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First published online June 6, 2002; 10.1105/tpc.000869 American Society of Plant Biologists The Homologous ABI5 and EEL Transcription Factors Function Antagonistically to Fine-Tune Gene Expression during Late Embryogenesis
a Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France 2 To whom correspondence should be addressed. E-mail francois.parcy{at}isv.cnrs-gif.fr; fax 33-1-69823695
In Arabidopsis, the basic leucine zipper transcription factor ABI5 activates several late embryogenesis–abundant genes, including AtEm1 and AtEm6. However, the expression of many other seed maturation genes is independent of ABI5. We investigated the possibility that ABI5 homologs also participate in the regulation of gene expression during seed maturation. We identified 13 ABI5-related genes in the Arabidopsis genomic sequence. RNA gel blot analysis showed that seven of these genes are active during seed maturation and that they display distinct expression kinetics. We isolated and characterized two mutant alleles of one of these genes, AtbZIP12/EEL. Unlike abi5, the eel mutations did not inhibit the expression of any of the maturation marker genes that we monitored. On the contrary, the accumulation of the AtEm1 and AtEm6 mRNAs was enhanced in eel mutant seeds compared with wild-type seeds. Gel mobility shift assays, combined with analysis of the genetic interactions among the eel and abi5 mutations, indicated that ABI5 and EEL compete for the same binding sites within the AtEm1 promoter. This study illustrates how two homologous transcription factors can play antagonistic roles to fine-tune gene expression.
In higher plants, seed development is divided roughly into two successive phases. Cell division and morphogenesis occur during the first phase. Once embryogenesis is completed, the seed enters a second phase called maturation, during which the embryo accumulates storage compounds, becomes dormant, and acquires desiccation tolerance (Wobus and Weber, 1999  ). All of these traits are of crucial importance for the survival of seed plant species, including Arabidopsis. At the molecular level, seed maturation is characterized by the coordinated expression of several classes of genes with distinct temporal patterns (Hughes and Galau, 1989; Parcy et al., 1994). The maturation MAT class includes genes that code for storage proteins such as 2S and 12S globulins in Arabidopsis (Pang et al., 1988; Guerche et al., 1990). The late embryogenesis–abundant (LEA) class comprises genes that code for LEA proteins that are thought to be involved in desiccation tolerance (Hoekstra et al., 2001).
Our knowledge of the mechanisms that control MAT and LEA gene expression is fragmentary. The phytohormone abscisic acid (ABA) accumulates during seed maturation and positively regulates the expression of genes in both the MAT and LEA classes (Koornneef et al., 1989
How the ABI3 protein regulates these genes is not known. The isolated basic domain B3 of the VP1 protein, the maize ortholog of ABI3, is able to bind DNA in vitro (Suzuki et al., 1997
Genetic evidence that a TRAB1-related bZIP protein is involved in the ABI3-dependent regulation of LEA genes came from the analysis of the abi5 mutant of Arabidopsis. The abi5 mutation leads to reduced ABA sensitivity during germination and to decreased expression of some LEA genes, including AtEm1 and AtEm6, during seed maturation (Finkelstein, 1993
The abi5 mutant has a markedly milder phenotype than abi3. In contrast to abi3, abi5 seeds are tolerant to desiccation and have a storage protein content similar to that of wild-type seeds (Finkelstein, 1994
An attractive possibility, therefore, is that additional ABI5-related bZIP proteins contribute to the ABI3-dependent regulation of gene expression during seed maturation and mediate the recruitment of ABI3 to distinct promoters. For instance, at least three different ABI5-related bZIP genes (DPBF-1, DPBF-2, and DPBF-3) are expressed in sunflower seed tissue. These three DPBF proteins are able to bind the ABRE-like motifs present in the promoter of the LEA gene DC3 from carrot (Kim et al., 1997
All of these genes are expressed in vegetative tissues, and at least several of them seem to be involved in ABA-dependent processes. ABF1, ABF2/AREB1, ABF3, and ABF4/AREB2 are induced differentially in response to abiotic stress or ABA treatment (Choi et al., 2000 In the present study, we performed an exhaustive search of the Arabidopsis genomic sequence and identified 13 ABI5-related genes, including 6 that had not been described previously. Seven of these bZIP genes are expressed during seed maturation. Two genes, including ABI5, displayed expression kinetics similar to that of the LEA genes. Remarkably, three other bZIP genes displayed expression kinetics similar to that of storage protein genes, suggesting that these bZIPs could regulate the expression of genes from the MAT class. We tested this hypothesis by isolating two insertional mutants for one of the three latter bZIP genes, AtbZIP12. The accumulation of storage protein mRNAs was not affected in the atbzip12 mutants. However, unexpectedly, the abundance of the LEA mRNAs AtEm1 and AtEm6 was increased in atbzip12, whereas the level of these same mRNAs was decreased in the abi5 mutant. We analyzed the genetic and molecular interactions between ABI5 and AtbZIP12 and propose a model of how these two homologous transcription factors play antagonistic roles to fine-tune LEA gene expression during Arabidopsis seed development.
The Arabidopsis ABI5/ABF/AREB Family Contains 13 Members In the complete sequence of the Arabidopsis genome, we identified 13 genes that encode proteins belonging to the ABI5/ABF/AREB family. Seven of these genes had been characterized previously and had been named ABI5, ABF1, ABF2/AREB1, ABF3, ABF4/AREB2, AREB3, and GBF4 (Menkens and Cashmore, 1994 ; Choi et al., 2000; Finkelstein and Lynch, 2000; Lopez-Molina and Chua, 2000; Uno et al., 2000). The six additional genes were designated AtbZIP12, AtbZIP13, AtbZIP14, AtbZIP15, AtbZIP27, and AtbZIP67, according to the unified nomenclature for AtbZIP genes proposed by Jacoby et al. (2002). The complete coding sequences of AtbZIP12, AtbZIP13, AtbZIP15, and AtbZIP67 were obtained by sequencing cDNA clones and assembling available EST sequences. In contrast, cDNA clones have not been identified for AtbZIP14 or AtbZIP27. Thus, the coding sequences proposed for these two genes are based solely on the results of gene-modeling programs and optimization of protein sequence alignments to other members of the ABI5/ABF/AREB family. Furthermore, because we were unable to detect their expression in the various tissues analyzed, we cannot exclude the possibility that AtbZIP14 and AtbZIP27 represent pseudogenes.
As shown in Figure 1
, the amino acid sequence of the basic region is extremely conserved between the 13 members of this bZIP subfamily. This region contains, in particular, the motifs M/K-I-K and Q-A-Y/Q, which are not found in any other Arabidopsis bZIP outside of this subfamily. The high degree of conservation of this DNA binding region suggests that all members of this subfamily recognize similar cis elements. As noted previously, three additional conserved domains are found in the N-terminal half of the proteins, and a fourth is found at their C-terminal end (Choi et al., 2000
The C1, C2, and C3 domains share some sequence similarity: for instance, they all contain stretches of two acidic residues (D or E) surrounded by nonpolar residues (F, I, L, M, or V). Finally, there is a fourth conserved small domain (C4) located at the C-terminal end of the proteins. The C1 to C4 domains contain consensus phosphorylation sites for various protein kinases that are well conserved in the different proteins. Interestingly, the four consensus phosphorylation sites for Ca2+-dependent protein kinase (R/K-X-X-S/T) are all found two residues C terminal to a conserved Leu (L-X-R/K-X-X-S/T), suggesting that they might be recognized by similar kinases. Figure 2 shows the degree of similarity between the various members of the Arabidopsis subfamily and related bZIP proteins from other plant species. It also illustrates the existence of several pairs of closely related Arabidopsis proteins that might have similar functional properties (ABF1 and ABF4/AREB2, AREB3 and AtbZIP12, GBF4 and AtbZIP13, and AtbZIP14 and AtbZIP27).
Expression Analysis during Seed Development As a first step toward the identification of bZIP genes involved in seed maturation, we analyzed which of the bZIP genes described above are expressed during silique development. For this purpose, we generated a gene-specific probe for each of the bZIP genes. We used a maximum level of 70% nucleotide identity over 100 bp as a specificity criterion. This criterion, however, could not be met for a few pairs of closely related bZIP genes, and we had to accept up to 79% identity between the AtbZIP14 probe and the corresponding region of AtbZIP27. In all cases, we also verified that the probe does not share any significant sequence similarity with any other predicted coding sequence of the Arabidopsis genome.
These probes were used to analyze the expression of the various bZIP genes during seed maturation. This developmental process is characterized by the existence of several temporal programs of gene expression. The storage protein At2S3 and LEA AtEm6 genes are representative members of the MAT and LEA programs, respectively (Parcy et al., 1994
AtbZIP13 mRNA was expressed at all stages analyzed, and like ABI5, its level increased sharply during the last 2 days of seed development. The AtbZIP12, AtbZIP67, and AREB3 mRNAs also were detectable as early as 6 to 7 DAP, but their abundance decreased toward the end of seed maturation. The expression kinetics of these three bZIPs is similar to that of the storage protein marker At2S3. It is unclear why two hybridizing mRNA bands were observed with the ABI5 and AtbZIP67 probes. Each of these probes shows a low level of sequence similarity to the other bZIP genes; thus, it is unlikely to cross-hybridize with another member of the gene family. Hence, the double bands may be indicative of alternative splicing in the ABI5 and AtbZIP67 genes. Finally, GBF4 mRNA was expressed uniformly throughout seed maturation, whereas ABF4 mRNA abundance was maximum at 6 DAP and then decreased progressively until the dry seed stage. Their distinctive expression kinetics suggest that the seven bZIP genes described above play different regulatory roles during seed maturation.
Isolation and Characterization of atbzip12/eel Mutants
Seeds from the eel-1 and eel-2 mutants did not display any morphological alteration. Seed development, defined as the period extending from pollination to the dry seed stage, lasted the same amount of time (16 days) as in the wild type, and the eel mutant seeds were as dormant as wild-type seeds. The eel mutants also showed wild-type sensitivity to the ABA inhibition of seedling establishment, whereas, as described previously (Finkelstein, 1994 ; Lopez-Molina et al., 2001), the abi5-1 mutant showed reduced ABA sensitivity in this bioassay (Figure 5)
.
The abi5 mutation leads to decreased expression of some LEA genes during seed maturation (Finkelstein and Lynch, 2000 ). We initially compared the abundance of the storage protein At2S3 mRNA and a few LEA mRNAs (AtEm1, AtEm6, M17, PAP38, and PAP140) in wild-type and eel-1 seeds. For At2S3, M17, PAP38, and PAP140, we occasionally observed a slight difference in mRNA abundance between wild-type and mutant seeds. However, these fluctuations were observed only once within a given experiment and were not reproducible. Hence, such variations were considered to be within the experimental fluctuations. They most likely result, at least in part, from the fact that staging and harvesting of the siliques inevitably vary slightly from one genotype to another and from one experiment to another. In contrast, the abundance of the AtEm1 and AtEm6 mRNAs was reproducibly higher in eel-1 than in the wild type (Figure 6)
. This observation was particularly surprising because, in marked contrast to eel-1, the abi5 mutation inhibits the expression of AtEm1 and AtEm6 (Finkelstein and Lynch, 2000).
We analyzed in more detail the expression of AtEm1, for which the eel phenotype seemed the clearest. In wild-type seed, AtEm1 mRNA was expressed during the last 4 days of seed maturation, and its level reached a maximum in the desiccated seeds (Figure 7) . The abundance of the AtEm1 mRNA was reduced approximately fourfold in abi5-1. In contrast, in eel-1 and eel-2, the AtEm1 mRNA level was approximately threefold higher than in the wild type, and the expression of this mRNA was detectable 1 day earlier than in the wild type (Figure 7B). Based on seven independent experiments (five with eel-1 and two with eel-2), the ratio between the AtEm1 level in eel and the wild type stands with 95% confidence between 3.3 and 6.5 at 13 DAP, between 2.2 and 4.7 at 14 DAP, and between 2.0 and 3.5 at 15 DAP. However, in dry seeds (16 DAP), in most cases the AtEm1 mRNA level was similar in the wild type and the eel mutants (Figure 7B).
We analyzed the spatial pattern of AtEm1 mRNA expression by in situ hybridization. In agreement with previous reports (Gaubier et al., 1993 ; Vicient et al., 2000), AtEm1 expression in the wild-type seeds was restricted mostly to the provascular tissue, the root tip, and the shoot apical meristem of the embryo (Figure 8A) . In abi5-1, AtEm1 mRNA was undetectable in most embryos (data not shown), but a faint hybridization signal was observed in a few cases (Figure 8B). In eel-1, the spatial distribution of AtEm1 mRNA was similar to that in wild-type seeds (Figures 8C and 8D). The increase in AtEm1 mRNA level detected by gel blot analysis was not apparent in the in situ hybridizations. Nevertheless, the combined results of gel blot and in situ hybridization analyses indicate that the eel mutations do not induce an ectopic accumulation of AtEm1 mRNA; rather, they increase the intensity of AtEm1 expression without altering its spatial distribution.
Genetic Interaction between ABI5 and EEL eel-1 and abi5-1 are null mutations that have opposite effects on AtEm1 expression. We tested the epistatic relationship between these mutations by analyzing AtEm1 expression in the abi5-1 eel-1 double mutant. The expression kinetics and abundance of the AtEm1 mRNA were similar in the abi5-1 eel-1 double mutant and the abi5-1 single mutant (Figure 7B). This result indicates that abi5 is epistatic to eel. In other words, the increased AtEm1 expression observed in the eel mutant is completely dependent on a functional ABI5 gene. A possible explanation for this finding could then be that EEL acts upstream of ABI5, for instance, by negatively regulating ABI5 expression. However, we analyzed the expression kinetics of the ABI5 mRNA from 11 to 16 DAP and found no obvious difference between wild-type and eel mutant seeds (data not shown). Another possibility is that EEL and ABI5 act at the same level to regulate AtEm1 expression. Consequently, we investigated whether these two transcription factors bind directly to the AtEm1 promoter.
ABI5 and EEL Bind to the Same Sites in the AtEm1 Promoter
In vitro–produced ABI5 and EEL proteins bound weakly, if at all, to oligonucleotide 0 containing ABRE0 (Figure 9C). In contrast, both proteins bound clearly to oligonucleotides 1 and 2, which contain ABRE1 and ABRE2, respectively (Figure 9C). No binding was observed when the ABRE motifs were mutated (Figure 9D). Thus, these data indicate that both ABI5 and EEL are able to bind to ABRE1 and ABRE2. Because bZIP proteins bind DNA as dimers, the shifted bands observed in Figure 9C correspond to complexes between the oligonucleotide and homodimers of either ABI5 or EEL. When EMSA was performed with cotranslated ABI5 and EEL proteins, an additional shifted band was observed that had mobility intermediate between those of the bands observed with ABI5 alone or EEL alone (Figure 9E, lanes 5 and 9). This result indicates that an ABI5-EEL heterodimer can form and can bind to ABRE1 and ABRE2.
Seven members of the ABI5 subfamily of bZIP genes are expressed during seed maturation. These genes, however, display various expression kinetics, suggesting that they play different roles in the control of gene expression programs during seed maturation. The ABI5 mRNA accumulates during the later stages of seed development, and ABI5 controls the expression of some LEA genes (Finkelstein and Lynch, 2000 ; Lopez-Molina and Chua, 2000). The expression kinetics of the AtbZIP12/EEL mRNA resembles that of storage protein mRNAs. Thus, we thought initially that AtbZIP12/EEL might be involved in regulating storage protein genes. However, using gel blot analysis and hybridization of macroarrays carrying 500 seed cDNA clones, we detected no effect of the eel mutations on the accumulation of napin and cruciferin mRNAs or of any other MAT mRNA. In contrast, the LEA mRNAs AtEm1 and AtEm6 were expressed at higher levels in eel mutant seeds than in wild-type seeds. Conversely, the AtEm1 and AtEm6 mRNA levels were lower in abi5 than in the wild type. Thus, genetically, these two homologous bZIPs have opposite roles in the control of AtEm1 and AtEm6: the expression of the AtEm genes is regulated positively by ABI5 and regulated negatively by EEL.
EEL function was investigated in more detail by studying AtEm1 expression. Double-mutant analysis showed that abi5 is epistatic to eel, indicating that the increased AtEm1 expression in eel mutant seeds is attributable to ABI5. Thus, genetically, EEL acts either upstream or at the same level as ABI5. It seems unlikely that EEL controls AtEm1 indirectly by inhibiting ABI5 expression, because we did not detect any increase in ABI5 mRNA level in the eel mutant. Moreover, overexpression of ABI5 results in supersensitivity to ABA inhibition of seed germination and seedling establishment (Lopez-Molina et al., 2001
Our gel retardation experiments suggest that both ABI5 and EEL regulate AtEm1 expression by binding directly to the AtEm1 promoter. The decreased abundance of AtEm1 mRNA in the abi5 mutant suggests that ABI5 is a transcriptional activator of AtEm1. In contrast, EEL seems unable to activate AtEm1 transcription: the AtEm1 mRNA level was higher in eel than in the wild type. Moreover, the abi5 single mutant and the abi5 eel double mutant had similar levels of AtEm1 mRNA, indicating that EEL is not responsible for the residual AtEm1 expression observed in abi5. EEL may be unable to activate AtEm1 transcription because, unlike ABI5 (Nakamura et al., 2001 In vitro, the EEL and ABI5 homodimers as well as the EEL-ABI5 heterodimer bind to the ABRE1 and ABRE2 elements of the AtEm1 promoter. The eel and abi5 mutant phenotypes then can be explained by assuming that, in the seed, EEL and ABI5 compete for these binding sites on the AtEm1 promoter. In eel mutant seeds, the EEL protein is absent and ABI5 can freely activate AtEm1 expression to maximum levels. In wild-type seeds, the EEL-ABI5 heterodimer and the EEL homodimer compete with the ABI5 homodimer for binding to the AtEm1 promoter, preventing ABI5 from optimally activating AtEm1. Consequently, AtEm1 expression is lower in wild-type seeds than in eel. In abi5 and abi5 eel mutant seeds, the ABI5 protein is absent and AtEm1 expression is reduced further.
The regulation of Em genes in monocots also might involve bZIP proteins with antagonistic functions. The bZIP EMBP-1 from wheat, a putative positive regulator of Em, binds in vitro to ABREs present in the wheat Em promoter. In the presence of a rice bZIP, osZIP-2a or osZIP-2b, heterodimerization between EmBP-1 and osZIP-2 prevents EmBP-1 from binding to the ABRE (Nantel and Quatrano, 1996
Competition between ABI5 and EEL could be a mechanism adopted by plants to tightly control the expression of LEA genes during seed development. RNA gel blot analysis suggests that until
The model described above for AtEm1 probably does not apply to all LEA genes. For instance, the abundance of the LEA mRNA M17 is increased (not decreased) in abi5 (Finkelstein and Lynch, 2000
Our macroarray analysis did not reveal any effect of the eel mutation on the accumulation of MAT mRNAs. However, EEL (AtbZIP12), AtbZIP67, and AREB3 display similar expression patterns and may have overlapping roles in regulating MAT genes. Testing this hypothesis will require the isolation of plants that combine mutations in EEL, AREB3, and AtbZIP67. Sequencing of the Arabidopsis genome revealed that, in this model dicotyledonous species, transcription factors exist as large families (Riechmann et al., 2000
Mutant analysis of several subgroups of MADS box genes underscored the existence of functional overlap between homologous transcription factors that leads to partial genetic redundancy (Jack, 2001
Plant Material and Culture Conditions eel-1, eel-2, and abi5-1 mutants are derived from the Arabidopsis thaliana Wassilewskija accession. eel-1 (line COU38) was isolated from the Versailles collection of T-DNA insertion lines (Bechtold et al., 1993 ). eel-2 (line CS11393) was isolated from the Feldmann collection of T-DNA insertion lines available at the ABRC (Ohio State University, Columbus). abi5-1 seeds (Finkelstein, 1994) were obtained from Ruth Finkelstein (University of California, Santa Barbara).
Plants were grown routinely in a greenhouse (22°C with a 16-h photoperiod) on soil irrigated with mineral nutrients. For aseptic growth, seeds were surface-sterilized and plated on medium containing the inorganic salts of Murashige and Skoog (1962)
bZIP Sequences DNA regions used as gene-specific probes were selected by pair-wise comparison of the bZIP sequences using Bestfit (GCG package; Genetics Computer Group, Madison, WI) and by BlastN comparison against all predicted Arabidopsis coding sequences at TAIR (http://www.arabidopsis.org). Gene-specific probes were prepared by PCR amplification, and the amplified DNA was subcloned into PCR-TOPO2.1 (Invitrogen, La Jolla, CA) or pGEM-T easy (Promega).
RNA Gel Blot Analysis
Mutant Isolation and Genotyping
Plant genotyping for the eel-1 mutation was performed by PCR using oligonucleotides oSR505 (5'-GCCACCAGCTGAGGAAGGGCTT-3'), oSR506 (5'-GGCTGATGATGATACTCAACCCATT-3'), and TAG3 (see above). Plant genotyping for the eel-2 mutation was performed by PCR using oligonucleotides oFP1080 (5'-CTCCACAATCTCTCCAGCTACTC-3'), oFP1108 (5'-GTTCGAGTGTAACAGTTTCTA-AGGC-3'), and oFPLB (see above). Plant genotyping for the abi5-1 mutation was performed by PCR using oligonucleotides oFP1081 (5'-GGTTATTGTTGTGTATATGATGCAGTTG-3') and oFP1082 (5'-CCACTACTCTTTTCCTTCCCC-3') followed by digestion with AvaII. Plant DNA was extracted as described by Edwards et al. (1991)
In Situ Hybridization
Electrophoretic Mobility Shift Assays
Overlapping oligonucleotides were annealed in 50 mM Tris-HCl, pH 8, 100 mM NaCl, and 10 mM MgCl2 from 85°C to room temperature, filled by T4 DNA polymerase with
Accession Numbers Accession numbers for EST and other sequences mentioned in Methods are as follows: EST sequences for AtbZIP13 (AV527966, 202M14T7, and AV549823), AtbZIP12/EEL partial cDNA (AJ420881), EST sequences for AtbZIP12 (BE529226, BE524624, and AV561543), DPBF-4 (AF334209), DPBF-2 (AF334207), and BAC clone T3K9 (AC004261). Accession numbers for the regions used as gene-specific probes are as follows: ABI5 (nucleotides 22 to 1074 in AF334206), ABF1 (1163 to 1428 in AF093544), ABF2 (550 to 1017 in AF093545), ABF3 (608 to 1053 in AF093546), ABF4 (1262 to 1592 in AF093547), AREB3 (63,805 to 64,439 in AL390921), AtbZIP12 (100 to 623 in AF334209), AtbZIP13 (69,270 to 69,846 in MRH10), AtbZIP14 (76,538 to 76,863 in AL161588), AtbZIP15 (545 to 1005 in AJ419599), AtbZIP27 (54,898 to 55,124 in AF024504), AtbZIP67 (154 to 626 in AJ419600), and GBF4 (70 to 370 in U01823).
We thank Ranjiv Khush and Michel Delseny for helpful comments on the manuscript, David Bouchez for access to the Versailles collection of T-DNA lines, Dominique Thomas for help with EMSA, Cristel Carles and Michel Delseny for the ABI5 expression clone, Monique Raynal for the M17 cDNA, Philippe Peynot for help with figures, Ruth Finkelstein for seeds, Erika Asamizu at Kazusa DNA Research Institute for cDNA clones, and the ABRC for seeds and DNA clones. This work was supported by the Centre National de la Recherche Scientifique and the European Community REGIA project (Grant QLG2-CT1999-00876). S.B. was the recipient of a fellowship from the Ministère de l'Education Nationale de la Recherche et de la Technologie.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.000869.
1 Current address: Laboratoire de Technologie Enzymatique, Université de Technologie de Compiègne, 60205 Compiègne Cedex, France. Received November 30, 2001; accepted February 27, 2002.
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Abstract
INTRODUCTION
-glucuronidase reporter gene (Delseny et al., 2001
13 DAP, EEL expression is highest, whereas ABI5 mRNA is barely detectable. We believe that, as a result, the AtEm1 gene is not expressed during this period. From 13 DAP onward, ABI5 expression increases but EEL expression decreases. This may explain the quick upregulation of AtEm1. AtEm1 mRNA reaches its maximum level at the end of seed maturation, when ABI5 also is highly expressed and EEL expression decreases. ABI5 might even contribute to the downregulation of EEL expression, because EEL mRNA level was higher in abi5 than in wild-type seeds (data not shown). Therefore, seeds appear to use two transcription factors based on the same DNA binding module to fine-tune the expression of LEA genes. 
-32P-dCTP, and purified on a column of Sephadex G-50 fine (Pharmacia, Uppsala, Sweden). Binding reactions were performed on ice for 30 min in 10 µL (final volume) of binding buffer (25 mM Hepes, pH 7.6, 60 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 µM DTT, and 5% glycerol) (Kuras et al., 1996
