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Preliminary crystallographic study of pyrimidine dimer-specific excision-repair enzyme from bacteriophage T4
J. Nol. Biol. (1988) 202, 683-684
Preliminary Crystallographic Study of Pyrimidine Dimer-specific Excision-Repair Enzyme from Bacteriophage T4 Bacteriophage T4 endonuclease V, which is an excision-repair enzyme specific to pyrimidine dimers within DNA, has been crystallized from polyethylene glycol 4000 solution by a vapour diffusion technique. The unit cell is monoclinic, space group P2,, with unit cell parameters: a=41+4 A, b=40*1 A, c=37*5 A, /?=90*01’. The unit cell contains two 16,000 M, molecules. The crystals diffract X-rays beyond 2.3 A resolution and are suitable for structural analysis at high resolution.
Irradiation with ultraviolet light causes the formation of pyrimidine dimers in DNA, which is lethal and mutagenic to the organism. The excision of the pyrimidine dimer involves a major repair mechanism which is, in general, inherent in all organisms. Bacteriophage T4 endonuclease V (T4 end V)?, which is encoded by the denV gene of bacteriophage T4, is responsible for the excision of pyrimidine dimers from DNA in bacteriophage T4infected Escherichia coli (Yasuda & Sekiguchi, 1970). In spite of it being a rather small protein consisting of 138 amino acid residues, this enzyme has two distinct catalytic activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic endonuclease (Nakabeppu & Sekiguchi, 1981). In order to elucidate its catalytic properties in terms of the tertiary structure, it is essential to determine the three-dimensional structure directly by X-ray structural analysis. Conveniently, a synthetic T4 denV gene has been expressed successfully in E. u&i, and this has enhanced the production of the T4 endV enzyme (Inaoka et al., 1986). We have been able to prepare an amount of protein sufficient for X-ray crystallographic analysis. Here, we describe the successful crystallization of T4 endV protein and its preliminary X-ray diffraction study. T4 endV was purified from E. coli mainly through phase partition and CM-Sephadex chromatography, as described (Nakabeppu & Sekiguchi, 1981). Various crystallizing conditions were surveyed with respect to salts, pH and precipitants using a vapour diffusion technique. The T4 endV protein is considerably unstable in acidic solution, at high temperatures, or in organic solvents such as 2-methylpentane-2,4-diol. The most suitable precipitant appears to be polyethylene glycol (PEG) 4000. The concentration of various salts and the pH were investigated systematically in order to refine the conditions for growing suitable crystals. The best crystals were drop contained grown when the crystallizing
10 mg protein/ml, 50 m&r-KCl, 8 mM-sodium cacodylate * HCl (pH 45) and 5% (w/v) PEG 4000. The equilibrating solution was initially 15% PEG at 4”C, the PEG concentration of the reservoir gradually increasing to 25%. The first crystals appeared in two or three weeks as thin plates with dimensions of O-7 mm x 0.3 mm x O-5 mm. These crystals were sensitive to slight changes of pH and salt concentrations, and seemed to be unsuitable for X-ray diffraction study. They were converted, however, after several weeks to a second crystal form, consisting of thick plates with stronger birefringence. This type of crystal was more stable and easier to handle for diffraction experiments. These crystals, in general, grew very slowly and finally reached a size of O-5 mm in length with sides of O-5 mm and more than 0.1 mm. The crystals were subjected to X-ray diffractuon study using a precession camera (Enraf-Nonius) operated with a rotating anode and a four-circle diffractometer (Enraf-Nonius, CAD4) in a sealed tube. The crystals belong to the P2, space group, with unit cell dimensions of a=41*4 A, b=40.1 A, c=37.5 A, /?=90*O1°. The unit cell volume (in A3/dalton: Matthews, 1968) is 1.95 assuming one molecule per unit. asymmetric Intensity data, to 2-2 A resolution, have been collected from two crystals on a CAD4 diffractometer with less than 2O”/b decay in the standard intensities. The crystals were reasonably stable to radiation. We thank Dr T. Higashi, Dr M. Matsushima, Dr T. Matsuzaki, 0. Matsumoto, M. Danno, K. Katayanagi & M. Miyagawa for their help in the X-ray diffraction experiments. Kosuke Morikawat Michiko Tsujimoto Mario Ikehara Protein Engineering Research Institute Kodenmacho-sako Building 12-5 Kodenmacho Nihonbashi Chuo-ku Tokyo 103, Japan
t Abbreviations used: T4 endV, bacteriophage T4 endonuclease V; PEG, polyethylene glycol. 9022-2836/88/150683-02
$03.00/O
$ Author to whom all correspondence
should be addressed.
683 0 1988 Academic Press Limited
K. Morikawa et al.
684
References
Tetsuya Inaoka Eiko Ohtsuka Faculty of Pharmaceutical Sciences Hokkaido University Kita-ku Sapporo 060, Japan
Inaoka, T., Miura, K. & Ohtsuka, E. (1986). Nucl. Acids symp. Series, 17, lOFhO8. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Nakabeppu, Y. & Sekiguchi, M. (1981). Proc. Nut. Acad.
Sci.. U.S.A. 78. 2742-2746. Received 14 January 16 March 1988
1988, and in revised form
Yasuda,‘S.
& Sekiguchi, M. (1970). Proc. Nat. Acad. Sci.,
U.S.A. 67, 1839-1845. Edited by R. Huber
Preliminary Crystallographic Study of Pyrimidine Dimer-specific Excision-Repair Enzyme from Bacteriophage T4 Bacteriophage T4 endonuclease V, which is an excision-repair enzyme specific to pyrimidine dimers within DNA, has been crystallized from polyethylene glycol 4000 solution by a vapour diffusion technique. The unit cell is monoclinic, space group P2,, with unit cell parameters: a=41+4 A, b=40*1 A, c=37*5 A, /?=90*01’. The unit cell contains two 16,000 M, molecules. The crystals diffract X-rays beyond 2.3 A resolution and are suitable for structural analysis at high resolution.
Irradiation with ultraviolet light causes the formation of pyrimidine dimers in DNA, which is lethal and mutagenic to the organism. The excision of the pyrimidine dimer involves a major repair mechanism which is, in general, inherent in all organisms. Bacteriophage T4 endonuclease V (T4 end V)?, which is encoded by the denV gene of bacteriophage T4, is responsible for the excision of pyrimidine dimers from DNA in bacteriophage T4infected Escherichia coli (Yasuda & Sekiguchi, 1970). In spite of it being a rather small protein consisting of 138 amino acid residues, this enzyme has two distinct catalytic activities, a pyrimidine dimer DNA glycosylase and an apurinic/apyrimidinic endonuclease (Nakabeppu & Sekiguchi, 1981). In order to elucidate its catalytic properties in terms of the tertiary structure, it is essential to determine the three-dimensional structure directly by X-ray structural analysis. Conveniently, a synthetic T4 denV gene has been expressed successfully in E. u&i, and this has enhanced the production of the T4 endV enzyme (Inaoka et al., 1986). We have been able to prepare an amount of protein sufficient for X-ray crystallographic analysis. Here, we describe the successful crystallization of T4 endV protein and its preliminary X-ray diffraction study. T4 endV was purified from E. coli mainly through phase partition and CM-Sephadex chromatography, as described (Nakabeppu & Sekiguchi, 1981). Various crystallizing conditions were surveyed with respect to salts, pH and precipitants using a vapour diffusion technique. The T4 endV protein is considerably unstable in acidic solution, at high temperatures, or in organic solvents such as 2-methylpentane-2,4-diol. The most suitable precipitant appears to be polyethylene glycol (PEG) 4000. The concentration of various salts and the pH were investigated systematically in order to refine the conditions for growing suitable crystals. The best crystals were drop contained grown when the crystallizing
10 mg protein/ml, 50 m&r-KCl, 8 mM-sodium cacodylate * HCl (pH 45) and 5% (w/v) PEG 4000. The equilibrating solution was initially 15% PEG at 4”C, the PEG concentration of the reservoir gradually increasing to 25%. The first crystals appeared in two or three weeks as thin plates with dimensions of O-7 mm x 0.3 mm x O-5 mm. These crystals were sensitive to slight changes of pH and salt concentrations, and seemed to be unsuitable for X-ray diffraction study. They were converted, however, after several weeks to a second crystal form, consisting of thick plates with stronger birefringence. This type of crystal was more stable and easier to handle for diffraction experiments. These crystals, in general, grew very slowly and finally reached a size of O-5 mm in length with sides of O-5 mm and more than 0.1 mm. The crystals were subjected to X-ray diffractuon study using a precession camera (Enraf-Nonius) operated with a rotating anode and a four-circle diffractometer (Enraf-Nonius, CAD4) in a sealed tube. The crystals belong to the P2, space group, with unit cell dimensions of a=41*4 A, b=40.1 A, c=37.5 A, /?=90*O1°. The unit cell volume (in A3/dalton: Matthews, 1968) is 1.95 assuming one molecule per unit. asymmetric Intensity data, to 2-2 A resolution, have been collected from two crystals on a CAD4 diffractometer with less than 2O”/b decay in the standard intensities. The crystals were reasonably stable to radiation. We thank Dr T. Higashi, Dr M. Matsushima, Dr T. Matsuzaki, 0. Matsumoto, M. Danno, K. Katayanagi & M. Miyagawa for their help in the X-ray diffraction experiments. Kosuke Morikawat Michiko Tsujimoto Mario Ikehara Protein Engineering Research Institute Kodenmacho-sako Building 12-5 Kodenmacho Nihonbashi Chuo-ku Tokyo 103, Japan
t Abbreviations used: T4 endV, bacteriophage T4 endonuclease V; PEG, polyethylene glycol. 9022-2836/88/150683-02
$03.00/O
$ Author to whom all correspondence
should be addressed.
683 0 1988 Academic Press Limited
K. Morikawa et al.
684
References
Tetsuya Inaoka Eiko Ohtsuka Faculty of Pharmaceutical Sciences Hokkaido University Kita-ku Sapporo 060, Japan
Inaoka, T., Miura, K. & Ohtsuka, E. (1986). Nucl. Acids symp. Series, 17, lOFhO8. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Nakabeppu, Y. & Sekiguchi, M. (1981). Proc. Nut. Acad.
Sci.. U.S.A. 78. 2742-2746. Received 14 January 16 March 1988
1988, and in revised form
Yasuda,‘S.
& Sekiguchi, M. (1970). Proc. Nat. Acad. Sci.,
U.S.A. 67, 1839-1845. Edited by R. Huber