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IL-3A virus infection of a Chlorella -like green alga induces a DNA restriction endonuclease with novel sequence specificity

IL-3A virus infection of a Chlorella -like green alga induces a DNA restriction endonuclease... Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 volume 15 Number 15 1987 Nucleic Acids Research IL-3A virus infection of a Chlorella-tike green alga induces a DNA restriction endonuclease with novel sequence specificity Yuannan Xia, Dwight E.Burbank, Lothar Uher1, Dietmar Rabussay1 and James L.Van Etten Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722 and 'Bethesda Research Laboratories, Molecular Biology Research and Development, Life Technologies, Inc., Gaithersburg, MD 20877, USA Received March 24, 1987; Revised and Accepted June 24, 1987 ABSTRACT A typ e I I restrictio n endonuclease, named fiviJI, was isolate d from a eukaryotic Chlorella-1ike green alga infected wit h the dsDNA containing virus IL-3A. £viJI is the first restrictio n endonuclease to recognize the sequence PuGCPy ; CviJI cleave s DNA between the G and C. Methylation of the cytosine in PuGCPy sequences prevents cleavage by CviJI. £viJI cleaved DNA int o smaller but defined fragments in the presence of ATP. This "star " activity was stimulated by dithiothreito l and/or S-adenosylmethionin e but did not occur under conditions which favor "star" activity of other restriction endonucleases. INTRODUCTION We have isolated and partiall y characterized 30 large polyhedral , dsDNA containing , plaque forming viruses which infect a unicellular , eukaryotic Chlorella-like green alga strai n NC64A (1 , 2, 3, **) . One unusual feature of these viruses , whose genomes are about 300 kbp in size and contain various amounts of methylate d bases [0.1 to 47% 5-methyldeoxycytosin e (m dC) and 0 t o 37% N -methyldeoxyadenosin e (m dA) (3 , 5)] , is that infection wit h at leas t some of them result s in the appearance of DNA methyltransferase s and type II DNA restrictio n endonucleases (6, 7 , 8). In fact the virus infected alga is the firs t source of typ e I I DNA restrictio n endonucleases from a eukaryotic system. The restrictio n endonucleases £viAI and £viBI appear after infectio n with the viruses PBCV-1 and NC-1A, respectively. £viAI , like the bacteria l restrictio n endonuclease Hbol recognize s the sequence GATC, cleaves DNA 5 ' to the G, and does no t cleave G ATC sequence s (7) . £viBI is similar to the bacteria l restriction endonuclease HinfI because i t recognizes th e sequence GANTC, cleaves DNA between the G and A, and does not cleav e G ANTC sequences (8). Thi s report describes a differen t restriction endonuclease, 11RL Press Limited, Oxford, England. 6075 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research named £viJI, which appears after infection of the alga with another of these viruses, IL-3A. £viJI is the first restriction endonuclease to recognize the sequence PuGCPy; cleavage occurs between the G and C. MATERIALS AND METHODS Growth and infection of the alga. The production and purification of IL-3A and the other viruses, and the growth of Chlorella strain NC64A on MBBM medium have been described (1, 3, 9) . Chlorella cells (1 to 2 x 10 cells/ml) were infected with IL-3A at a multiplicity of infection of 10 and cells were collected by centrifugation at 6 to 7 hr post infection and used immediately or frozen at -80 C. Enzyme extracts. Five to 10 x 10 infected cells were suspended in 15 ml of buffer A [0.01 M Tris-HCl (pH 7.9), 0.01 M 2-mercaptoethanol and 50 yg/ml phenylmethylsulfonyl fluoride] plus 10 gm of 0.3 mm glass beads and homogenized for 80 sec at 4000 rpm in a Bronwill MSK mechanical homogenizer with cooling from a CO tank. The homogenate was centrifuged at 10,000 X g for 20 min and protein was precipitated from the supernatant at 70% saturation with (NH ) SO . The precipitate was dissolved in buffer A and treated with dextran T500 and polyethylene glycol 6000 as described (7, 10). The resulting upper aqueous phase was adjusted to 20$ (v/v) glycerol and the sample was dialyzed overnight at 4 C against three changes of buffer B [20 mM. Tris-HCl (pH 7.5), 0.5 mM sodium EDTA, 7 mM 2-mercaptoethanol and 10% glycerol]. The dialysate was diluted 5 fold with buffer B and applied to a heparin sepharose (Pharmacia) column; the column was eluted with a 0 to 1.2 M gradient of NaCl in buffer B. £viJI activity was pooled and adjusted to 0.1 M NaCl with buffer C [10 mM KPO (pH 7.4), 0.5 mM EDTA, 7 mM 2-mercaptoethanol, 10$ glycerol]. The sample was applied to a phosphocellulose (Whatman P11) column and eluted with a 0.2 to 1.2 M gradient of KC1 in buffer C. Pooled CviJI activity was adjusted to 0.1 M KC1 with buffer C and applied to a hydroxylapatite (Bio-Gel HTP, Biorad) column. C_viJI was eluted with a 0.2 to 1.2 M KPO gradient; the pooled active fractions were dialyzed against buffer B containing 50 mM KC1 and 50$ glycerol and then stored at -20 C. Enzyme assays. Unless noted otherwise, CviJI was assayed at 37 C for 2 hr in 20 v1 reaction mixtures containing core buffer [50 mM Tris-HCl 6076 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research (pH 8.0) , 10 mM MgCl , 50 mM NaCl] , 1 ug substrate DNA and 1 t o 3 vl of enzyme extract . The reaction products were electrophoresed eithe r on 2% agaros e gels in 90 mM Tris-HCl , 90 mM bori c acid, 2 mM EDTA, pH 8. 0 (11) or on 7.5 to 15% linea r gradient polyacrylamid e gels in 40 mM Tris-acetate , pH 8.3 , 20 mM sodium acetate , 2 mM EDTA as described (7, 12). One uni t of enzyme is th e amount required to completely digest 1 vg o f unmethylated lambda DNA i n 1 hr at 37 C i n 20 vl of assay buffer. Determinatio n of restrictio n endonuclease cleavage sit e bv DNA sequencing . Dideox y chain terminatio n sequencing reaction s were conducte d on alkali-denature d plasmid pBR322 DNA preannealed with 5'-end-labelle d pBR322 Sai l site sequencing primer (New England Biolabs) . The 15 base sequence 5 ' -AGTCATGCCCCGCGC- 3 ' complementary to the pBR322 region at position 700 to 714 served as a prime r for sequencing the DNA i n a counterclockwise direction . The oligonucleotid e primer was labeled at the 5'-end by the method of McGraw (13) in 10 vl reactio n mixtures containing : 70 mM Tris-HC l (pH 7.6) , 10 mM MgCl , 120 uC (24 pmoDy - P ATP, 100 ng (20 pmol) of primer DNA and 5 unit s of T4 polynucleotid e kinase (Bethesda Research Labs). The mixtur e was incubate d at 37 C fo r 60 min and the reactio n stopped by adding 20 vl formamide dye mix (8). The sample was electrophoresed on a 25? polyacrylamide gel at 47 volts/cm (1800 volt s total ) and the ge l was autoradiographed for 2 min on Kodak XAR-5 film . The oligonucleotid e primer, identifie d by autoradiography, was cut ou t and purifie d over a NACS prepac column (Bethesda Research Labs) . The specific activit y of the DNA was about 10 cpm/pmol. The pBR322 DNA (5 vg) was denatured wit h alkal i at 30°C for 5 min in 50 Pi of 0.2 M NaOH and 0.2mM sodium EDTA (14) . The solutio n was neutralize d wit h 5 vl of 2 M ammonium acetate (pH 4.5 ) and the DNA was precipitate d with ethanol, washed, dried and suspended in 20 vl of water. Five ul of thi s solution was mixed wit h 10 ng of the 51- P-Sal1 sit e sequencing primer and the solutio n was adjusted to 12.5 mM Tris-HC l (pH 8) , 12.5 mM MgCl , and 37 mM NaCl in a tota l volume of 16 ul . The mixtur e was incubate d at 35 C fo r 1 hr , cooled on ice , and 2 ul of 0.1 M dithiothreito l and 2 unit s (2 1 ) of the large fragment of DNA polymerase I (Klenow fragment) were added. Dideoxynucleotid e sequencing reactions were carrie d out as describe d in the Bethesda Research Lab M13 Cloning and Sequencing 6077 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research Manual , 1986 edition . DNA primed synthesis products for restrictio n endonuclease digestions were prepared in the same way as the "A reaction" except that dideoxyATP was omitted . After th e chase reaction with unlabeled dATP, the mixture (mixture AA) comprisin g 14 yl was heated for 15 min at 65 C. Mixture AA was added to 2 y l of 10 x core buffer and incubated with 0.4 units CviJ I (tota l volume of 20 l) at 37 C fo r 15 min for partial digestion . The reaction was stopped wit h 40 yl of the formamide-dye mixture. The samples were electrophoresed on 8% polyacrylamide/ 7 M urea sequencing gels adjacent to the products o f a set of the dideoxynucleotide DNA sequencing reactions and treate d as described previously (8). Mis. Hos t nuclear and viru s DNAs were isolated as described previousl y (5). Unmethylated pBR322 plasmid DNA was propagated i n dam , d_c_m L, c_oii strai n GM2163 (15) as described (5). Plasmid pUC19 was propagated in L. col i strain JM83. SV40 DNA (Form I ) isolated from BSC-1 monkey cells , 0X174 DNA (Form I) , and adenovirus type 2 DNA isolate d from HeLa cell s were from Bethesda Research Labs. Other procedures. The DNAs were treated with methylases (New England Biolabs) or with restrictio n endonucleases as suggested by the suppliers. RESULTS Properties of IL-3A. A one step growth curve of IL-3A revealed that the virus was first released about 5 hr after infection and virus release was complete at about 10 to 11 hr after infection. Like infection by the virus PBCV-1 (16), IL-3A infection resulted in the degradation of the host nuclear DNA beginning between 1 and 2 hr after infection; IL-3A DNA synthesis also started at about 1 hr after infection (unpublished results). Restriction endonuclease activity. Enzyme extracts were prepared from uninfected Chlorella cells and cells at 7 hr after IL-3A infection and assayed for DNA restriction endonuclease activity. Enzyme activity (named CviJI) was detected in extracts from the virus infected cells but not from healthy cells. A single peak of restriction endonuclease activity appeared after chromatography on heparin sepharose, phosphocellulose , and hydroxylapati te columns. £viJI activity 6078 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nuclei c Acid s Research 123 4 5 678 9 1374- 6 65 - 358 - 258 - 207 - 105 - 75 - 46 - Figure 1. Ability of £viJi to digest unmethy1 a ted pBR322 plasmid DNA. Lane 1 contains untreated pBR322 DNA, lane 2 contains pBR322 DNA after a 2 hr incubation with £viJI, lane 3 is the same as lane 2 only the enzyme reaction was incubated for 16 hr, lane 4 is the same as lane 3 but incubated an additional ? hr with a second aliquot of CviJI, lane 5 is the same as lane 2 but MgCl_ was omitted from the assay, lane 6 is the same as lane 2 except that £viJI was heated to 65 C for 10 min prior to adding it to the DNA. £viJI was incubated in core buffer for 4 hr, 8 hr and 16 hr (lanes 7, 8, and 9, respectively) at 37°C before adding pBR322 DNA and incubated an additional 2 hr. eluted at 0.6 to 0.8 M NaCl from heparin sepharose, at 0.7 to 0.9 M KC1 from phosphocellulose, and at 0.5 to 0.7 M KPO from hydroxylapatite columns (data not shown). The purification steps increased the £viJI specific activity greater than 100-fold. The specific activity of the purified enzyme was estimated at about 10 units/mg of protein and about 2000 units of activity were obtained per liter (2 x 10 cells) of infected cells. As shown in Fig. 1 (lane 2) £viJI cleaved u nmeth y 1 a ted pBR322 DNA into many discrete fragments. The size of the DNA fragments did not change upon exhaustive incubation with £viJI 6079 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research Figure 2. Ability of £viJI to degrade various DNAs. (A) Untreated pBR322 DNA (lane 1), pBR322 DNA treated with IsflT, Mhsl, Hinfl, and CviJI (lanes 2 to 5, respectively). (B) Lane 1 contains pBR322 DNA digested with Mkfil as standards, lanes 2 to 6 contain 0X174, SV40, adenovirus, IL-3A, and host Chlorella nuclear DNAs, respectively, after digestion with CviJI. Lane 7 contains untreated host nuclear DNA. (Fig. 1, lanes 3 and 4) . Thus £viJI was essentially free of nonspecific nucleases and cleaved pBR322 DNA at discrete sites. CviJI required Mg (Fig. 1, lane 5) for activity. The enzyme was most active in 50 mM Tris-HCl (pH 8.0) , 10 mM MgCl , and 50 mM NaCl. The temperature optimum was 37 C. £viJI activity was measured after incubating the enzyme in the assay mixture at 37 C for various intervals prior to adding substrate DNA. The enzyme was still active after a 4 hr incubation but much less active after a 16 hr incubation (Fig. 1, lanes 7 to 9) . Heating CviJI to 65 C for 10 min reduced activity more than 95% (Fig. 1, lane 6) . £viJI can be stored at -20 C in buffer B containing 50 mM KC1 and 50 % glycerol for at least 1 year without loss of activity. Mapping of CviJI cleavage sites. The CviJI cleavage products of pBR322, 0X174 , SV40 and adenovirus DNAs were examined on 7.5 to 15% linea r gradient polyacrylamide gels (Fig. 2) . Plasmid pBR322 DNA was cleaved into at least 20 fragments; however, only 5 of these fragments 6080 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 i 4 5 6 Figur e 3. Effect of Hafilll methylase modificatio n of pUC19 DNA o n cleavag e by CviJI . Untreated pUC19 DNA (lan e 1), Haelll digeste d pUC19 DNA (lane 2) , £viJ I digested pUC19 DNA (lan e 3), Ha£lII/£viJ I digested pUC19 DNA (lan e 4) , Ha^II I digestio n of H_a_e.III methylas e treate d pUC19 DNA (lan e 5) , and £viJ I digestio n of Haell l methylase treate d pUC19 DNA (lane 6) . Note: (i ) the identica l sizes of the DNA fragment s in lanes 3 and 4; (ii ) the siz e of the "A" fragment of pUC19 DNA increase d after methylatio n wit h £La_e_III methylas e (compare lanes 3 and 6, arrowheads) . wer e large r than 140 bp (Fig . 2A, lane 5) . Comparing the size of thes e 5 large r pBR322 DNA fragment s wit h those produced by known restrictio n endonucleases suggested tha t C_viJI had a unique specificit y (17, 18). The number and size s o f the cleavage fragment s obtained by CviJ I digestio n of 0X174 and SV40 DNAs supporte d thi s conclusion (Fig . 2B). IL-3 A viru s DNA i s resistan t to bacteria l restriction endonuclease s tlindlll , Afia.1, Alul , Haell , and tLagll l (data not shown ) as wel l as CviJ I (Fig . 2B, lane 5) . The base sequence recognize d by each of th'e bacteria l enzymes contain s a GC sequence ; cytosin e methylatio n in thi s GC sequenc e prevents activit y of each of the enzymes (18 , 19). Therefore, we assumed tha t the recognitio n and cleavag e sit e of £viJ I contained GC. 6081 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 3 4 5 6 Figure 4. Effect of Alul methylase modification of pUC19 DNA on cleavage by CviJI. Untreated pl)C19 DNA (lane 1), Alul digested pUC19 DNA (lane 2) , CviJI digested pUC19 DNA (lane 3) , AiljI/£viJI digested pUC19 DNA (lane 4) , Alul digested Alul methylase treated pUC19 DNA (lane 5), and CviJI digested ALuI methylase treated pUC19 DNA (lane 6) . Note: (i) the identical sizes of the DNA fragments in lanes 3 and 1; (ii) the size of the CviJI "B" fragment of pUC19 DNA increased after methylation with Alul methylase (compare lanes 3 and 6, arrowheads). This assumption led to the successful identification of CviJI cleavage sites on pUC19 DNA. One of the £viJI cleavage sites was located by digesting pUC19 DNA with CviJI and/or iLaelll; tLa_gIII recognizes the sequence GGCC. As shown in Fig. 3, the CviJI "A" fragment (lane 3) and Cv iJI/Haelll "A" fragment (lane H) are identical in size to the 587 bp Haelll "A" fragment (lane 2) [located at bases 2088 to 2675 (20)]. Methylation of pUC19 DNA with M.tia£.III prior to CviJI digestion gave a 680 bp fragment in place of the 587 bp fragment (Fig. 3, compare lanes 3 and 6) . These results suggest that CviJI recognizes and cleaves GGCC sequences. Another CviJI cleavage site was determined by digesting pUC19 DNA with £viJI and AIM I . As shown in Fig. 4 (lanes 3 and .608.2 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 4), DNA digested with both enzymes or with £viJI alone produced identical fragments. DNA digested with either Alul or CviJI alone (lanes 2 and 3) produced some overlapping fragments. For example, there are regions of overlap between the 679 bp Alul "A" fragment (located at bases 2050 to 43) and £viJI "A" fragment (about 580 bp) and the 521 bp Alul "B" fragment (located at bases 1366 to 1887) and CviJI "B" fragment (about 370 bp). Methylation of the DNA with M.AluI followed by CviJI digestion gave a 400 bp fragment in place of a 370 bp fragment (Fig. 4, compare lanes 3 and 6) . This implied that M.AluI, which methylates cytosines in AGCT sequences, protects one end of the 370 bp £viJI "B" fragment from digestion by CviJI and we conclude that CviJI also cleaves the sequence AGCT. To determine the location of the AGCT site on the 370 bp CviJI "B" fragment the DNA was double digested with either CviJI/MboII or CviJI/Bgll. pUC19 DNA contains two MboII sites, located at bases 1456 and 1547, and one Bell site, located at base 1813. The £viJI "B" fragment contained 2 MboII sites and no Ball site (data not shown) and so the C_viJI "B" fragment is located between bases 1366 and 1740 and contains an AGCT site at base 1366. The ends of the 400 bp £viJI "B" fragment of M.AXuI methylated pUC19 DNA should then be near positions 1340 and 1740. These regions were examined and the two sequences AGCC at 1337 and GGCT at 1729 were identified. This allowed us to propose that CviJI recognized (A/G)GC(C/T) sequences. A computer search was conducted to determine the number and locations of (A/G)GC(C/T) sequences in several DNAs. The computer derived number of £viJI cleavage sites were 72 for PBR322 DNA, 69 for 0X174 DNA, 88 for SV40 DNA, and 692 for lambda DNA. Fragments of 587, 363, 258, and 199 bases were the four largest fragments predicted from the location of the C_viJI cleavage sites in pBR322 DNA. The predicted sizes of these fragments matched the experimental data (Fig. 2A, lane 5) . Likewise, the predicted sizes of pUC19 and 0X174 fragments agreed perfectly with the experimental determined sizes. Determination of CviJI cleavage site. The cleavage sites were confirmed by sequencing a stretch of pBR322 DNA and determining the position of the fragments generated by £viJI. Plasmid pBR322 DNA contains all four subsets of the sequence (A/G)GC(C/T) , i.e. AGCT, AGCC, GGCC, and GGCT, within 120 bp of the Sail site (at position 651). A 5'-end 6083 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research NACGT N NACQTN >o Fiaur e 5. £viJI cleavage site s of pBR322 DNA. A stretc h of pBR322 DNA was sequenced in counterclockwise direction by the dideoxy sequencing method using a 15-base 5'-end labeled pBR322 Sai l site sequencing primer annealed to linearized alkali-denature d pBR322 DNA at positio n 700 to 7 14 (lanes A,C,G,T) . 3 P-labeled pBR322 DNA prepared analogously to the sample in the "A" lane (except that dideoxy ATP was omitted) was partiall y digested with C_viJI (lanes N). In gel 1 the bromophenol blue marker migrated 30 cm; gel 2 i s the same as ge l 1 but bromophenol blue migrated 50 cm. The bases recognize d by C_viJI are indicated by the bars. The bands in lane s N which coincided with the G where cleavage occurs are indicate d by an arrowhead. labele d pBR322 Sai l site primer was extended through and beyond th e four suspected £viJ I sites . The extended oligonucleotides were partiall y digested with CviJI under conditions which yielded al l possible complete and partia l products. Four products with increasin g sizes co-migrated with bands in the G channel of the sequencing reactions (Fig. 5). Thus C_viJI cleaved DNA between G and C i n al l four sequences AGCT, GGCT, AGCC, and GGCC and create d restrictio n fragments with blunt ends. The intensit y of th e G band at the AGCC sit e was less than at the other 3 site s which may reflec t reduced activit y at this site . Two additional observation s indicate that CviJI produces fragments with blunt ends. (i) Conditions for ligatin g blunt-ended fragments were .6Q84 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 3 4 5 Figure 6. Effect of ATP on £viJI specificity. Lane 1 contains untreated pUC19 DNA, lane 2 is pUC19 DNA after a 2 hr incubation with CviJI under standard conditions, lane 3 is the same as lane 2 except that the assay mixture contained 2 raM ATP and 20 mM dithiothreitol, lane it is the same as lane 3 except that the reaction time was 16 hr, and lane 5 is the same as lane 4 but with an additional 2 hr incubation after adding a second aliquot of CviJI. required to ligate C_viJI restriction fragments (data not shown), (ii) No end filling was observed with £viJI restriction fragments on the sequencing gel (Fig. 5, lane N) . This indicates that CviJI creates blunt or 5' recessed ends (7). Thus, the CviJI recognition and cleavage site is (A/G)G+C(C/T). ATP influences specificity of CviJI. Blunt end ligation of £viJI restriction fragments, followed by recleavage with CviJI, produced more fragments than expected. This led to the surprising finding that the size of the C_viJI restriction fragments was affected by ATP. That is, C_viJI cleaved pUC19 DNA into smaller but defined fragments when 1 to 2 mM ATP was present in the assay buffer (Fig. 6, compare lanes 2 and 3) . The reaction was stimulated by reducing agents such as 6085 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 i 4 5 6 7 8 9 10 11 12 1J Figur e 7. Time course of pUC19 DNA digestio n by C.viJI under standar d and "sta r " conditions . Lane 1 contain s untreated pUC19 DNA, lanes 2 and 8, 3 and 9, 4 and 10, 5 and 11, 6 and 12, and 7 and 13 are DNAs afte r incubation with £viJI for 1, 5, 10, 30, 60, and 120 min, respectively. The DNAs in lanes 2 to 7 were incubated with .CviJI under standard conditions and in lane s 8 to 13 under "star" conditions (see legend of Fie. 6). The reactions were stopped by heating at 65 C for 10 min. dithiothreito l or 2-mercaptoethanol and/or S-adenosylmethionine. Adenosine-5'- 0 (1-thiotriphosphate ) (ATP-a-S), adenosine-5 '- 0 (3-thiotriphosphate ) (ATP-Y-S), dATP, and ADP substituted , albeit poorly , for ATP whereas, GTP, CTP, UTP, and non hydrolyzable analogue s adenylyl-imido-diphosphate (AMP-PNP) and adenylyl (g, y -methylene)-diphosphonat e (AMP-PCP) did not. Reactio n conditions influence substrate specificit y of severa l bacterial type II restriction endonucleases. Altered activitie s are referred to as "star (•) activity " (21, 22). By analog y the ATP-requiring specificity of CviJI is referred to as £viJI» . However, £viJI # activit y is not observed under condition s which favor "star" activity of other restriction endonucleases ; i.e . products of £viJI* activit y were not obtained by adding glycerol [up to 30$ (v/v) ] or NaCl (up to 0.15 M) to th e reaction mixture, varying the pH of the reaction mixture from ++ ++ ++ 6 to 9, or replacing Mg with Mn or Zn (data not shown). The optimum conditions for £viJI* activit y were the same as fo r £viJI except that 1 mM ATP, 20 mM dithiothreitol , and 100 yM S-adenosylmethionin e were added to the core buffer. £viJI# 808t Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research produced the same cleavage fragments if pUC19 DNA was incubated with the enzyme for 2 hr, 16 hr, or if the DNA was treated with a second aliquot of enzyme for 2 additional hrs after a 16 hr incubation (Fig. 6, lanes 3 to 5) . Thus, like CviJI, £viJI* cleaved pUC19 DNA at a limited number of sites. CviJI* does not cleave IL-3A DNA (data not shown); therefore, the presence of m dC in the sequence PuGCPy inhibits its activity. Several reasons suggest that £viJI and CviJI* activities result from the same protein. First, CviJI and CviJI* activities co-eluted from heparin sepharose, phosphoce11u 1 o s e , and hydroxylapatite columns. Second, comparison of the time courses of £viJI and CviJI* digestion indicates that £viJI* cleaves at the same sites as CviJI as well as at additional sites. As seen in Fig. 7, digestion of pUC19 DNA by £viJI was still incomplete after a 10 min incubation (lane 4) but was complete after 30 min (lane 5) . However, digestion under "star" conditions produced an almost complete £viJI cleavage pattern within 5 min of incubation (Fig. 7, lane 9) . With longer incubation times the pattern shifted towards the complete £viJI* pedigree (Fig. 7, lanes 10 to 13). If pUC19 DNA was digested first under standard £viJI conditions and then used as a substrate for £viJI», the final cleavage pattern was identical to that of pUC19 DNA cleaved directly by £viJI* (data not shown). These results indicate that CviJI and CviJI* share common cleavage sites and that £viJI cleavage sites are more susceptible to £viJI* cleavage than the £viJI* specific sites. DISCUSSION This report establishes that IL-3A infection of Chlorella strain NC64A induces the synthesis of a DNA restriction endonuclease, £viJI, which recognizes the sequence PuGCPy and cleaves between the G and C; £viJI does not cleave PuG CPy sequences. £viJI is the first DNA restriction endonuclease to recognize this site (17, 18). £viJI should cleave DNAs more frequently than any known restriction endonuclease since, it recognizes on a probability basis, the equivalent of a three base sequence. I1-3A is one of six viruses that we have grouped in class 4 which infect Chlorella NC64A (3). Enzyme extracts prepared from Chlorella cells infected with each of these viruses (i.e. CA-1A, CA-2A, I1-2A, IL-2B, and I1-3D) cleaved pBR322 DNA into the same 6087 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research siz e fragments as C_viJI and al l exhibited star activity under the condition s described above (unpublished data). In each case the restrictio n endonuclease cleaves host DNA i n vitr o but not th e viru s DNA. The function of these restriction endonucleases has not been establishe d unequivocally. However, circumstantial evidence suggest s that these enzymes are involved in the breakdown of host DNA and might thus facilitate virus replication (7, 8) . If this i s true , these viral enzymes would serve a functio n exactly opposit e of what is considered the main function of bacterial typ e II restrictio n endonucleases, namely the preventio n of successfu l infection by bacteria l viruses. In thi s sense the Chlorell a virus-induced restriction endonucleases would function as "facilitating " rather than "restriction" enzymes. Restrictio n endonucleases are classifie d into three types based on thei r cofactor requirements and other criteria . Type I enzymes, such as EcoK and EcoB have stringen t requirements for ATP, S-adenosylmethionine and Mg (23, 24, 25) . Type II enzymes such as EcoRI and HindIII , require only Mg for activit y (22) . Type II I enzymes, such as EcoPI and EcoP15. require only ATP and Mg for activity ; however, S-adenosylmethionine stimulates activit y (25, 26, 27) . In the absence of ATP, CviJI cleaves DNA a t al l PuGCPy site s and only requires Mg for activity . Thus, £viJ I functions as a typica l type II restrictio n endonuclease. However, £viJI obviously recognizes and cleave s a smaller base sequence when ATP i s included in the reactio n mixture. Assuming tha t CviJI and CviJI * activitie s are associate d with the same protein , the £viJI * activit y differs from "star" activities of othe r type II restrictio n endonucleases (22). First, CviJI1 activit y does not occur under conditions which favor "star" activit y of other restriction endonucleases, e.g. high enzyme concentration , addition of glycerol, variations of pH and ionic + + ++ strength , or replacement of Mg with Mn . Second, £viJI* activit y absolutely requires ATP; relate d compounds such as ADP, dATP, ATP-a-S, ATP-y-S induce some sta r activit y whereas, GTP, CTP, UTP, AMP-PNP, and AMP-PCP do not . £viJI« activit y is stimulate d by reducin g agents and S-adenosylmethionine ; these compound s by themselve s do no t induc e "star " activity . Therefore , £viJI* resembles a type II I restrictio n enzyme in ++ respect to its dependence on Mg and ATP and its stimulation by S-adenosylmethionine. 6088 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research The rol e of ATP in the £viJI « activit y is als o not clear. I t will be interestin g to determin e if ATP change s the conformatio n of £viJ I as i t does with type I and typ e III restrictio n endonucleases and if CviJI has ATPase activit y like Type I restrictio n endonucleases. The successful purification of CviJ I wil l help to answer these questions. ACKNOWLEDGMENTS We thank Robert Blakesley, Russel Meints, Les Lane, and Myron Brakke for many helpful discussions. This investigation was supported, in part, by Public Health Service grant GM-32441 from the National Institute of General Medicine and grant DE-ACO2-82ER12086 from the Department of Energy. Published with the approval of the Director as paper no. 8247, Journal Series, Nebraska Agricultural Research Division. REFERENCES 1. Van Etten, J.L., Burbank, D.E., Schuster, A.M. and Meints, R.H. (1985) Virology 140, 135-143. 2. Meints, R.H., Schuster, A.M. and Van Etten, J.L. (1985) Plant Mol. Biol. Rept. 3, 180-187. 3. Schuster, A.M., Burbank, D.E., Meister, B., Skrdla, M.P., Meints, R.H., Hattman, S., Swinton, D. and Van Etten, J.L. (1986) Virology 150, 170-177. 4. Van Etten, J.L., Xia, Y. and Meints, R.H. (1987) in Plant-Microbe Interaction, Kosuge, T. and Nester, E.W. Eds., Vol II. pp. 307-325, Macmillan Publishing Co., New York. 5. Van Etten, J.L., Schuster, A.M., Girton, L., Burbank, D.E., Swinton, D. and Hattman, S. (1985) Nucl. Acid. Res. 13, 3471-3478. 6. Xia, Y. and Van Etten, J.L. (1986) Mol. Cell Biol. 6, 1440-1445. 7. Xia, Y., Burbank, D.E., Uher, L., Rabussay, D. and Van Etten, J.L. (1986) Mol. Cell. Biol. 6, 1430-1439. 8. Xia, Y., Burbank, D.E. and Van Etten, J.L. (1986) Nucl. Acid. Res. 14, 6017-6030. 9. Van Etten, J.L., Burbank, D.E., Xia, Y. and Meints, R.H. (1983) Virology 126, 117-125. 10. Schleif, R. (1980) Methods in Enzymology, Grossman, L. and Moldave, K. Eds., Vol. 65, pp. 19-23, Academic Press, New York. 11. Peacock, A.C. and Dingman, C.W. (1967) Biochemistry 6, 1818-1827. 12. Jeppesen, P.G.N. (1980) Methods in Enzymology, Grossman, L. and Moldave, K. Eds., Vol. 65, pp. 305-319, Academic Press, New York. 13. McGraw, R.A. (1984) Anal. Biochem. 143, 298-303. 14. Chen, E.Y. and Seeburg, P.H. (1985) DNA 4, 165-170. 15. Marinus, M.G., Carraway, M., Frey, A.Z., Brown, L. and Arraj, J.A. (1983) Mol. Gen. Genet. 192, 288-289. 6089 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 16. Van Etten, J.L., Burbank, D.E., Joshi, J. and Meints, R.H. (1981) Virology 134, 443-419. 17. Roberts, R.J. (1985) Nucl. Acid. Res. 13, r165-r200. ' 18. Kessler, C , Neumaier, T.S. and Wolf, W. (1985) Gene 33, * 1-102. 19. McClelland, M. and Nelson, M. (1985) Nucl. Acid. Res. 13, - r201-r207. 20. Yanisch-Perron, C , Vieira, J. and Messing, J. (1985) Gene 33, 103-119. 21. Polisky, B., Greene, P., Garfin, D.E., McCarthy, B.J., Goodman, H.M. and Boyer, H.W. (1975) Proc. Natl. Acad. Sci. USA 72, 3310-3314. 22. Modrich, P. and Roberts, R.J. (1982) in Nucleases, Linn, S.M. and Roberts, R.J. Eds. pp. 109-154, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Boyer, H., Scibienski, E., Slocum, H. and Roulland-Dussoix, D. (1971) Virology 46, 703-710. 24. Bickle, T.A. (1982) in Nucleases, Linn, S.M. and Roberts, R.J. Eds. pp. 85-108, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 25. Yuan, R. and Hamilton, D.L. (1984) in DNA Methylation - Biochemistry and Biological Significance, Razin, A., Cedar, H. and Riggs, A.D. Eds, pp. 11-38, Springer-Verlag, New York. 26. Kauc, L. and Piekarowicz, A. (1978) Eur. J. Biochem. 92, 417-426. 27. Bachi, B., Reiser, J. and Pirrotta, V. (1979) J. Mol. Biol. 128, 143-163. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

IL-3A virus infection of a Chlorella -like green alga induces a DNA restriction endonuclease with novel sequence specificity

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Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 volume 15 Number 15 1987 Nucleic Acids Research IL-3A virus infection of a Chlorella-tike green alga induces a DNA restriction endonuclease with novel sequence specificity Yuannan Xia, Dwight E.Burbank, Lothar Uher1, Dietmar Rabussay1 and James L.Van Etten Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722 and 'Bethesda Research Laboratories, Molecular Biology Research and Development, Life Technologies, Inc., Gaithersburg, MD 20877, USA Received March 24, 1987; Revised and Accepted June 24, 1987 ABSTRACT A typ e I I restrictio n endonuclease, named fiviJI, was isolate d from a eukaryotic Chlorella-1ike green alga infected wit h the dsDNA containing virus IL-3A. £viJI is the first restrictio n endonuclease to recognize the sequence PuGCPy ; CviJI cleave s DNA between the G and C. Methylation of the cytosine in PuGCPy sequences prevents cleavage by CviJI. £viJI cleaved DNA int o smaller but defined fragments in the presence of ATP. This "star " activity was stimulated by dithiothreito l and/or S-adenosylmethionin e but did not occur under conditions which favor "star" activity of other restriction endonucleases. INTRODUCTION We have isolated and partiall y characterized 30 large polyhedral , dsDNA containing , plaque forming viruses which infect a unicellular , eukaryotic Chlorella-like green alga strai n NC64A (1 , 2, 3, **) . One unusual feature of these viruses , whose genomes are about 300 kbp in size and contain various amounts of methylate d bases [0.1 to 47% 5-methyldeoxycytosin e (m dC) and 0 t o 37% N -methyldeoxyadenosin e (m dA) (3 , 5)] , is that infection wit h at leas t some of them result s in the appearance of DNA methyltransferase s and type II DNA restrictio n endonucleases (6, 7 , 8). In fact the virus infected alga is the firs t source of typ e I I DNA restrictio n endonucleases from a eukaryotic system. The restrictio n endonucleases £viAI and £viBI appear after infectio n with the viruses PBCV-1 and NC-1A, respectively. £viAI , like the bacteria l restrictio n endonuclease Hbol recognize s the sequence GATC, cleaves DNA 5 ' to the G, and does no t cleave G ATC sequence s (7) . £viBI is similar to the bacteria l restriction endonuclease HinfI because i t recognizes th e sequence GANTC, cleaves DNA between the G and A, and does not cleav e G ANTC sequences (8). Thi s report describes a differen t restriction endonuclease, 11RL Press Limited, Oxford, England. 6075 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research named £viJI, which appears after infection of the alga with another of these viruses, IL-3A. £viJI is the first restriction endonuclease to recognize the sequence PuGCPy; cleavage occurs between the G and C. MATERIALS AND METHODS Growth and infection of the alga. The production and purification of IL-3A and the other viruses, and the growth of Chlorella strain NC64A on MBBM medium have been described (1, 3, 9) . Chlorella cells (1 to 2 x 10 cells/ml) were infected with IL-3A at a multiplicity of infection of 10 and cells were collected by centrifugation at 6 to 7 hr post infection and used immediately or frozen at -80 C. Enzyme extracts. Five to 10 x 10 infected cells were suspended in 15 ml of buffer A [0.01 M Tris-HCl (pH 7.9), 0.01 M 2-mercaptoethanol and 50 yg/ml phenylmethylsulfonyl fluoride] plus 10 gm of 0.3 mm glass beads and homogenized for 80 sec at 4000 rpm in a Bronwill MSK mechanical homogenizer with cooling from a CO tank. The homogenate was centrifuged at 10,000 X g for 20 min and protein was precipitated from the supernatant at 70% saturation with (NH ) SO . The precipitate was dissolved in buffer A and treated with dextran T500 and polyethylene glycol 6000 as described (7, 10). The resulting upper aqueous phase was adjusted to 20$ (v/v) glycerol and the sample was dialyzed overnight at 4 C against three changes of buffer B [20 mM. Tris-HCl (pH 7.5), 0.5 mM sodium EDTA, 7 mM 2-mercaptoethanol and 10% glycerol]. The dialysate was diluted 5 fold with buffer B and applied to a heparin sepharose (Pharmacia) column; the column was eluted with a 0 to 1.2 M gradient of NaCl in buffer B. £viJI activity was pooled and adjusted to 0.1 M NaCl with buffer C [10 mM KPO (pH 7.4), 0.5 mM EDTA, 7 mM 2-mercaptoethanol, 10$ glycerol]. The sample was applied to a phosphocellulose (Whatman P11) column and eluted with a 0.2 to 1.2 M gradient of KC1 in buffer C. Pooled CviJI activity was adjusted to 0.1 M KC1 with buffer C and applied to a hydroxylapatite (Bio-Gel HTP, Biorad) column. C_viJI was eluted with a 0.2 to 1.2 M KPO gradient; the pooled active fractions were dialyzed against buffer B containing 50 mM KC1 and 50$ glycerol and then stored at -20 C. Enzyme assays. Unless noted otherwise, CviJI was assayed at 37 C for 2 hr in 20 v1 reaction mixtures containing core buffer [50 mM Tris-HCl 6076 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research (pH 8.0) , 10 mM MgCl , 50 mM NaCl] , 1 ug substrate DNA and 1 t o 3 vl of enzyme extract . The reaction products were electrophoresed eithe r on 2% agaros e gels in 90 mM Tris-HCl , 90 mM bori c acid, 2 mM EDTA, pH 8. 0 (11) or on 7.5 to 15% linea r gradient polyacrylamid e gels in 40 mM Tris-acetate , pH 8.3 , 20 mM sodium acetate , 2 mM EDTA as described (7, 12). One uni t of enzyme is th e amount required to completely digest 1 vg o f unmethylated lambda DNA i n 1 hr at 37 C i n 20 vl of assay buffer. Determinatio n of restrictio n endonuclease cleavage sit e bv DNA sequencing . Dideox y chain terminatio n sequencing reaction s were conducte d on alkali-denature d plasmid pBR322 DNA preannealed with 5'-end-labelle d pBR322 Sai l site sequencing primer (New England Biolabs) . The 15 base sequence 5 ' -AGTCATGCCCCGCGC- 3 ' complementary to the pBR322 region at position 700 to 714 served as a prime r for sequencing the DNA i n a counterclockwise direction . The oligonucleotid e primer was labeled at the 5'-end by the method of McGraw (13) in 10 vl reactio n mixtures containing : 70 mM Tris-HC l (pH 7.6) , 10 mM MgCl , 120 uC (24 pmoDy - P ATP, 100 ng (20 pmol) of primer DNA and 5 unit s of T4 polynucleotid e kinase (Bethesda Research Labs). The mixtur e was incubate d at 37 C fo r 60 min and the reactio n stopped by adding 20 vl formamide dye mix (8). The sample was electrophoresed on a 25? polyacrylamide gel at 47 volts/cm (1800 volt s total ) and the ge l was autoradiographed for 2 min on Kodak XAR-5 film . The oligonucleotid e primer, identifie d by autoradiography, was cut ou t and purifie d over a NACS prepac column (Bethesda Research Labs) . The specific activit y of the DNA was about 10 cpm/pmol. The pBR322 DNA (5 vg) was denatured wit h alkal i at 30°C for 5 min in 50 Pi of 0.2 M NaOH and 0.2mM sodium EDTA (14) . The solutio n was neutralize d wit h 5 vl of 2 M ammonium acetate (pH 4.5 ) and the DNA was precipitate d with ethanol, washed, dried and suspended in 20 vl of water. Five ul of thi s solution was mixed wit h 10 ng of the 51- P-Sal1 sit e sequencing primer and the solutio n was adjusted to 12.5 mM Tris-HC l (pH 8) , 12.5 mM MgCl , and 37 mM NaCl in a tota l volume of 16 ul . The mixtur e was incubate d at 35 C fo r 1 hr , cooled on ice , and 2 ul of 0.1 M dithiothreito l and 2 unit s (2 1 ) of the large fragment of DNA polymerase I (Klenow fragment) were added. Dideoxynucleotid e sequencing reactions were carrie d out as describe d in the Bethesda Research Lab M13 Cloning and Sequencing 6077 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research Manual , 1986 edition . DNA primed synthesis products for restrictio n endonuclease digestions were prepared in the same way as the "A reaction" except that dideoxyATP was omitted . After th e chase reaction with unlabeled dATP, the mixture (mixture AA) comprisin g 14 yl was heated for 15 min at 65 C. Mixture AA was added to 2 y l of 10 x core buffer and incubated with 0.4 units CviJ I (tota l volume of 20 l) at 37 C fo r 15 min for partial digestion . The reaction was stopped wit h 40 yl of the formamide-dye mixture. The samples were electrophoresed on 8% polyacrylamide/ 7 M urea sequencing gels adjacent to the products o f a set of the dideoxynucleotide DNA sequencing reactions and treate d as described previously (8). Mis. Hos t nuclear and viru s DNAs were isolated as described previousl y (5). Unmethylated pBR322 plasmid DNA was propagated i n dam , d_c_m L, c_oii strai n GM2163 (15) as described (5). Plasmid pUC19 was propagated in L. col i strain JM83. SV40 DNA (Form I ) isolated from BSC-1 monkey cells , 0X174 DNA (Form I) , and adenovirus type 2 DNA isolate d from HeLa cell s were from Bethesda Research Labs. Other procedures. The DNAs were treated with methylases (New England Biolabs) or with restrictio n endonucleases as suggested by the suppliers. RESULTS Properties of IL-3A. A one step growth curve of IL-3A revealed that the virus was first released about 5 hr after infection and virus release was complete at about 10 to 11 hr after infection. Like infection by the virus PBCV-1 (16), IL-3A infection resulted in the degradation of the host nuclear DNA beginning between 1 and 2 hr after infection; IL-3A DNA synthesis also started at about 1 hr after infection (unpublished results). Restriction endonuclease activity. Enzyme extracts were prepared from uninfected Chlorella cells and cells at 7 hr after IL-3A infection and assayed for DNA restriction endonuclease activity. Enzyme activity (named CviJI) was detected in extracts from the virus infected cells but not from healthy cells. A single peak of restriction endonuclease activity appeared after chromatography on heparin sepharose, phosphocellulose , and hydroxylapati te columns. £viJI activity 6078 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nuclei c Acid s Research 123 4 5 678 9 1374- 6 65 - 358 - 258 - 207 - 105 - 75 - 46 - Figure 1. Ability of £viJi to digest unmethy1 a ted pBR322 plasmid DNA. Lane 1 contains untreated pBR322 DNA, lane 2 contains pBR322 DNA after a 2 hr incubation with £viJI, lane 3 is the same as lane 2 only the enzyme reaction was incubated for 16 hr, lane 4 is the same as lane 3 but incubated an additional ? hr with a second aliquot of CviJI, lane 5 is the same as lane 2 but MgCl_ was omitted from the assay, lane 6 is the same as lane 2 except that £viJI was heated to 65 C for 10 min prior to adding it to the DNA. £viJI was incubated in core buffer for 4 hr, 8 hr and 16 hr (lanes 7, 8, and 9, respectively) at 37°C before adding pBR322 DNA and incubated an additional 2 hr. eluted at 0.6 to 0.8 M NaCl from heparin sepharose, at 0.7 to 0.9 M KC1 from phosphocellulose, and at 0.5 to 0.7 M KPO from hydroxylapatite columns (data not shown). The purification steps increased the £viJI specific activity greater than 100-fold. The specific activity of the purified enzyme was estimated at about 10 units/mg of protein and about 2000 units of activity were obtained per liter (2 x 10 cells) of infected cells. As shown in Fig. 1 (lane 2) £viJI cleaved u nmeth y 1 a ted pBR322 DNA into many discrete fragments. The size of the DNA fragments did not change upon exhaustive incubation with £viJI 6079 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research Figure 2. Ability of £viJI to degrade various DNAs. (A) Untreated pBR322 DNA (lane 1), pBR322 DNA treated with IsflT, Mhsl, Hinfl, and CviJI (lanes 2 to 5, respectively). (B) Lane 1 contains pBR322 DNA digested with Mkfil as standards, lanes 2 to 6 contain 0X174, SV40, adenovirus, IL-3A, and host Chlorella nuclear DNAs, respectively, after digestion with CviJI. Lane 7 contains untreated host nuclear DNA. (Fig. 1, lanes 3 and 4) . Thus £viJI was essentially free of nonspecific nucleases and cleaved pBR322 DNA at discrete sites. CviJI required Mg (Fig. 1, lane 5) for activity. The enzyme was most active in 50 mM Tris-HCl (pH 8.0) , 10 mM MgCl , and 50 mM NaCl. The temperature optimum was 37 C. £viJI activity was measured after incubating the enzyme in the assay mixture at 37 C for various intervals prior to adding substrate DNA. The enzyme was still active after a 4 hr incubation but much less active after a 16 hr incubation (Fig. 1, lanes 7 to 9) . Heating CviJI to 65 C for 10 min reduced activity more than 95% (Fig. 1, lane 6) . £viJI can be stored at -20 C in buffer B containing 50 mM KC1 and 50 % glycerol for at least 1 year without loss of activity. Mapping of CviJI cleavage sites. The CviJI cleavage products of pBR322, 0X174 , SV40 and adenovirus DNAs were examined on 7.5 to 15% linea r gradient polyacrylamide gels (Fig. 2) . Plasmid pBR322 DNA was cleaved into at least 20 fragments; however, only 5 of these fragments 6080 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 i 4 5 6 Figur e 3. Effect of Hafilll methylase modificatio n of pUC19 DNA o n cleavag e by CviJI . Untreated pUC19 DNA (lan e 1), Haelll digeste d pUC19 DNA (lane 2) , £viJ I digested pUC19 DNA (lan e 3), Ha£lII/£viJ I digested pUC19 DNA (lan e 4) , Ha^II I digestio n of H_a_e.III methylas e treate d pUC19 DNA (lan e 5) , and £viJ I digestio n of Haell l methylase treate d pUC19 DNA (lane 6) . Note: (i ) the identica l sizes of the DNA fragment s in lanes 3 and 4; (ii ) the siz e of the "A" fragment of pUC19 DNA increase d after methylatio n wit h £La_e_III methylas e (compare lanes 3 and 6, arrowheads) . wer e large r than 140 bp (Fig . 2A, lane 5) . Comparing the size of thes e 5 large r pBR322 DNA fragment s wit h those produced by known restrictio n endonucleases suggested tha t C_viJI had a unique specificit y (17, 18). The number and size s o f the cleavage fragment s obtained by CviJ I digestio n of 0X174 and SV40 DNAs supporte d thi s conclusion (Fig . 2B). IL-3 A viru s DNA i s resistan t to bacteria l restriction endonuclease s tlindlll , Afia.1, Alul , Haell , and tLagll l (data not shown ) as wel l as CviJ I (Fig . 2B, lane 5) . The base sequence recognize d by each of th'e bacteria l enzymes contain s a GC sequence ; cytosin e methylatio n in thi s GC sequenc e prevents activit y of each of the enzymes (18 , 19). Therefore, we assumed tha t the recognitio n and cleavag e sit e of £viJ I contained GC. 6081 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 3 4 5 6 Figure 4. Effect of Alul methylase modification of pUC19 DNA on cleavage by CviJI. Untreated pl)C19 DNA (lane 1), Alul digested pUC19 DNA (lane 2) , CviJI digested pUC19 DNA (lane 3) , AiljI/£viJI digested pUC19 DNA (lane 4) , Alul digested Alul methylase treated pUC19 DNA (lane 5), and CviJI digested ALuI methylase treated pUC19 DNA (lane 6) . Note: (i) the identical sizes of the DNA fragments in lanes 3 and 1; (ii) the size of the CviJI "B" fragment of pUC19 DNA increased after methylation with Alul methylase (compare lanes 3 and 6, arrowheads). This assumption led to the successful identification of CviJI cleavage sites on pUC19 DNA. One of the £viJI cleavage sites was located by digesting pUC19 DNA with CviJI and/or iLaelll; tLa_gIII recognizes the sequence GGCC. As shown in Fig. 3, the CviJI "A" fragment (lane 3) and Cv iJI/Haelll "A" fragment (lane H) are identical in size to the 587 bp Haelll "A" fragment (lane 2) [located at bases 2088 to 2675 (20)]. Methylation of pUC19 DNA with M.tia£.III prior to CviJI digestion gave a 680 bp fragment in place of the 587 bp fragment (Fig. 3, compare lanes 3 and 6) . These results suggest that CviJI recognizes and cleaves GGCC sequences. Another CviJI cleavage site was determined by digesting pUC19 DNA with £viJI and AIM I . As shown in Fig. 4 (lanes 3 and .608.2 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 4), DNA digested with both enzymes or with £viJI alone produced identical fragments. DNA digested with either Alul or CviJI alone (lanes 2 and 3) produced some overlapping fragments. For example, there are regions of overlap between the 679 bp Alul "A" fragment (located at bases 2050 to 43) and £viJI "A" fragment (about 580 bp) and the 521 bp Alul "B" fragment (located at bases 1366 to 1887) and CviJI "B" fragment (about 370 bp). Methylation of the DNA with M.AluI followed by CviJI digestion gave a 400 bp fragment in place of a 370 bp fragment (Fig. 4, compare lanes 3 and 6) . This implied that M.AluI, which methylates cytosines in AGCT sequences, protects one end of the 370 bp £viJI "B" fragment from digestion by CviJI and we conclude that CviJI also cleaves the sequence AGCT. To determine the location of the AGCT site on the 370 bp CviJI "B" fragment the DNA was double digested with either CviJI/MboII or CviJI/Bgll. pUC19 DNA contains two MboII sites, located at bases 1456 and 1547, and one Bell site, located at base 1813. The £viJI "B" fragment contained 2 MboII sites and no Ball site (data not shown) and so the C_viJI "B" fragment is located between bases 1366 and 1740 and contains an AGCT site at base 1366. The ends of the 400 bp £viJI "B" fragment of M.AXuI methylated pUC19 DNA should then be near positions 1340 and 1740. These regions were examined and the two sequences AGCC at 1337 and GGCT at 1729 were identified. This allowed us to propose that CviJI recognized (A/G)GC(C/T) sequences. A computer search was conducted to determine the number and locations of (A/G)GC(C/T) sequences in several DNAs. The computer derived number of £viJI cleavage sites were 72 for PBR322 DNA, 69 for 0X174 DNA, 88 for SV40 DNA, and 692 for lambda DNA. Fragments of 587, 363, 258, and 199 bases were the four largest fragments predicted from the location of the C_viJI cleavage sites in pBR322 DNA. The predicted sizes of these fragments matched the experimental data (Fig. 2A, lane 5) . Likewise, the predicted sizes of pUC19 and 0X174 fragments agreed perfectly with the experimental determined sizes. Determination of CviJI cleavage site. The cleavage sites were confirmed by sequencing a stretch of pBR322 DNA and determining the position of the fragments generated by £viJI. Plasmid pBR322 DNA contains all four subsets of the sequence (A/G)GC(C/T) , i.e. AGCT, AGCC, GGCC, and GGCT, within 120 bp of the Sail site (at position 651). A 5'-end 6083 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research NACGT N NACQTN >o Fiaur e 5. £viJI cleavage site s of pBR322 DNA. A stretc h of pBR322 DNA was sequenced in counterclockwise direction by the dideoxy sequencing method using a 15-base 5'-end labeled pBR322 Sai l site sequencing primer annealed to linearized alkali-denature d pBR322 DNA at positio n 700 to 7 14 (lanes A,C,G,T) . 3 P-labeled pBR322 DNA prepared analogously to the sample in the "A" lane (except that dideoxy ATP was omitted) was partiall y digested with C_viJI (lanes N). In gel 1 the bromophenol blue marker migrated 30 cm; gel 2 i s the same as ge l 1 but bromophenol blue migrated 50 cm. The bases recognize d by C_viJI are indicated by the bars. The bands in lane s N which coincided with the G where cleavage occurs are indicate d by an arrowhead. labele d pBR322 Sai l site primer was extended through and beyond th e four suspected £viJ I sites . The extended oligonucleotides were partiall y digested with CviJI under conditions which yielded al l possible complete and partia l products. Four products with increasin g sizes co-migrated with bands in the G channel of the sequencing reactions (Fig. 5). Thus C_viJI cleaved DNA between G and C i n al l four sequences AGCT, GGCT, AGCC, and GGCC and create d restrictio n fragments with blunt ends. The intensit y of th e G band at the AGCC sit e was less than at the other 3 site s which may reflec t reduced activit y at this site . Two additional observation s indicate that CviJI produces fragments with blunt ends. (i) Conditions for ligatin g blunt-ended fragments were .6Q84 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 3 4 5 Figure 6. Effect of ATP on £viJI specificity. Lane 1 contains untreated pUC19 DNA, lane 2 is pUC19 DNA after a 2 hr incubation with CviJI under standard conditions, lane 3 is the same as lane 2 except that the assay mixture contained 2 raM ATP and 20 mM dithiothreitol, lane it is the same as lane 3 except that the reaction time was 16 hr, and lane 5 is the same as lane 4 but with an additional 2 hr incubation after adding a second aliquot of CviJI. required to ligate C_viJI restriction fragments (data not shown), (ii) No end filling was observed with £viJI restriction fragments on the sequencing gel (Fig. 5, lane N) . This indicates that CviJI creates blunt or 5' recessed ends (7). Thus, the CviJI recognition and cleavage site is (A/G)G+C(C/T). ATP influences specificity of CviJI. Blunt end ligation of £viJI restriction fragments, followed by recleavage with CviJI, produced more fragments than expected. This led to the surprising finding that the size of the C_viJI restriction fragments was affected by ATP. That is, C_viJI cleaved pUC19 DNA into smaller but defined fragments when 1 to 2 mM ATP was present in the assay buffer (Fig. 6, compare lanes 2 and 3) . The reaction was stimulated by reducing agents such as 6085 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 1 2 i 4 5 6 7 8 9 10 11 12 1J Figur e 7. Time course of pUC19 DNA digestio n by C.viJI under standar d and "sta r " conditions . Lane 1 contain s untreated pUC19 DNA, lanes 2 and 8, 3 and 9, 4 and 10, 5 and 11, 6 and 12, and 7 and 13 are DNAs afte r incubation with £viJI for 1, 5, 10, 30, 60, and 120 min, respectively. The DNAs in lanes 2 to 7 were incubated with .CviJI under standard conditions and in lane s 8 to 13 under "star" conditions (see legend of Fie. 6). The reactions were stopped by heating at 65 C for 10 min. dithiothreito l or 2-mercaptoethanol and/or S-adenosylmethionine. Adenosine-5'- 0 (1-thiotriphosphate ) (ATP-a-S), adenosine-5 '- 0 (3-thiotriphosphate ) (ATP-Y-S), dATP, and ADP substituted , albeit poorly , for ATP whereas, GTP, CTP, UTP, and non hydrolyzable analogue s adenylyl-imido-diphosphate (AMP-PNP) and adenylyl (g, y -methylene)-diphosphonat e (AMP-PCP) did not. Reactio n conditions influence substrate specificit y of severa l bacterial type II restriction endonucleases. Altered activitie s are referred to as "star (•) activity " (21, 22). By analog y the ATP-requiring specificity of CviJI is referred to as £viJI» . However, £viJI # activit y is not observed under condition s which favor "star" activity of other restriction endonucleases ; i.e . products of £viJI* activit y were not obtained by adding glycerol [up to 30$ (v/v) ] or NaCl (up to 0.15 M) to th e reaction mixture, varying the pH of the reaction mixture from ++ ++ ++ 6 to 9, or replacing Mg with Mn or Zn (data not shown). The optimum conditions for £viJI* activit y were the same as fo r £viJI except that 1 mM ATP, 20 mM dithiothreitol , and 100 yM S-adenosylmethionin e were added to the core buffer. £viJI# 808t Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research produced the same cleavage fragments if pUC19 DNA was incubated with the enzyme for 2 hr, 16 hr, or if the DNA was treated with a second aliquot of enzyme for 2 additional hrs after a 16 hr incubation (Fig. 6, lanes 3 to 5) . Thus, like CviJI, £viJI* cleaved pUC19 DNA at a limited number of sites. CviJI* does not cleave IL-3A DNA (data not shown); therefore, the presence of m dC in the sequence PuGCPy inhibits its activity. Several reasons suggest that £viJI and CviJI* activities result from the same protein. First, CviJI and CviJI* activities co-eluted from heparin sepharose, phosphoce11u 1 o s e , and hydroxylapatite columns. Second, comparison of the time courses of £viJI and CviJI* digestion indicates that £viJI* cleaves at the same sites as CviJI as well as at additional sites. As seen in Fig. 7, digestion of pUC19 DNA by £viJI was still incomplete after a 10 min incubation (lane 4) but was complete after 30 min (lane 5) . However, digestion under "star" conditions produced an almost complete £viJI cleavage pattern within 5 min of incubation (Fig. 7, lane 9) . With longer incubation times the pattern shifted towards the complete £viJI* pedigree (Fig. 7, lanes 10 to 13). If pUC19 DNA was digested first under standard £viJI conditions and then used as a substrate for £viJI», the final cleavage pattern was identical to that of pUC19 DNA cleaved directly by £viJI* (data not shown). These results indicate that CviJI and CviJI* share common cleavage sites and that £viJI cleavage sites are more susceptible to £viJI* cleavage than the £viJI* specific sites. DISCUSSION This report establishes that IL-3A infection of Chlorella strain NC64A induces the synthesis of a DNA restriction endonuclease, £viJI, which recognizes the sequence PuGCPy and cleaves between the G and C; £viJI does not cleave PuG CPy sequences. £viJI is the first DNA restriction endonuclease to recognize this site (17, 18). £viJI should cleave DNAs more frequently than any known restriction endonuclease since, it recognizes on a probability basis, the equivalent of a three base sequence. I1-3A is one of six viruses that we have grouped in class 4 which infect Chlorella NC64A (3). Enzyme extracts prepared from Chlorella cells infected with each of these viruses (i.e. CA-1A, CA-2A, I1-2A, IL-2B, and I1-3D) cleaved pBR322 DNA into the same 6087 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research siz e fragments as C_viJI and al l exhibited star activity under the condition s described above (unpublished data). In each case the restrictio n endonuclease cleaves host DNA i n vitr o but not th e viru s DNA. The function of these restriction endonucleases has not been establishe d unequivocally. However, circumstantial evidence suggest s that these enzymes are involved in the breakdown of host DNA and might thus facilitate virus replication (7, 8) . If this i s true , these viral enzymes would serve a functio n exactly opposit e of what is considered the main function of bacterial typ e II restrictio n endonucleases, namely the preventio n of successfu l infection by bacteria l viruses. In thi s sense the Chlorell a virus-induced restriction endonucleases would function as "facilitating " rather than "restriction" enzymes. Restrictio n endonucleases are classifie d into three types based on thei r cofactor requirements and other criteria . Type I enzymes, such as EcoK and EcoB have stringen t requirements for ATP, S-adenosylmethionine and Mg (23, 24, 25) . Type II enzymes such as EcoRI and HindIII , require only Mg for activit y (22) . Type II I enzymes, such as EcoPI and EcoP15. require only ATP and Mg for activity ; however, S-adenosylmethionine stimulates activit y (25, 26, 27) . In the absence of ATP, CviJI cleaves DNA a t al l PuGCPy site s and only requires Mg for activity . Thus, £viJ I functions as a typica l type II restrictio n endonuclease. However, £viJI obviously recognizes and cleave s a smaller base sequence when ATP i s included in the reactio n mixture. Assuming tha t CviJI and CviJI * activitie s are associate d with the same protein , the £viJI * activit y differs from "star" activities of othe r type II restrictio n endonucleases (22). First, CviJI1 activit y does not occur under conditions which favor "star" activit y of other restriction endonucleases, e.g. high enzyme concentration , addition of glycerol, variations of pH and ionic + + ++ strength , or replacement of Mg with Mn . Second, £viJI* activit y absolutely requires ATP; relate d compounds such as ADP, dATP, ATP-a-S, ATP-y-S induce some sta r activit y whereas, GTP, CTP, UTP, AMP-PNP, and AMP-PCP do not . £viJI« activit y is stimulate d by reducin g agents and S-adenosylmethionine ; these compound s by themselve s do no t induc e "star " activity . Therefore , £viJI* resembles a type II I restrictio n enzyme in ++ respect to its dependence on Mg and ATP and its stimulation by S-adenosylmethionine. 6088 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research The rol e of ATP in the £viJI « activit y is als o not clear. I t will be interestin g to determin e if ATP change s the conformatio n of £viJ I as i t does with type I and typ e III restrictio n endonucleases and if CviJI has ATPase activit y like Type I restrictio n endonucleases. The successful purification of CviJ I wil l help to answer these questions. ACKNOWLEDGMENTS We thank Robert Blakesley, Russel Meints, Les Lane, and Myron Brakke for many helpful discussions. This investigation was supported, in part, by Public Health Service grant GM-32441 from the National Institute of General Medicine and grant DE-ACO2-82ER12086 from the Department of Energy. Published with the approval of the Director as paper no. 8247, Journal Series, Nebraska Agricultural Research Division. REFERENCES 1. Van Etten, J.L., Burbank, D.E., Schuster, A.M. and Meints, R.H. (1985) Virology 140, 135-143. 2. Meints, R.H., Schuster, A.M. and Van Etten, J.L. (1985) Plant Mol. Biol. Rept. 3, 180-187. 3. Schuster, A.M., Burbank, D.E., Meister, B., Skrdla, M.P., Meints, R.H., Hattman, S., Swinton, D. and Van Etten, J.L. (1986) Virology 150, 170-177. 4. Van Etten, J.L., Xia, Y. and Meints, R.H. (1987) in Plant-Microbe Interaction, Kosuge, T. and Nester, E.W. Eds., Vol II. pp. 307-325, Macmillan Publishing Co., New York. 5. Van Etten, J.L., Schuster, A.M., Girton, L., Burbank, D.E., Swinton, D. and Hattman, S. (1985) Nucl. Acid. Res. 13, 3471-3478. 6. Xia, Y. and Van Etten, J.L. (1986) Mol. Cell Biol. 6, 1440-1445. 7. Xia, Y., Burbank, D.E., Uher, L., Rabussay, D. and Van Etten, J.L. (1986) Mol. Cell. Biol. 6, 1430-1439. 8. Xia, Y., Burbank, D.E. and Van Etten, J.L. (1986) Nucl. Acid. Res. 14, 6017-6030. 9. Van Etten, J.L., Burbank, D.E., Xia, Y. and Meints, R.H. (1983) Virology 126, 117-125. 10. Schleif, R. (1980) Methods in Enzymology, Grossman, L. and Moldave, K. Eds., Vol. 65, pp. 19-23, Academic Press, New York. 11. Peacock, A.C. and Dingman, C.W. (1967) Biochemistry 6, 1818-1827. 12. Jeppesen, P.G.N. (1980) Methods in Enzymology, Grossman, L. and Moldave, K. Eds., Vol. 65, pp. 305-319, Academic Press, New York. 13. McGraw, R.A. (1984) Anal. Biochem. 143, 298-303. 14. Chen, E.Y. and Seeburg, P.H. (1985) DNA 4, 165-170. 15. Marinus, M.G., Carraway, M., Frey, A.Z., Brown, L. and Arraj, J.A. (1983) Mol. Gen. Genet. 192, 288-289. 6089 Downloaded from https://academic.oup.com/nar/article/15/15/6075/2358850 by DeepDyve user on 21 August 2020 Nucleic Acids Research 16. Van Etten, J.L., Burbank, D.E., Joshi, J. and Meints, R.H. (1981) Virology 134, 443-419. 17. Roberts, R.J. (1985) Nucl. Acid. Res. 13, r165-r200. ' 18. Kessler, C , Neumaier, T.S. and Wolf, W. (1985) Gene 33, * 1-102. 19. McClelland, M. and Nelson, M. (1985) Nucl. Acid. Res. 13, - r201-r207. 20. Yanisch-Perron, C , Vieira, J. and Messing, J. (1985) Gene 33, 103-119. 21. Polisky, B., Greene, P., Garfin, D.E., McCarthy, B.J., Goodman, H.M. and Boyer, H.W. (1975) Proc. Natl. Acad. Sci. USA 72, 3310-3314. 22. Modrich, P. and Roberts, R.J. (1982) in Nucleases, Linn, S.M. and Roberts, R.J. Eds. pp. 109-154, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 23. Boyer, H., Scibienski, E., Slocum, H. and Roulland-Dussoix, D. (1971) Virology 46, 703-710. 24. Bickle, T.A. (1982) in Nucleases, Linn, S.M. and Roberts, R.J. Eds. pp. 85-108, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 25. Yuan, R. and Hamilton, D.L. (1984) in DNA Methylation - Biochemistry and Biological Significance, Razin, A., Cedar, H. and Riggs, A.D. Eds, pp. 11-38, Springer-Verlag, New York. 26. Kauc, L. and Piekarowicz, A. (1978) Eur. J. Biochem. 92, 417-426. 27. Bachi, B., Reiser, J. and Pirrotta, V. (1979) J. Mol. Biol. 128, 143-163.

Journal

Nucleic Acids Research – Oxford University Press

Published: Aug 11, 1987

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