A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence
A structure-specific endonuclease from cauliflower ( Brassica oleracea var. botrytis) inflorescenceSeisuke Kimura, Mihoko Kai, Hiroyuki Kobayashi1, Atsushi Suzuki1, Hiroshi Morioka1, Eiko Otsuka1 and Kengo Sakaguchi*
Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba-ken 278, Japan and 1Faculty of Pharmaceutical Science, Hokkaido University, Sapporo, Japan
Received August 26, 1997;Revised and Accepted October 20, 1997
ABSTRACT
A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower (Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5' -> 3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.
DNA replication, recombination and repair are key processes to maintain integrity of the genome. We are interested in proteins that are associated with these processes in higher plants, basidiomycetes and Drosophila (1 -10 ). Repair of some types of DNA double-strand breaks is thought to proceed through flap DNA structure intermediates and, consequently, repair requires a DNA structure-specific endonuclease to identify and cleave the flap structure (11 ). Structure-specific endonucleases perform structure-specific cleavage at the junction between the 5' unannealed tail and that of a primer annealed to a template and play a important role in these processes (11 ,12 ). Several different branched DNA structures, such as the flap structure, have been prepared and structure-specific nucleases such as mammalian flap endonuclease-1 (FEN-1) have been found (11 ). FEN-1 is essential for maturation of Okazaki fragments in DNA replication (13 ,14 ) and recognizes a specific 5'-flap structure. Homologs of FEN-1 have been identified in budding (15 ,16 ) and fission yeast (17 ). The yeast genetic studies provide evidence that the FEN-1 homologs are involved in DNA replication, repair and, possibly, recombination. FEN-1 is a homolog of a Saccharomyces cerevisiae gene, RAD2. XPG-RAD2 are essential for the incision steps of nucleotide excision repair (NER) of UV-damaged DNA (12 ,18 -20 ). We recently found a structure-specific endonuclease that recognizes a UV-induced (6-4) photoproduct lesion in DNA molecules in Drosophila (7 , Kai et al., in preparation). It was also thought to have an important role in the (6-4) photoproduct repair process. Deoxyinosine 3'-endonuclease, an Escherichia coli repair enzyme that recognizes and cleaves DNA containing deoxyinosine and base mismatches, could also cleave the 5'-single-stranded tails of flap and pseudo-Y structures (21 ). These results indicate a variety of structure-specific endonucleases, each of which recognizes one or more damaged specific DNA structures. Each of the newly found structure-specific endonucleases could, therefore, be a key to study unknown repair reactions in which damaged DNA structures are different.
We have extensively screened the structure-specific endonucleases in the branches of the evolutionary tree. Our primary concern in this study was to identify a structure-specific endonuclease in higher plants. Although plants are constantly bombarded by UV light (22 ), little is known about plant DNA structure-specific endonucleases involved in the UV lesion repair process. We chose cauliflower inflorescence as a study material because it is a promising biochemical material in higher plants. Cauliflower inflorescence can be easily collected year round and exhibits hypertrophic differentiation with continuous growth by mitotic division (23 ). Secondly, the apical portion of the inflorescence consists of small cells and has a much higher DNA content than the underlying axial tissues (24 ,25 ). Finally, the cauliflower has a close relationship to Arabidopsis thaliana, which has become a model for plant molecular genetics (26 ).
In the present study we found a structure-specific endonuclease in cauliflower inflorescence. The cauliflower structure-specific endonuclease, like FEN-1 and XPG-RAD2, cleaves the single-stranded tail in 5'-flap structures. Saccharomyces cerevisiae RAD1-RAD10, which are respectively homologs of XPF-ERCC1, are also thought to be a structure-specific endonuclease. RAD1-RAD10 or XPF-ERCC1 form a heterodimeric complex having structure-specific endonuclease activity with a polarity opposite to XPG-RAD2 (27 ). The polarity of this cauliflower structure-specific endonuclease suggests that it is in the FEN-1 family. We describe herein our findings and the first extensive purification of a plant structure-specific endonuclease.
The Bradford reagents and molecular weight markers for SDS-PAGE were purchased from BioRad. AF-Blue Toyopearl 650ML was purchased from Tosoh. Sephadex G-25, Mono Q HR 5/5, Mono S HR 5/5, HiTrap desalting column and Superose 6 were from Pharmacia. Double-stranded DNA-Sepharose was made in our laboratory according to the instruction manual for HiTrap affinity columns (Pharmacia). DE52 was from Whatman Biosystem Ltd. Centricon-10 was purchased from Amicon. [[gamma]-32P]ATP and [[alpha]-32P]ddATP (3000 Ci/mmol) were purchased from Amersham. T4 polynucleotide kinase (PNK), calf thymus terminal deoxynucleotidyl transferase (TdT) and bacterial alkaline phosphatase (BAP) were obtained from TaKaRa. All other reagents were from Sigma or Wako.
The substrates used in this study were prepared according to the methods described by Harrington et al. (28 ) with minor changes. Radiolabeled oligonucleotide C was annealed to oligonucleotides A and B to generate the 5'-flap structure. Radiolabeled oligonucleotide C was annealed to oligonucleotide B to generate the 5'-pseudo-Y structure. Radiolabeled oligonucleotide E was annealed to oligonucleotides D and F to generate the 3'-flap structure. Radiolabeled oligonucleotide E was annealed to oligonucleotide F to generate the 3'-pseudo-Y structure. To make the stem-loop structure oligonucleotide H was labeled and annealed. Radiolabeled oligonucleotide F was used as single-stranded DNA. Radiolabeled oligonucleotide F was annealed to oligonucleotide G to produce double-stranded DNA. Radiolabeled oligonucleotide I or J was annealed to oligonucleotides A and B to generate the 5'-flap structure with a 5 or 1 nt strand. The annealing reactions were carried out by mixing the oligonucleotides in an annealing buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl), followed by incubation at 95°C for 2.5 min, then slow cooling to room temperature.
The structure-specific endonuclease activity was measured in a 20 µl reaction containing 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM DTT and 0.5 pmol 32P-labeled DNA substrate (10 000-50 000 c.p.m.). After incubation at 37°C for 60 min the reaction was stopped by adding 10 µl gel loading buffer (90% formamide, 5 mM EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol) and heated at 95°C for 5 min. Then the reaction was loaded onto a 20% polyacrylamide gel containing 7 M urea in TBE buffer and electrophoresis was performed. The reaction products were visualized by autoradiography or quantified using a BAS2000 Bio imaging analyzer (Fuji). One unit of activity was defined as the amount of enzyme required to cleave 1 pmol 5'-flap DNA substrate in 30 min under standard structure-specific endonuclease assay conditions.
Buffer A contained 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM 2-mercaptoethanol and 15% glycerol. Buffer B was identical to buffer A except that 0.01% NP-40 was added. Buffer C was identical to buffer B except that the glycerol concentration was decreased to 10%.
Purification of the cauliflower structure-specific endonuclease was performed at 4°C by the following operations. Unless stated otherwise all buffers contained three different types of protease inhibitor: 1 mM PMSF and 1 mg/ml each leupeptin and pepstatin.
Approximately 30 g fresh cauliflower inflorescence tissues were suspended in 20 ml buffer A containing 0.4 M KCl after being ground with a mortar and pestle. The suspension was fully disrupted with a French press and sonication (20 kHz, 3 × 20 s), then centrifuged at 30 000 g for 15 min. The supernatant was desalted through Sephadex G-25 (Fraction I). Fraction I was loaded onto a DEAE-cellulose column equilibrated with buffer A. The column was washed with a sufficient amount of buffer A and fractions were collected with a 200 ml linear gradient of 0-0.5 M KCl in the same buffer. The structure-specific endonuclease activity was eluted as a broad peak centered at 0.2 M KCl. The active fraction was pooled and dialyzed against buffer B (Fraction II). Fraction II was loaded onto an AF-Blue Toyopearl column equilibrated with buffer B. The column was washed with 100 ml buffer B, then eluted using 130 ml of a 0-0.5 M KCl gradient in buffer B. The structure-specific endonuclease activity was eluted at 0.2 M KCl. The active fraction was pooled and dialyzed against buffer C (Fraction III). Fraction III was loaded on a Mono Q HR 5/5 column equilibrated with buffer C and then the column was washed with 10 ml buffer C. Fractions were collected with 30 ml of a 0-0.4 M NaCl gradient in buffer C. The active fraction eluted at 0.1 M NaCl was collected and desalted on a HiTrap desalting column equilibrated with buffer C (Fraction IV). Fraction IV was loaded on a Mono S HR 5/5 column equilibrated with buffer C. The column was washed with 10 ml buffer C, then eluted with a 20 ml linear gradient of 0-0.5 M NaCl in the same buffer. The structure-specific endonuclease activity was eluted at 0.25 M NaCl as a tight peak. The active fraction was collected and desalted on a HiTrap desalting column equilibrated with buffer C (Fraction V). A final purification step was achieved using dsDNA-Sepharose column chromatography and the structure-specific endonuclease activity was eluted at 0.2 M NaCl in 10 ml of a 0-0.5 M NaCl gradient in buffer C. The combined active fraction was dialyzed against buffer C (Fraction IV) and used in the subsequent experiments.
SDS-PAGE was performed with a 10% gel using the standard Laemmli methods (29 ). After electrophoresis the gel was silver stained as described by Wray et al. (30 ). The protein concentration was determined using the BioRad protein assay with [gamma]-globulin as the standard.
The structure-specific endonuclease was purified from cauliflower inflorescence as described in Materials and Methods. FEN-1 was found using the 5'-flap structure as substrate (11 ). We also used it in the first place to assay the column fractions. Purification was by column chromatography using DEAE-cellulose, AF-Blue Toyopearl, Mono Q HR 5/5, Mono S HR 5/5 and finally a dsDNA-Sepharose (HiTrap affinity column) column. The chromatographic behavior of the cauliflower structure-specific endonuclease was monitored using the 5'-flap structure (Fig. 1 ) as substrate under standard assay conditions. The results of purification are summarized in Table 1 .The structure-specific endonuclease activity before Mono Q column chromatography could not be measured precisely.
The active fractions from the dsDNA-Sepharose column were combined, temporarily designated Fraction VI and then analyzed by SDS-PAGE. As shown in Figure 2 A, Fraction VI was found to be composed of a single polypeptide with a molecular mass of 40 kDa and the enzyme was sufficiently pure as judged by the results of SDS-PAGE analysis. To determine the native molecular mass of the structure-specific endonuclease Fraction VI was concentrated using Centricon-10 and then loaded onto a Superose 6 gel filtration column for FPLC. The cauliflower structure-specific endonuclease was eluted at a position representing a calculated molecular mass of 40 kDa (Fig. 2 B), indicating that the enzyme is a 40 kDa monopolypeptide.
In accordance with the methods described by Harrington et al. (11 ), we hybridized 5'-32P-labeled oligonucleotide C or 3'-32P-labeled oligonucleotide C to the non-radiolabeled oligonucleotides A and B (Figs 1 and 3 A). This structure, called the 5'-flap structure, contains 19 nt of the 5' overhanging single-stranded sequence (Fig. 1 ). We similarly hybridized 5'- or 3'-radiolabeled oligonucleotide E to non-radiolabeled oligonucleotides D and F to form the 3'-flap structure, which contains a 3' overhanging single-stranded sequence (Fig. 1 ). The 5'-flap structure terminates with a 5' single-stranded end and, conversely, the 3'-flap structure has a flap strand that terminates with a 3' single-stranded end.
The 5'-flap structure-dependent biochemical properties of the cauliflower structure-specific endonuclease are shown in Table 2 . Mg2+ ions were quite important for the reaction, but not essential (Table 2 ). Compared with the complete reaction, activity without Mg2+ ions was reduced to 30%. Ca2+ ions were unable to support activity, but Mn2+ ions could support moderate activity. The cleavage pattern in the presence of Mn2+ ionswas the same as that in the presence of Mg2+ ions (data not shown). In the presence of 1 mM ATP (lanes 6 and 12 in Fig. 3 ) the structure-specific endonuclease activity of the enzyme was reduced to 85% compared with the complete reaction (Table 2 ). Since ATP can chelate divalent metal ions, activity might be reduced. The enzyme is thus ATP independent.
As shown in Figure 4 A, the cauliflower structure-specific endonuclease could not cleave single-stranded DNA, indicating that it has no detectable single-stranded DNA-dependent endonuclease or exonuclease activity. However, the cauliflower structure-specific endonuclease when incubated with double-stranded DNA produced a monomer band of the 32P-labeled 5'-end nucleotide (Fig. 4 B). This indicates that the cauliflower structure-specific endonuclease has 5' -> 3' double-stranded DNA-specific exonuclease activity and this function is also known in FEN-1 (11 ).
XPG-RAD2 and FEN-1 efficiently cleave the 5' overhanging single-strand tail in the flap structures at and around the single strand-duplex junction, but FEN-1 cannot cleave 5'- or 3'-pseudo-Y structures (see Fig. 1 ). XPG-RAD2 has been shown to recognize and cleave such a pseudo-Y structure. To test this we also made pseudo-Y DNA structures. When the 5'-32P-radiolabeled oligonucleotide C was hybridized to the partially homologous non-radiolabeled oligonucleotide B it gave the 5'-pseudo-Y structure (Fig. 1 ). The 5'-pseudo-Y structure has a 5' overhanging single-stranded tail in its structure. In addition to the 5'-pseudo-Y structure we hybridized the 5'-radiolabeled oligonucleotide E to the non-labeled oligonucleotide F to form the 3'-pseudo-Y structure, which contains a 3' overhanging single-stranded tail (Fig. 1 ). The two pseudo-Y structures were incubated with the cauliflower structure-specific endonuclease under the reaction conditions employed for cleavage of the flap structures. As shown in Figure 5 , the cauliflower structure-specific endonuclease was able to cleave the 5'-pseudo-Y structure (lanes 1-3 in Fig. 5 ), although not efficiently. The enzyme could not cleave the 3'-flap or 3'-pseudo-Y structures (lanes 4-9 in Fig. 5 ). Using the 3'-pseudo-Y structure no detectable cleavage of the 3' overhanging single-strand was observed, even in the presence of excess cauliflower structure-specific endonuclease (data not shown). Thus the inability of the cauliflower structure-specific endonuclease to cleave the 3'-flap and 3'-pseudo-Y structures further confirms its specificity for the 5'-flap structure.
To determine the cut ends of the oligonucleotide generated by the cauliflower structure-specific endonuclease we analyzed it by the following methods (Fig. 6 A). The non-labeled 5'-flap structure, which is composed of oligonucleotides A-C, was incubated with the enzyme under standard conditions. Oligonucleotide C (33 nt) could be separated into two fragments, 19mer of the 5'-end side and 14mer of the 3'-end side, because the cleavage site is at the junction of the single-stranded and duplex DNA. Next, the mixture of oligonucleotides A-C after cleavage was incubated with or without bacterial alkaline phosphatase (BAP), which can remove the phosphate from both the 5'- and 3'-end of the the DNA strand. Then the BAP was removed by phenol extraction. The 3'-ends of the oligonucleotides were then labeled with [[alpha]-32P]ddATP and calf thymus terminal deoxytransferase or the 5'-ends of the oligonucleotides were labeled with [[gamma]-32P]ATP and T4 polynucleotide kinase. The products were analyzed by 20% PAGE and autoradiography. The 3'-end-labeled 19mer and the 5'-end-labeled 14mer could be visualized (Fig. 6 B). If the phosphate is not present at the termini of the oligonucleotides only the termini will be labeled by 32P. As shown in Figure 6 B, the 19mer was labeled by 32P with or without BAP treatment, suggesting that the 3'-end generated by the enzyme has no phosphate. In contrast, the 14mer was labeled only when the BAP treatment was performed, suggesting that the 5'-end generated by the enzyme has a phosphate. These results indicate that the cauliflower structure-specific endonuclease leaves 5'-PO4 and 3'-OH on the 5'-flap DNA.
Based on the data of this study, the cleavage sites on various substrates of the cauliflower structure-specific endonuclease are summarized in Figure 7 . The cauliflower structure-specific endonuclease could not cleave either the 3'-flap or 3'-pseudo-Y structures but could cleave the 5'-flap and 5'-pseudo-Y structures, indicating that the cauliflower structure-specific endonuclease acts on the 5' overhanging single-stranded tail in both the 5'-flap and 5'-pseudo-Y structures but does not act on the 3' overhanging region in the 3'-flap or 3'-pseudo-Y structures. The cauliflower structure-specific endonuclease can also cleave the stem-loop structure on the 3'-side of the junction of unpaired and duplex DNA. The cut ends of the oligonucleotides generated by the cauliflower structure-specific endonuclease are the 5'-PO4 and 3'-OH termini.
This study represents the first report and the first extensive purification of a structure-specific endonuclease in higher plants. Although it has not often been pointed out, higher plants like the vegetable used here are bombarded by severe sunlight containing UV for much longer periods than are animals or yeast. They cannot avoid this bombardment or escape from it. This plant enzyme may therefore help to elucidate the biochemistry of the DNA repair system for UV-damaged DNA. Using the purified cauliflower structure-specific endonuclease we have tried to clone the gene and expect to do so soon, along with monoclonal antibodies. In this report we describe the purification and the structure-specific properties of this new plant structure-specific endonuclease as a first step in a series of plant UV lesion repair studies.
We thank Mr Hirokazu Seto of our Department for his technical advice concerning cauliflower cultivation. This work was supported in part by a Grant-in-Aid (no. 362-0163-08277223) from the Ministry of Education, Science and Culture (Japan).
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