to the 30-mer template. Increasing amounts of dpoleta Delta C were incubated with these primed templates. The results of a set of three reactions for each A, C, G, and T primer are shown. Each set contained three reactions with different amounts of enzyme; the one shown on the left (-) contained no enzyme, and the middle and right ones contained increasing amounts of enzyme; 1 and 5 fmol (A and C) and 2.5 and 12.5 fmol (B and D). The template containing no damage (A and C) or TT-CPD (B and D), respectively. The autoradiograms of the gels are shown.

Next, such primers were annealed to TT-(6-4)PP or TC-(6-4)PP to examine whether they could be extended by dpoleta Delta C. On a template with TT-(6-4)PP and a 17-mer primer whose 3'-terminal nucleotide pairs with the 3'-T of the lesion, dpoleta Delta C elongated the DNA chain more efficiently from a primer with a 3'-terminal G (G17) than from a primer with a 3'-terminal A (A17) (Fig. 6A). This makes a sharp contrast with the above result that in the case of TT-CPD, dpoleta Delta C carried out the elongation more efficiently from the A17 primer than from the G17 primer (Fig. 5B). To examine which base is incorporated after the G residue opposed the 3'-T of TT-(6-4)PP, 18-mer oligomers with GN (N is any base) opposite the lesion were synthesized and examined for the extension by dpoleta Delta C. The result showed that the enzyme elongated the DNA chain most efficiently from the primer with GA opposite the lesion (Fig. 6B). However, on a template with TC-(6-4)PP, dpoleta did not show selective elongation from a specific 3' end; it elongated the DNA chain with equal efficiency from 17-mer oligomers with a 3'-terminal A or G opposite the 3'-C of TC-(6-4)PP and from 18-mer oligomers terminating with AA, AG, GA, or GG opposite the lesion (data not shown). Thus, the ability of dpoleta to recognize the correctly inserted base opposite a lesion site is lost more severely in TT-(6-4)PP and TC-(6-4)PP than in TT-CPD.


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Fig. 6.   Ability of dpoleta Delta C to elongate DNA chains past TT-(6-4)PP. Sets of 5'-32P-labeled17-mers (N17-mer, A) or 18-mers (GN18-mer, B) were annealed to the 30-mer template. In GN18-mer, G is opposite the 3'-T of (6-4)PP, and N is opposite the 5'-T of (6-4)PP. Conditions are the same as in Fig. 5, except for the amount of enzyme; middle and right lanes contain 50 and 250 fmol, respectively.

Direct Analysis of Bypass Products Synthesized by dpoleta Delta C and dpoliota Delta C-- The above results suggest that dpoleta Delta C incorporates a significant level of incorrect nucleotides during bypass of (6-4)PP; and in contrast, correct nucleotides are selectively incorporated opposite CPD by dpoleta Delta C and dpoliota Delta C. To know whether dpoleta Delta C and dpoliota Delta C the incorporate correct nucleotide opposite each damage, the bypass products were analyzed directly (Fig. 7). For this experiment, three kinds of oligonucleotides containing TT-CPD, TT-(6-4)PP, or TC-(6-4)PP were synthesized. TT-CPD and TT-(6-4)PP were positioned at the TT of the TTAA sequence, and TC-(6-4)PP was positioned at the TC of the TCGA sequence; these are the recognition sequences for the restriction enzymes MseI and TaqI, respectively. If the bypass products by dpoleta Delta C or dpoliota Delta C have the correct AA or GA sequence, then they should be cleaved by MseI or TaqI after rehybridization with a nondamaged oligonucleotide; the product of this cleavage reaction can be readily detected as a 19- or 14-base fragment, respectively (Fig. 7A). Almost all of the bypass products of TT-CPD by dpoleta Delta C or dpoliota Delta C were cleaved by MseI (Fig. 7B, lanes 9-12); more than 90% of the bypass products have the AA sequence (Table I). On the other hand, only some of the bypass products of either TT-(6-4)PP or TC-(6-4)PP were susceptible to restriction enzyme digestion (Fig. 7B, lanes 15, 16, 19, and 20). The ratio of the correct base incorporated opposite (6-4)PP is much lower than for the CPD. Of the bypass products of TT-(6-4)PP by dpoleta Delta C, 22.6% are susceptible to MseI digestion, and 36% of the TC-(6-4)PP bypass products are susceptible to TaqI digestion (Table I), indicating that bypass of (6-4)PP by dpoleta is highly error prone.


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Fig. 7.   MseI or TaqI cleavage assay of the TLS bypass products by dpoleta Delta C and dpoliota Delta C. A, a schematic drawing of the experimental protocol of the MseI cleavage assay is shown. In TaqI assay, a 30-mer substrate was used, and a cleavage of the substrate produced a 14-base fragment. B, the 49-mer oligomer with no lesion (lanes 1-6), a TT-CPD (lanes 7-12), a TT-(6-4)PP (lanes 13-16), or the 30-mer oligomer with TC-(6-4)PP (lanes 17-20) were annealed to a 5'-32P-labeled primer and then were mixed with Klenow fragment (2 units in lanes 1, 2, 7, 8, 13, 14, 17, and 18), dpoleta Delta C (500 fmol in lanes 3, 4, 9, 10, 15, and 16 or 1 nmol in lanes 19 and 20), or dpoliota Delta C (500 fmol in lanes 5, 6, 11, and 12). The reaction products were denatured by heating at 95 °C for 5 min, and then excess amounts of the 49-mer oligonucleotide (lanes 1-16) or the 30-mer oligonucleotide (lanes 17-20), which does not contain a DNA lesion, were added and annealed to the newly synthesized DNA. The resultant duplex DNA was cleaved with MseI or TaqI, respectively, denatured in the presence of formamide, and then electrophoresed on a sequencing gel. The autoradiograms of the gels are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Damage-induced mutagenesis is an important biological process whose molecular mechanism is not yet understood and which is the subject of much current research. Recently, the proteins of the UmuC/DinB/Rev1/Rad30 superfamily have been isolated and characterized biochemically, and it was shown that these proteins have a nonprocessive DNA polymerase activity that can bypass DNA lesions. Thus, these enzymes are central to the process of damage-induced mutagenesis. In this study, Drosophila homologues of this polymerase superfamily, dpoleta and dpoliota , were identified and characterized with respect to their ability to bypass UV-induced DNA lesions.

Saccharomyces cerevisiae and human poleta are considered to bypass TT-CPD in a mostly error-free manner (7, 15, 21). Similarly, dpoleta Delta C and dpoliota Delta C incorporate AA opposite TT-CPD more efficiently than other nucleotides (Fig. 4A), and the elongation starts selectively from a primer ending in A opposite the lesion (Fig. 5, B and D). These results suggest that dpoleta Delta C and dpoliota Delta C bypass TT-CPD in a error-free manner as in the case of hpoleta . In addition, direct analysis of the products of lesion bypass also indicates that dpoleta Delta C and dpoliota Delta C carry out error-free bypass of TT-CPD (Fig. 7). Thus, for these enzymes, TT-CPD maintains a structure that directs correct Watson-Crick base-pairing despite the base modification. In fact, Lawrence et al. (38) argue that the CPD must be an instructive lesion by virtue of its high coding specificity in E. coli, and structural studies show that the Watson-Crick base pair is still intact at the CPD site (39-41). Therefore, the TLS polymerases bypass this lesion by incorporating the correct bases on this instructional lesion.

However, evidence also indicates that dpoleta Delta C does not carry out error-free bypass of (6-4)PP lesions. On templates with TT-(6-4)PP, dpoleta Delta C did not show selective elongation from the correct base paired primer/template (Figs. 6A). Furthermore, the bypass products of TT-(6-4)PP by dpoleta Delta C showed high levels of misincorporation (Fig. 7B). dpoleta Delta C elongated DNA efficiently from the 17-mer end with a mismatched G opposite the 3'-T of TT-(6-4)PP (Fig. 6A) and preferentially from the 18-mer with GA opposite the lesion (Fig. 6C). The favored elongation from the mismatched GA/TT-(6-4)PP terminus is consistent with structural studies. An NMR study of a duplex DNA with (6-4)PP revealed that TT-(6-4)PP greatly distorts the DNA structure and prevents base-pairing with A in the complementary strand (41). However, a mismatched base pair between the 3'-T of TT-(6-4)PP and G forms hydrogen bonds and stabilizes the helix (42, 43). Furthermore, the G/T mispair at the 3'-T of TT (6-4)PP retains the normal Watson-Crick-type base-pairing between the 5'-T of the TT-(6-4)PP and an opposed A, and the resultant GA/TT-(6-4)PP duplex can assume the typical B-form-DNA conformation (44). Our results also fit well with the specificity of base alteration of UV-induced mutations in which a G residue is preferentially inserted opposite the 3'-T of the 6-4-photoproduct during TLS in E. coli (19). These results indicate that the TT-(6-4) photoproduct can be classified as a mis-instructional lesion.

Despite the biological relevance of this DNA lesion, the tertiary structures of oligonucleotide duplexes, which include TC-(6-4)PP, have not been well characterized. Fujiwara and Iwai (42) show that the duplex containing G opposite the 3'-C of TC-(6-4)PP is thermodynamically more stable than another duplex (42). Horsfall and Lawrence (20) studied the mutagenicity of TC-(6-4)PP in E. coli cells and found that 66% of the bypass products have the correct TC sequence, 28% of the mutants having the TT sequence (20). Our observation that both G and A are incorporated opposite the 3'-C of TC-(6-4)PP (Fig. 4B) fit well with these results. On the other hand, the nucleotide incorporated opposite the 5'-T of TC-(6-4)PP is not consistent with the mutation spectrum. Although the in vivo mutation spectrum indicates that A is incorporated preferentially opposite the 5'-T of TC-(6-4)PP, our in vitro results indicated that dpoleta Delta C elongated the DNA chain equally well from the 18-mer primer with A or G opposite the 5'-T (data not shown). It will be necessary to determine the sequence of the bypass products directly or perform steady-state kinetics to clarify this point.

The mutagenic properties of dpoleta Delta C and dpoliota Delta C fit well with the mutation spectrum obtained using a defined photoproduct introduced into SOS-induced E. coli (19, 38). The mutation spectrum reported in those studies reflects the mutagenic properties of pol V (UmuD·2C complex). Thus, the mutagenic properties of dpoleta Delta C on TT-CPD and TT-(6-4)PP are very similar to those of pol V. Both enzymes bypass TT-CPD in an error-free manner and incorporate GA opposite TT-(6-4)PP cells (Ref. 8 and this study). The similarity in the nucleotides incorporated suggests that structural information in the altered bases contribute to nucleotide selection during incorporation opposite these lesions by these polymerases.

In the Rad30 protein family, dpoleta Delta C seems to be an unusual member of the Rad30 family in having the ability to bypass (6-4)PP, although we cannot rule out a possibility that having an N-terminal GST tag and a C-terminal truncation have affected the TLS specificity and activity; we also have to keep in mind that we cannot draw a quantitatively definitive conclusion since the observations present in this study are qualitative. It should also be noted that the DNA repair capacity of Drosophila cells is somewhat different from that of human cells. The most striking difference is that Drosophila cells have a 6-4-photolyase, which specifically repairs (6-4)PP in a light-dependent manner, but human cells lack this enzyme (45). The (6-4)PP lesions are removed rapidly by nucleotide excision repair. However, a small amount of the lesion might remain that would be toxic because (6-4)PP is highly mutagenic. The remaining (6-4)PP lesions can be removed by the 6-4-photolyase in Drosophila cells, and thus, the error-prone bypass activity of dpoleta might not be so deleterious. On the other hand, human cells lack the 6-4-photolyase, and thus, more lesions might remain, so that error-prone bypass of this lesion would have a deleterious effect. Thus, hpoleta might have lost the ability to bypass (6-4)PP because that loss enhances genetic stability. Paradoxically, we have recently shown that in human cells, mutations are produced at the (6-4)PP site, although the frequency is low (46). Thus, in human cells, some DNA polymerases bypass the (6-4)PP in an error-prone manner. Another enzyme, for example hpoliota , might bypass (6-4)PP in vivo, in conjunction with polzeta (27).

In this study, TT (6-4)PP was bypassed by dpoleta Delta C but not by dpoliota Delta C. The TLS activity of dpoleta Delta C and dpoliota Delta C was also examined on chemically modified damage, and all of the other lesions tested were bypassed by both enzymes (data not shown). Thus, at present we cannot assign any specific biological function to dpoliota . Further analyses of the substrate specificity of these polymerases by screening various types of DNA lesions may reveal differences in their substrate specificity. Another possibility is that each polymerase interacts with other protein differently in vivo. The recombinant protein used in this study has a C-terminal deletion. The deleted C-terminal portion might have an important biological interaction with other proteins in vivo, whereas it is not required for polymerase activity in vitro. It is also possible that dpoleta and dpoliota have different in vivo functions in Drosophila that have not been detected by in vitro studies carried out to date. One enzyme may be important for the defense against DNA damage, and another may be involved in some biological process that requires DNA replication on a primer-template complex containing an unusual mismatch. Detailed analyses of the expression pattern of each gene at the tissue and cellular levels will provide clues to the function of each gene. Furthermore, the phenotypes of transgenic flies that overexpress these genes and mutant flies in which each gene is disrupted by an insertion or repressed by RNA interference (RNAi) will clarify the biological roles of these proteins in Drosophila.

    ACKNOWLEDGEMENT

We thank E. Ohashi for technical assistance and for kindly providing oligonucleotides used as primer and template and T. Ogi for the phylogenetic analysis to construct the tree (Fig. 1C).

    FOOTNOTES

* This work was supported by Grants-in-aid from the ministry of Education, Science, Sports, and Culture of Japan 09308020, 11146206, 11480140, 11878093 and by the REIMEI Research Resources of Japan Atomic Energy Research Institute.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(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB049435 (DmRev1), AB049433 (DmRAD30A), and AB049434 (DmRAD30B).

§§ To whom correspondence should be addressed: Radiation Biology Center, Kyoto University, Yoshidakonoe-cho, Sakyoku, Kyoto 606-8501, Japan. Tel.: 81-75-753-7555; Fax: 81-75-753-7564; E-mail: todo@house.rbc.kyoto-u.ac.jp.

Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009822200

    ABBREVIATIONS

The abbreviations used are: TLS, translesion DNA synthesis CPD, cis-syn-cyclobutane pyrimidine dimer; (6-4)PP, pyrimidine-pyrimidone 6-4-photoproduct; GST, glutathione S-transferase; TT, thymine-thymine; CT, thymine-cytosine; pol, polymerase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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