Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2687-2691, July 1977 Biochemistry A site-specific single-strand endonuclease from the eukaryote Chlamydomonas* (DNA/endodeoxyribonuclease/enzymatic cleavage) WILLIAM G. BURTONt§, RICHARD J. ROBERTS*, PHYLLIS A. MYERS*, AND RUTH SAGERt t Sidney Farber Cancer Institute and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115; and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Communicated by Arthur B. Pardee, March 16, 1977 ABSTRACT We have found a unique deoxyribonuclease the initial cleavage as well as subsequent development of the in extracts of the eukaryotic green alga Chlamydomonas. When gap may be due to the action of a single enzymatic moiety. incubated with viral DNA from adenovirus-2, this enzyme produces discrete fragments that form bands upon electropho- MATERIALS AND METHODS resis in an agarose gel. Site specificity of the enzymatic cleavage, examined by identifying the 5'-terminal nucleotides in cleaved The enzyme was obtained from vegetative cells of Chlamy- adenovirus-2 DNA and by studies with synthetic polynucleotides domonas reinhardi, strain 21 gr, mating type plus. Cells were of defined sequence, indicates that the initial endonucleolytic grown in liquid culture under continuous light on acetate- cleavage occurs at a site containing a deoxythymidine residue. Electron microscopy of cleaved adenovirus-2 DNA revealed supplemented minimal medium (11) at 250, harvested in late single-strand segments within duplex DNA. We propose that logarithmic phase of growth (6 to 7 X 106 cells per ml), washed the enzyme acts by making initial site-specific single-strand twice with ice-cold 10 mM 2-mercaptoethanol/10 mM Tris- incisions, followed by subsequent excision on the same strand, HCI (pH 7.9)i and stored as a pellet at -700. producing a gapped duplex molecule; and that double-strand Isolation of Endonuclease. Five to ten grams of frozen cells scissions result from limited occurrence of overlapping single- were thawed and resuspended in two volumes of cold extraction strand gaps on complementary strands. buffer (10 mM 2-mercaptothanol/10 mM Tris-HCl, pH 7.9). Specific endonucleases are important components of DNA All subsequent steps were at 20. The cells were disrupted by metabolism in both prokaryotes and eukaryotes. For example, sonication (12 times for 30 sec each) and the sonicated cells were endonucleases that detect structural distortions in a DNA helix centrifuged at 100,000 X g for 90 min. The supernatant was and remove mismatched bases have been implicated in DNA adjusted to 1 M NaCI, and was applied to a column of Sephadex repair (1) and gene conversion (2). In bacteria, modification- G-100 (100 X 2.5 cm) equilibrated with 1 M NaCl/10 mM 2- restriction systems consist of site-specific DNA methylases that mercaptoethanol/10 mM Tris-HCI (pH 7.9). Elution was with protect sites by methylation and the corresponding endonu- the same buffer; column fractions (4 ml) were assayed for en- cleases that recognize the same sites and cleave unmethylated donuclease activity as described below using 2 to 4-,l aliquots DNA (3). In eukaryotes, a wide range of biological processes, of column fractions, incubated for 5-16 hr. All fractions con- such as the selective silencing of particular DNAs (4) and certain taining endonuclease activity were combined, and solid am- aspects of differentiation (5), have been postulated to utilize monium sulfate was added to 75% saturation. The precipitate specific methylases and endonucleases in restriction-modifi- was recovered by centrifugation, resuspended in 25-30 ml of cation processes analogous to those of bacteria, but no such DEAE-buffer (10 mM 2-mercaptoethanol/0.1 mM EDTA/10 enzymes have been described. mM potassium phosphate (pH 7.4)/10% glycerol), and dialyzed In the sexual alga Chlamydomonas, inheritance of the against this buffer (three changes of buffer, 2 liters each). The chloroplast genome is non-Mendelian (maternal) as a result of sample was then applied to a column of DEAE-cellulose the selective degradation of the chloroplast DNA of paternal (Whatman DE 52,25 X 1.2 cm) that had been equilibrated with origin that occurs in zygotes (6, 7); we have postulated that the DEAE-buffer. The column was washed with 25 ml of DEAE- molecular mechanism of this degradation is a modification- buffer and was eluted with 200 ml of a linear KCI gradient restriction process (6, 7). In vegetative cells, the chloroplast (0-0.2 M) in DEAE-buffer. If required (see Results), fractions genes undergo nonreciprocal recombination events at a high of the KCI eluate that contained endonuclease activity were frequency (8, 9), suggesting that the molecular events may further purified by a second cycle of DEAE-cellulose chro- involve specific nucleases of the kind described in fungi (2, 10). matography. Thus, the genetic evidence, suggesting that the chloroplast DNA Enzyme Assays. Assay mixtures for the Chlamydomonas of Chlamydomonas is acted upon by an array of site-specific endonuclease contained the following in a volume of 0.05 ml: endonucleases, led us to examine the endonucleolytic activities 6 mM MgCl2/6 mM 2-mercaptoethanol/6 mM Tris-HCl (pH of this organism. 7.9) (standard assay buffer), and 2 ,ug of DNA substrate. After We report here the discovery and preliminary character- incubation at 370 for times indicated in the figure legends, the ization of site-specific endonucleolytic activity in a eukaryote. reaction was terminated by the addition of 5 ,l of 0.1 M This enzymatic activity, in extracts of Chlamydomonas, pro- Na2EDTA (pH 7). Nonradioactive digestion products were duces single-strand gaps starting at specific sites in homoduplex analyzed by agarose slab gel electrophoresis (12). The digestion DNA. The ability of this nuclease is unique in that products of 32P-labeled synthetic polynucleotides were analyzed gap-forming by (a) measurement of perchloric acid solubility, (b) high- voltage electrophoresis on DEAE-cellulose paper (13), and (c) Abbreviation: Ad-2, adenovirus-2. two-dimensional fractionation by electrophoresis on cellulose * Presented in part at the 67th Annual Meeting of the American Society acetate and homochromatography (14). of Biological Chemists in San Francisco, CA., June 6-10, 1976. Substrates. Adenovirus-2 (Ad-2) DNA was isolated as de- § To whom reprint requests should be addressed. scribed (15). Polydeoxyribonucleotides were purchased from 2687 Downloaded by guest on September 29, 2021 2688 Biochemistry: Burton et al. Proc. Natl. Acad. Sci. USA 74 (1977) KCO molarity x 102 0 5 Ia Table 1. Purification of Chlamydomonas endonuclease Fraction no. 8 16 24 32 40 48 56 C.. 7,' 4 12 20 28 36 44 52 6i0" C5 Specific A activity,t Fraction Protein, Endpoint units/ Total (ml) mg/ml titer* mg protein units Sonication (31) 23.8 High-speed supernate (21) 13.5 Sephadex G-100 eluate (138) 1.51 5-2-6t 22 6.9 X 103 Eluate of first DEAE-cellulose column (104) 0.075 5-2-6 4.44 X 102 3.47 X 103 Eluate of second DEAE-cellulose column (25.6) 0.0044 5-2-6 7.58 X 103 8.53 X 102 * The sequence of numbers under this heading refers to gl ofenzyme, Mg of DNA, and hours of incubation, respectively, required to obtain a limit digest (see Results). t With exonuclease-free enzyme fractions, a linear reciprocal rela- tionship was observed between volume of enzyme and length of incubation. The same degree of digestion was obtained when 1 Ml of enzyme was incubated for 5 hr or 5 Al ofenzyme for 1 hr. One unit FIG. 1. Assay of DEAE-cellulose column eluates. An aliquot from of activity: 1 Al of enzyme degrades 2 Mg of Ad-2 DNA to a limit di- each of the indicated column fractions was assayed as described in gest (see Results) in 1 hr at 37°. Materials and Methods. Fraction numbers are indicated above each In the qualitative agarose gel assay, contaminating exonuclease(s) gel channel. For both columns, after the sample was loaded, the col- degrade discrete DNA fragments produced by the endonuclease. umns were washed with loading (sample) buffer (fractions 6-20) and Thus, the endpoint titers assigned to eluates from the Sephadex and the gradient was started while fraction 21 was being collected. (A) first DEAE-cellulose columns are approximate. First DEAE-cellulose column. (B) Second DEAE-cellulose column. was bound resulted in the co-elution of specific and nonspecific PL Biochemicals, Inc., and were labeled in vitro by nick- nucleases upon application of the KCI gradient. In some enzyme translation (16). preparations, prolonged incubation of aliquots from fractions 5'-Terminal Nucleotide Analysis of Cleaved Ad-2 DNA. eluted by KCI indicated the presence of contaminating exo- Ad-2 DNA (20 ,tg) was digested with Chiamydomonas endo- nuclease(s). These preparations were further purified by re- nuclease. Digested DNA was recovered from the reaction chromatography of the initial KCI eluate on DEAE-cellulose. mixture either by phenol extraction and ethanol precipitation In enzyme preparations that required the additional cycle of or after electrophoresis on a 1.4% agarose gel (12) to separate DEAE-cellulose chromatography, it was the endonuclease small, single-strand excision products from cleaved and gapped activity in the flow-through, rather than the KC1 eluate, that DNA. The digested DNA was then treated with bacterial al- appeared to be exonuclease free (Fig. 1B). The purification kaline phosphatase (obligatory step) prior to labeling the 5' scheme is summarized in Table 1. The experiments presented termini using polynucleotide kinase (17). The 5'-terminal nu- here used exonuclease-free fractions from either the first or the cleotides were then analyzed after complete digestion with second DEAE-cellulose chromatography steps.