Biochit,lica et Biophysica Acta, 1079(19ql) 57-64
57
© 1991 ElsevierSciencePublishersB.V. 0167-4838/ql/$03.50 ADONIS 0167483891002536 BBAPRO 33980
A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization Shuji Seki, Shogo Ikeda *, Sekiko Watanabe, Masao Hatsushika, Ken Tsutsui, Kosuke Akiyama and Bo Zhang Department of Molecular Biology. Institute o[ Cellular and Molecular Biolo~', Okayama Uniuersiry Medical School. Okayama ¢Japan)
(Received 18 March 1991)
Key words: DNA repair enzyme;Exonuclease:Apurinic/apyrimidinicendoauclease;DNA phosphatase;Aminoacid sequc0ce; Primingenzyme
A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chramatograpbies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), ~phadex G.100, single.stranded DNA cellulose and hydroxyapatite. The purified enzyme has an M, of 34000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escher/ch/a co/i exonuclease !I!. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.
Introduction The DNA damage that occurs daily as a consequence of spontaneous hydrolysis under physiological conditions or by chemical, physical or biological stresses is substantial and of various types [I]. Among the types of damage, apurinic/apyrimidinic (AP} sites are generated at several thousand residues per genome per day in a mammalian cell [2]. Single-strand DNA breaks with 3' termini blocked I%' nucleotide fragments are also known to be frequently induced by free radical pathways initiated by ionizing radiation, bleomycin and other sources of oxygen radicals [1,3-6]. In our studies on DNA repair synthesis after bleomycin-induced dam-
* Present address: Departmentof Bu~logicalChcmi,try, Facully ¢~[ Science.Okayama Univer~a~of Science.Okayama 7(10.~'apan. Correspondence: S. Seki. Department of Molecular BioloK,. Institute of Cellular and Molecular Biolog'y,Oka~ama UniversilyMedical School. 2-5-1. Shikata-cho.Okayama 711~).Japan
age in mammalian cells, we found and partially purified a priming enzyme (an exonuclease) that can initiate repair of bleomycin-induced and X-ray-induced single-strand breaks [7-11]. This enzyme also showed priming activiD' for DNA polymerase on acid-depurinated DNA and micrococcal nuclease-treated DNA, .suggesting that it is a multifunctional enD'rae having exonuclease. AP endonuclease and phosphatase act vities [9.12]. Thus this enzsme exhibits properties sir dlar to those of E. coli ,:xonuclea~ !I1 and yeast DNA 3'-repair diesterase [IA3,1a]. Mammalian AP endonucleases possibly involved in the repair of AP sites have been purified from various species including mice [15-171. rats [181, cattle [t9] and humans [20-221. As far as we know, however, none of the previously reported mammalian AP endonucleases possessed exonuclease actMty. In the present paper we describe the purification, characterization and partial amino acid sequences of the mouse repair enzyme (designated as APEX nuelease) having exonuclease and AP endonuclease activities.
58 Materials and Methods
Materials The reagents used in these experiments were obtained from the following sources: [~H]dTTP from Amersham Japan, Tokyo, Japan: [a~'P]phos~hate and [a-3-'P]dCTP from ICN Biochemicals, CA, U.S.A.; ribonucleotides {NTPs) and deoxyribonuclcotides (dNTPs) from Seikagakv. Kngyou, Tokyo, Jap,m: E. colt exonuclease Ill, DNA polymerase I and the large fragment (Klenow polymcrase) of DNA polymerase I from Takara Shuzo. Kyoto, Japan; calf thymus DNA and DNA alternate copolymer poly(dA-dT)-poly(dAdT) from Pharmacia, Uppsala, Sweden. The other reagents used were obtained from the previously described sources [Q]. Mouse ascites sarcoma [SRC3H/He) cells were obtained and maintained as described previously [23]. pUC18 DNA was prepared as described previously [2,1]. Bleomycin-Fc(ll)-trcated ¢calf thymus and pUCI8) DNAs and X-irradiated DNA were preparcd as described previously [9-11]. Preparation of acid-depurinated DNA and assay for AP endonuclease actiei.,y The superhelical (form 1) pUCIS DNA and calf thymus DNA were depurinated by i~lcubating them in 3 vol. of 50 mM sodium citrate (pH 3.5) at 60°C for 15 rain essentially as described by Niwa and Moses [9.25]. After the incubation, the mixture was chilled to 0°C and dialysed against 50 mM Tris-HCI (pH 7.5) for 3 h and t,en against distilled water overnight. The dialysed DNA solution was used to measure AP endonucleasc activity. The acid treatment produced approximately six alkali-sensitive sites per pUCI8 DNA molecule, as determined by alkaline gel eleetrophoresis [26]. The assay mixture (15 tal final vol.) co:retained 0.25 ~,g (0.14 pmol) acid-depurinated pUCI8 DNA and 0-7.5 ~tl of an appropriate dilution of the enzyme in Triton-buffer B (0.0175% Triton X-ID0. 0.25 M sucrose, 10 mM Tris-HCl, 4 mM MgCI:, [ mM EDTA and 6 mM 2-mereaptoethano[, pH 8JI adjusted at 25°C1. After incubating the assay raixture at 37°C for 20 rain, the reaction was stopped by chilling it to 0°C and then adding 3 tal of 6-fold-concentrated gel loading buffer (0.25% bromphenol blue, 0.25% xylenc cyanol and 30% glycerol in H20). The mixture was loaded into a slot of a submerged 0.8% agarose gel. Electrophoretic analyses of conformation of pUC18 DNA were conducted as described previously [10,11,24]. Assay of the enzyme that enhances the template-primer actit'i~" of bleomycin-damaged, X-irradiated or acid.depurinated DNA for DNA polymemse 13 or Klenow polymerase In the present paper the term "template-primer activity' of DNA is used to indicate the ability to ser~c as
a template-primer for DNA polymerase /~ or Klenow polymerase. The term 'priming activity (or enzyme)" on bteomycin-damaged, X-irradiated or acid-depurinated DNA is used to denote the activity (or enzyme) that enhances the template-primer activity of bleomycindamaged, X-irradiated or acid-dcpurinated DNA by removing the 3' tags or by introducing single-strand breaks. The priming activity was measured by the twostep- {priming and repair DNA synthesis step-} method [U,ll]. For measuring the priming activity on bleomycin-damaged DNA, for example, 6 tag calf thymus DNA was incubated at 37°C for 30 min with 20 #M Fe(ll), 0.2 ~.g bleomycin and an appropriate an~ount of a priming enzyme fraction in a 39-/~I reaction mixture made up with Tritor,-buffer B. After the incubation, the reaction mixture was incubated at 60°C fo~ I0 min to inactivate enzymes in the priming enzyme fraction and then chilled to 0°C. A 21-~1 aliquot of the substrate mixture (labeled substrate: [~H]dTTP) for DNA synthesis supplemented with 0.04 unit of Klenow polymerase or DNA polymerase /3 was added to the reaction mixture, DNA synthesis carried out at 37°C for 30 rain. The ractioactivity incorporated into acid-insoluble materials was mcasured by a disc method [91. One unit of pri,ning enzyme in the present assay is defined as the amount of the enzyme causing 1 nmol of [3H]dTMP incorporation per 30 rain into bleomyeindamaged DNA under the standard assay conditions.
Assay of DNA double-stranded exonuclease and phosphatase acticitws Radioactive DNA for exonuelease assays was prepared by growing Escherichia colt HBI01 in LuriaBertani medium (125 ml) supplemented with [3H] thvmidine (i m C i / l ~ ml, 50 Ci/mmol). When the cclls reached the late log phase, they were harvested and their DNA was purified by standard phenol extraction techniques. The radioactivit~ of the labeled DNA was 744 cpm/nmol of nucleotide. Bleomycin-, micrococcal nuclcase- or DNase l-treatment of the labeled DNA was conducted as described previously [9,27]. Exonuclease activity of the priming enzyme was measured using the bleomycin-, micrococcal nuclease- or DNasc l-treated, ~H-labeled DNA essentially as described [9]. To demonstrate 3'-5' exonuclease activity, the alternate copolymer poly(dA-dT) - poly(dA-dT) was 3'-end-labeled by incubating it with [3H]d'i~P and Klenow polymerase. The purified, 3'-end-labeled copolymers were incubated at 37°C for varying times with the priming enzyme in Triton-buffer B. Nucleotides released by exonucleolytic digestion were analyzed by polyethylene imine-cclluiose thin layer chromatography [28]. To measure DNA 3' phosphatase activity, 3'[3ZP]phosphoryl-terminated DNA (specific activity: 8.9.104 cpm/nmol of phosphate) was prepared by partially digesting ~:P-labeled E. colt DNA
5O with micrococcal nucleasc [29,30]. The priming enzyme was incubated at 37°C with the 3'-[~:P]phosphor3l terminated DNA and released [':P]inorganic phosphate (acid-soluble. Norit-non-adsorbable ":P) ~as measured as dcscribcd pre~4ously [31)].
Detennhmtion of partial amim~ acid sequences o[ the enzyme When the purified priming enzyme 1,APEX nuclease) was treated at 37°C for 12.5 h with 70(~ formic acid. which hydrolyzes peptidc bonds between aspartate and proline, the enzyme was cleaved into two fragments with molecular masses of 30 and 4 kDa. ]'he amino-terminal amino acid sequence of the en~'me and that of the large formic acid fragment purified by SDS-polyaerylamide gel elcctrophoresis (SDS-PAGE) were determined by an Applied Biosystems Model 477A automated protein sequencer. Other methods DNA polymerase/3 was purified [9,31] and assayed [9] as described previously. Agmose gel electrophoresis and SDS-PAGE were performed as described previously [10-12,32]. Repair of pUCI8 DN,'~ with bleomycin-, peplomycin- or X-ray-induced single-strand breaks was measured by monitoring conformational changes of the plasmid DNA [10,11,26]. Protein concentrations were determined by the BCA Protein Assay (Pierce, IIfinois. U.S.A), using bovine serum albumin as the standard. The amino acid composition of the enzyme was determined using an Applied Biosysterns Derivatizer. Model 420A. Results
Purification of a mouse repair enzj'me for bleomvcindamaged DNA Partial purification of a repair cnz,yme having exonucleolytic activity on bleomycin-damaged DNA and thereby providing priming sites for DNA polymerase was reported previously [9]. In the present stud}', the enzyme was further purified to apparent homogeneity. The purification was followed by measuring its DNA polymerase /3 prim!rig activity on bleomycin-damaged DNA. The enzyme was extracted from permeable mouse ascites sarcoma cells with 0.2 M potassium phosphate (KPi) buffer (pH 7.5) [9]. After adjustment of the KPi concentration to 0.1 M, the extract (fraction Nil was mixed with packed phosphtx;ellulose equilibrated with 0.1 M KPi. Enzymes bound to phosphocellulose were eluted with (;.3 M KPi. The eluant (fraction N 2) was passed through DEAE-celluk)se mainly to remove nuelcic acid as described previously 19,31]. Material (fraction N3) not retained by lhe DEAE-cellulose column was loaded onto tne ~cond phosphocellulose column equilibrated with 0.15 M KPi
J~
3d.
,
10 [-
0
J
13 30
40
70
50 60 FIq'ACT ION
MW 44 46 48 50 52 54 56 58 60
8 kDQ
29~
2/.. ~.
~
~.-
~
~:.
.
.
~--
~
~-
.
Fig I Second phosphocellukvse chromatogtzphs ot {he enzymc (priming IacIoL APEX n u c l e a r ) having DNA polvmerasc/~ primin~ actKily on bleomycin-dama~ed DNA. Panel A: the enzyme ,aas eluted ~tlh a linear gri~ienl eft potassium phosphate buffer (KPi. pit 75~ fmra IF 15 to 0 3 5 M. Priming acti',.t b v,a,, measured m the presence ( B t . M + . ~1 or absence (BLM - . .) cl bteom,F:m. :J- indl cared in Malerials and Methods The DNA ~x~L'~n3crase ~cttvtb ~1+ ot t:ach fraclion ",,,,as measured n,,ing aCli','~tcd DNA as a t,~mplale primer. Panel B: a pq~rti~m (33 ,allof eac,~ Waction (";(~ 44 6qi '.',a'., ei¢c~[,,lphorescd on a SI)S-Ix~t~a~.ryt-~mide gel and d~e gt:l ~as .,laJncd ~ l l h ('t~ma' ~e brilhant blue R-25it M,Jtkcl pr
57
© 1991 ElsevierSciencePublishersB.V. 0167-4838/ql/$03.50 ADONIS 0167483891002536 BBAPRO 33980
A mouse DNA repair enzyme (APEX nuclease) having exonuclease and apurinic/apyrimidinic endonuclease activities: purification and characterization Shuji Seki, Shogo Ikeda *, Sekiko Watanabe, Masao Hatsushika, Ken Tsutsui, Kosuke Akiyama and Bo Zhang Department of Molecular Biology. Institute o[ Cellular and Molecular Biolo~', Okayama Uniuersiry Medical School. Okayama ¢Japan)
(Received 18 March 1991)
Key words: DNA repair enzyme;Exonuclease:Apurinic/apyrimidinicendoauclease;DNA phosphatase;Aminoacid sequc0ce; Primingenzyme
A mouse repair enzyme having priming activity on bleomycin-damaged DNA for DNA polymerase was purified to apparent homogeneity and characterized. The enzyme extracted from permeabilized mouse ascites sarcoma (SR-C3H/He) cells with 0.2 M potassium phosphate buffer (pH 7.5) was purified by successive chramatograpbies on phosphocellulose, DEAE-cellulose, phosphocellulose (a second time), ~phadex G.100, single.stranded DNA cellulose and hydroxyapatite. The purified enzyme has an M, of 34000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatical studies indicated that it is a multifunctional enzyme having exonuclease, apurinic/apyrimidinic endonuclease and phosphatase activities, similar to Escher/ch/a co/i exonuclease !I!. This enzyme is tentatively designated as APEX nuclease for apurinic/apyrimidinic endonuclease and exonuclease activities. The amino acid composition, amino-terminal amino acid sequence and an internal amino acid sequence of APEX nuclease are determined.
Introduction The DNA damage that occurs daily as a consequence of spontaneous hydrolysis under physiological conditions or by chemical, physical or biological stresses is substantial and of various types [I]. Among the types of damage, apurinic/apyrimidinic (AP} sites are generated at several thousand residues per genome per day in a mammalian cell [2]. Single-strand DNA breaks with 3' termini blocked I%' nucleotide fragments are also known to be frequently induced by free radical pathways initiated by ionizing radiation, bleomycin and other sources of oxygen radicals [1,3-6]. In our studies on DNA repair synthesis after bleomycin-induced dam-
* Present address: Departmentof Bu~logicalChcmi,try, Facully ¢~[ Science.Okayama Univer~a~of Science.Okayama 7(10.~'apan. Correspondence: S. Seki. Department of Molecular BioloK,. Institute of Cellular and Molecular Biolog'y,Oka~ama UniversilyMedical School. 2-5-1. Shikata-cho.Okayama 711~).Japan
age in mammalian cells, we found and partially purified a priming enzyme (an exonuclease) that can initiate repair of bleomycin-induced and X-ray-induced single-strand breaks [7-11]. This enzyme also showed priming activiD' for DNA polymerase on acid-depurinated DNA and micrococcal nuclease-treated DNA, .suggesting that it is a multifunctional enD'rae having exonuclease. AP endonuclease and phosphatase act vities [9.12]. Thus this enzsme exhibits properties sir dlar to those of E. coli ,:xonuclea~ !I1 and yeast DNA 3'-repair diesterase [IA3,1a]. Mammalian AP endonucleases possibly involved in the repair of AP sites have been purified from various species including mice [15-171. rats [181, cattle [t9] and humans [20-221. As far as we know, however, none of the previously reported mammalian AP endonucleases possessed exonuclease actMty. In the present paper we describe the purification, characterization and partial amino acid sequences of the mouse repair enzyme (designated as APEX nuelease) having exonuclease and AP endonuclease activities.
58 Materials and Methods
Materials The reagents used in these experiments were obtained from the following sources: [~H]dTTP from Amersham Japan, Tokyo, Japan: [a~'P]phos~hate and [a-3-'P]dCTP from ICN Biochemicals, CA, U.S.A.; ribonucleotides {NTPs) and deoxyribonuclcotides (dNTPs) from Seikagakv. Kngyou, Tokyo, Jap,m: E. colt exonuclease Ill, DNA polymerase I and the large fragment (Klenow polymcrase) of DNA polymerase I from Takara Shuzo. Kyoto, Japan; calf thymus DNA and DNA alternate copolymer poly(dA-dT)-poly(dAdT) from Pharmacia, Uppsala, Sweden. The other reagents used were obtained from the previously described sources [Q]. Mouse ascites sarcoma [SRC3H/He) cells were obtained and maintained as described previously [23]. pUC18 DNA was prepared as described previously [2,1]. Bleomycin-Fc(ll)-trcated ¢calf thymus and pUCI8) DNAs and X-irradiated DNA were preparcd as described previously [9-11]. Preparation of acid-depurinated DNA and assay for AP endonuclease actiei.,y The superhelical (form 1) pUCIS DNA and calf thymus DNA were depurinated by i~lcubating them in 3 vol. of 50 mM sodium citrate (pH 3.5) at 60°C for 15 rain essentially as described by Niwa and Moses [9.25]. After the incubation, the mixture was chilled to 0°C and dialysed against 50 mM Tris-HCI (pH 7.5) for 3 h and t,en against distilled water overnight. The dialysed DNA solution was used to measure AP endonucleasc activity. The acid treatment produced approximately six alkali-sensitive sites per pUCI8 DNA molecule, as determined by alkaline gel eleetrophoresis [26]. The assay mixture (15 tal final vol.) co:retained 0.25 ~,g (0.14 pmol) acid-depurinated pUCI8 DNA and 0-7.5 ~tl of an appropriate dilution of the enzyme in Triton-buffer B (0.0175% Triton X-ID0. 0.25 M sucrose, 10 mM Tris-HCl, 4 mM MgCI:, [ mM EDTA and 6 mM 2-mereaptoethano[, pH 8JI adjusted at 25°C1. After incubating the assay raixture at 37°C for 20 rain, the reaction was stopped by chilling it to 0°C and then adding 3 tal of 6-fold-concentrated gel loading buffer (0.25% bromphenol blue, 0.25% xylenc cyanol and 30% glycerol in H20). The mixture was loaded into a slot of a submerged 0.8% agarose gel. Electrophoretic analyses of conformation of pUC18 DNA were conducted as described previously [10,11,24]. Assay of the enzyme that enhances the template-primer actit'i~" of bleomycin-damaged, X-irradiated or acid.depurinated DNA for DNA polymemse 13 or Klenow polymerase In the present paper the term "template-primer activity' of DNA is used to indicate the ability to ser~c as
a template-primer for DNA polymerase /~ or Klenow polymerase. The term 'priming activity (or enzyme)" on bteomycin-damaged, X-irradiated or acid-depurinated DNA is used to denote the activity (or enzyme) that enhances the template-primer activity of bleomycindamaged, X-irradiated or acid-dcpurinated DNA by removing the 3' tags or by introducing single-strand breaks. The priming activity was measured by the twostep- {priming and repair DNA synthesis step-} method [U,ll]. For measuring the priming activity on bleomycin-damaged DNA, for example, 6 tag calf thymus DNA was incubated at 37°C for 30 min with 20 #M Fe(ll), 0.2 ~.g bleomycin and an appropriate an~ount of a priming enzyme fraction in a 39-/~I reaction mixture made up with Tritor,-buffer B. After the incubation, the reaction mixture was incubated at 60°C fo~ I0 min to inactivate enzymes in the priming enzyme fraction and then chilled to 0°C. A 21-~1 aliquot of the substrate mixture (labeled substrate: [~H]dTTP) for DNA synthesis supplemented with 0.04 unit of Klenow polymerase or DNA polymerase /3 was added to the reaction mixture, DNA synthesis carried out at 37°C for 30 rain. The ractioactivity incorporated into acid-insoluble materials was mcasured by a disc method [91. One unit of pri,ning enzyme in the present assay is defined as the amount of the enzyme causing 1 nmol of [3H]dTMP incorporation per 30 rain into bleomyeindamaged DNA under the standard assay conditions.
Assay of DNA double-stranded exonuclease and phosphatase acticitws Radioactive DNA for exonuelease assays was prepared by growing Escherichia colt HBI01 in LuriaBertani medium (125 ml) supplemented with [3H] thvmidine (i m C i / l ~ ml, 50 Ci/mmol). When the cclls reached the late log phase, they were harvested and their DNA was purified by standard phenol extraction techniques. The radioactivit~ of the labeled DNA was 744 cpm/nmol of nucleotide. Bleomycin-, micrococcal nuclcase- or DNase l-treatment of the labeled DNA was conducted as described previously [9,27]. Exonuclease activity of the priming enzyme was measured using the bleomycin-, micrococcal nuclease- or DNasc l-treated, ~H-labeled DNA essentially as described [9]. To demonstrate 3'-5' exonuclease activity, the alternate copolymer poly(dA-dT) - poly(dA-dT) was 3'-end-labeled by incubating it with [3H]d'i~P and Klenow polymerase. The purified, 3'-end-labeled copolymers were incubated at 37°C for varying times with the priming enzyme in Triton-buffer B. Nucleotides released by exonucleolytic digestion were analyzed by polyethylene imine-cclluiose thin layer chromatography [28]. To measure DNA 3' phosphatase activity, 3'[3ZP]phosphoryl-terminated DNA (specific activity: 8.9.104 cpm/nmol of phosphate) was prepared by partially digesting ~:P-labeled E. colt DNA
5O with micrococcal nucleasc [29,30]. The priming enzyme was incubated at 37°C with the 3'-[~:P]phosphor3l terminated DNA and released [':P]inorganic phosphate (acid-soluble. Norit-non-adsorbable ":P) ~as measured as dcscribcd pre~4ously [31)].
Detennhmtion of partial amim~ acid sequences o[ the enzyme When the purified priming enzyme 1,APEX nuclease) was treated at 37°C for 12.5 h with 70(~ formic acid. which hydrolyzes peptidc bonds between aspartate and proline, the enzyme was cleaved into two fragments with molecular masses of 30 and 4 kDa. ]'he amino-terminal amino acid sequence of the en~'me and that of the large formic acid fragment purified by SDS-polyaerylamide gel elcctrophoresis (SDS-PAGE) were determined by an Applied Biosystems Model 477A automated protein sequencer. Other methods DNA polymerase/3 was purified [9,31] and assayed [9] as described previously. Agmose gel electrophoresis and SDS-PAGE were performed as described previously [10-12,32]. Repair of pUCI8 DN,'~ with bleomycin-, peplomycin- or X-ray-induced single-strand breaks was measured by monitoring conformational changes of the plasmid DNA [10,11,26]. Protein concentrations were determined by the BCA Protein Assay (Pierce, IIfinois. U.S.A), using bovine serum albumin as the standard. The amino acid composition of the enzyme was determined using an Applied Biosysterns Derivatizer. Model 420A. Results
Purification of a mouse repair enzj'me for bleomvcindamaged DNA Partial purification of a repair cnz,yme having exonucleolytic activity on bleomycin-damaged DNA and thereby providing priming sites for DNA polymerase was reported previously [9]. In the present stud}', the enzyme was further purified to apparent homogeneity. The purification was followed by measuring its DNA polymerase /3 prim!rig activity on bleomycin-damaged DNA. The enzyme was extracted from permeable mouse ascites sarcoma cells with 0.2 M potassium phosphate (KPi) buffer (pH 7.5) [9]. After adjustment of the KPi concentration to 0.1 M, the extract (fraction Nil was mixed with packed phosphtx;ellulose equilibrated with 0.1 M KPi. Enzymes bound to phosphocellulose were eluted with (;.3 M KPi. The eluant (fraction N 2) was passed through DEAE-celluk)se mainly to remove nuelcic acid as described previously 19,31]. Material (fraction N3) not retained by lhe DEAE-cellulose column was loaded onto tne ~cond phosphocellulose column equilibrated with 0.15 M KPi
J~
3d.
,
10 [-
0
J
13 30
40
70
50 60 FIq'ACT ION
MW 44 46 48 50 52 54 56 58 60
8 kDQ
29~
2/.. ~.
~
~.-
~
~:.
.
.
~--
~
~-
.
Fig I Second phosphocellukvse chromatogtzphs ot {he enzymc (priming IacIoL APEX n u c l e a r ) having DNA polvmerasc/~ primin~ actKily on bleomycin-dama~ed DNA. Panel A: the enzyme ,aas eluted ~tlh a linear gri~ienl eft potassium phosphate buffer (KPi. pit 75~ fmra IF 15 to 0 3 5 M. Priming acti',.t b v,a,, measured m the presence ( B t . M + . ~1 or absence (BLM - . .) cl bteom,F:m. :J- indl cared in Malerials and Methods The DNA ~x~L'~n3crase ~cttvtb ~1+ ot t:ach fraclion ",,,,as measured n,,ing aCli','~tcd DNA as a t,~mplale primer. Panel B: a pq~rti~m (33 ,allof eac,~ Waction (";(~ 44 6qi '.',a'., ei¢c~[,,lphorescd on a SI)S-Ix~t~a~.ryt-~mide gel and d~e gt:l ~as .,laJncd ~ l l h ('t~ma' ~e brilhant blue R-25it M,Jtkcl pr
