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International Journal of Radiation Biology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/irab20

Enhanced Excision Repair Activity in Mammalian Cells After Ionizing Radiation a

b

a

b

R. Bases , W.A. Franklin , T. Moy & F. Mendez a

Department of Radiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue Bronx, New York, NY, 10461, USA b

Department of Radiation Oncology, Albert Einstein College of Medicine, 1300 Morris Park Avenue Bronx, New York, NY, 10461, USA Published online: 24 May 2015.

To cite this article: R. Bases, W.A. Franklin, T. Moy & F. Mendez (1992) Enhanced Excision Repair Activity in Mammalian Cells After Ionizing Radiation, International Journal of Radiation Biology, 62:4, 427-441 To link to this article: http://dx.doi.org/10.1080/09553009214552311

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INT . I . RADIAT . BIOL .,

1992,

VOL .

62,

NO .

4, 427-441

Enhanced excision repair activity in mammalian cells after ionizing radiation R . BASES*t, W . A . FRANKLINt, T . MOYt and F . MENDEZt

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(Received 10 March 1992 ; Revised 8 May 1992; accepted 10 May 1992) .

Abstract. Monkey CV-1 cells which had received 5 Gy 12 h before harvesting lysates from their cell cultures contained approximately three times as much DNA excision repair enzyme activity as unirradiated cells . The activity was determined in crude cell lysates by the release of intermediate mobility DNA fragments and fragments with 3'-phosphoryl ends from 5'- 32 P-end labelled irradiated 95 by ocDNA. Different 3'-termini endow the fragments with differing mobilities, signifying steps in the processing of radiation damaged DNA . Similar results were obtained when Krebs II mouse tumour cells growing in mice as ascites received 5 Gy 12 h before harvest . The enzyme activities from CV-1 cells and from Krebs II cells were partially purified as 60-70 kDa proteins on Superose 12 or Ultrogel AcA-54 columns . Divalent cations were not required for enzyme activity . A 23 nucleotide long defined duplex oligodeoxynucleotide substrate containing a single 8-oxodG residue was also very actively cleaved by the partially purified cell enzymes . 8-oxoguanine is a major product of ionizing radiation's action on DNA and was recognized by the enzymes described here . The mechanism by which radiation increased excision repair activity of cellular enzymes is not understood .

1. Introduction DNA bases damaged by ionizing radiation or oxidizing agents can be repaired in cells ; one of the pathways is the so called two-step excision enzyme process (reviewed by Wallace 1988, Doetsch and Cunningham 1990) . First, glycosylase activities remove the damaged base and then the corresponding sugar is released by strand scission . Redoxyendonucleases can leave a sugar remnant attached to the 3'-phosphoryl . Since only a 3'-OH can serve as the acceptor for newly replaced nucleotides by a DNA polymerase, removal of these inappropriate groups by enzymes such as endonuclease IV of E . coli or exonuclease III of E. coli is required . Endonuclease III of E . coli is a paradigm for mammalian redoxyendonucleases . It incises Xirradiated DNA or DNA from X-irradiated cells (Bases et al. 1990), liberating fragments which have 3'-sugar remnants ; presumably they are c/fl unsaturated aldehydes . The mobility of DNA fragments *Author for correspondence . tDepartments of Radiology, and $Radiation Oncology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA

(intermediate mobility fragments) ending in these groups is less than those ending with 3'-phosphoryl or 3'-phosphoglycolate because they have a lower net negative charge . However, fl-elimination by heat and piperidine can remove sugar remnants, exposing 3'-phosphoryl ends. In this way the intermediate mobility fragments can be converted to more mobile species . We did not detect intermediate mobility DNA fragments in cDNA prepared from monkey CV-1 cells during post-radiation repair after a single dose of 300 Gy (Feingold et al . 1988) . Instead, DNA fragments with the 3'-phosphoryl and 3'phosphoglycolate termini at each nucleotide sequence position were found, as expected from results with DNA irradiated in vitro (Henner et al . 1983, Bases et al . 1986) . The fragments disappeared progressively when the irradiated cells were incubated in growth medium during post-radiation repair. However, if cDNA from the irradiated CV-1 cells was digested with endonuclease III of E. coli, abundant intermediate mobility fragments could still be released (Bases et al. 1990) . Although the intermediate mobility DNA fragments did not accumulate in detectable amounts during normal processing in cells, the presence of large amounts of their precursors in DNA of irradiated cells could be unmasked by endonuclease III of E. coli . By contrast, intermediate mobility cDNA fragments were readily detectable in DNA of CV-1 cells if they had received pretreatment doses of 0 . 2-6-6 Gy several hours before the 320 Gy dose (Bases et al. 1990, Bases and Mendez, 1992) . Therefore, in the experiments to be described here we tested the influence of 5 Gy alone on excision repair activity in lysates of monkey and mouse cells . We reasoned that the relatively small pre-irradiation doses of the earlier studies had somehow increased the activity of cellular redoxyendonucleases, leading to abundant accumulation of the intermediate mobility species . Alternatively, the pre-irradiation may have interfered with normal processing, causing inappropriate accumulation of DNA fragments with phosphosugar termini . In the experiments described above we studied ocDNA, a highly repetitive 172 by DNA of

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primates which is associated with centromere antigens of human cells (Masumoto et al . 1989) . We had also studied intermediate mobility fragments in cDNA recovered from rat NRK cell cultures which had been transfected with heavily irradiated plasmids containing a DNA . Intermediate mobility cDNA fragments were readily detected in cell nuclei 5-15 min after transfection with the irradiated plasmids . Pre-irradiating the rat cells with 5 Gy 12 h before transfection delayed net accumulation of intermediate mobility cDNA fragments and the pre-irradiation prolonged the time during which they could be found . Pre-irradiation may have enhanced the overall activity of excision enzymes and DNA repair enzymes in the rat cells; other explanations could not be excluded . Since it seemed likely that small amounts of ionizing radiation enhanced the level of redoxyendonuclease activity in cells we assayed lysates from irradiated cells to determine their ability to release intermediate mobility DNA fragments from irradiated cDNA substrates . The activity was partially purified by Superose 12 and acrylamide/agarose Ultrogel filtration . A preliminary account of this work has been reported (Bases et al . 1991) . Here we report that 12 h after 5 Gy to CV-1 cells or to Krebs II mouse ascites tumour cells, the activity was approximately three fold greater than in unirradiated cells . Moreover, the constitutive and the enhanced enzyme activity very actively recognized 8oxoguanine, one of the major radiation products in DNA of cells (Kasai et al . 1986, Dizdaroglu and Bergtold 1986) and a pro-mutagenic lesion (Shibutani et al. 1991) . The in vivo potential of 8oxoguanine for mutagenesis has been found (Wood et al. 1990, Moriya et al . 1991, Cheng et al. 1992, Michaela et al. 1991) . 2. Materials and methods 2. 1 . Cell cultures, irradiation Monkey CV-1 cells were grown in Eagle's minimal essential medium with 10% fetal bovine serum in 10cm plastic Petri dishes at 37 °C in 5% CO 2 . Before harvesting the cells for enzyme preparation, they were trypsinized, given 5 Gy with 137 Cs at 1 .25 Gy/min, and cultured for 12 h at 5 x 10 6 cells/ dish . Dishes were then rinsed twice with tris buffered saline at 4 °C and the monolayers were harvested by scraping with Teflon covered razor blades using ice cold 50 mm Tris buffered 0-15N NaCl containing I mm EDTA . Unirradiated cells were harvested identically.

Krebs II mouse ascites tumour cells were grown in Swiss mice; 5-7 days after intraperitoneal inoculation with 1 x 10 7 cells, when the ascites cells were in log phase growth, the mice received 5 Gy of total body irradiation from a 137 Cs source (Atomic Energy of Canada) at 1 .1 Gy/min . The mice were killed 12 h after the irradiation and their ascites cells were collected and rinsed by low speed centrifugation in buffered saline . 2.2 . Cell lysates CV-1 cells were harvested from 10 cm Petri dishes, pelleted by low speed centrifugation, resuspended in 0.5 ml of 100 mm NaCl, 50 mm Tris, pH 7 .4, 5 mm dithiothreitol (DTT), 1 mm EDTA, 10 µM Aprotinin (a protease inhibitor, purchased from Boehringer Mannheim) and sonicated at 4°C with eight strokes of a Branson sonicator probe powered at 60 W . Cell debris was removed by centrifugation at 15 x 10 3g for 15 min . From 1 .0 x 108 cells, 0 .5 ml of crude lysate containing - 8 mg/ml of protein was recovered ; it was stored in 10% glycerol at -80°C . The protein concentrations of cell lysates and column fractions were determined by the method of Bradford (1976) . Lysates from Krebs II mouse ascites tumour cells were similarly prepared . 2.3 . Enzyme assays 5'- 32P-end-labelled 95 by aDNA substrates were prepared as described previously (Bases et al. 1986, Feingold et al . 1988) . Irradiated substrate DNA received 400 Gy in 10 mm KI to minimize strand breaks, while producing base damage (Bases et al . 1990, Bases and Mendez 1992) . Enzyme activities in lysate fractions were tested by incubating 10µl of the prepared lysate fraction for 30 min at 37 ° C in 100 yl volumes of reaction buffer : 50 mm Tris/HCl, pH 7 .6, 100 mm KCl, 1 mm EDTA and 0 . 1 mm DTT containing 100 ng of labelled DNA substrate. Endonuclease III of E. coli, a gift of Dr Richard Cunningham, State University of New York at Albany (Cunningham and Weiss 1985), was assayed as described previously (Bases et al. 1990) . 2 .4 . Autoradiography and quantitative densitometry

Separation of DNA fragments by electrophoresis was done in 38 cm 20% polyacrylamide gels at 2000 V for ' 7 h . Exposure of these sequencing gels were at -80 ° C for 1-14 days with intensifiers, using



Radiation-enhanced excision repair Kodak XAR-5 film . The gel autoradiograms were scanned by a Pharmacia LKB 2222-020 laser densitometer in the linear response range of the instrument. Specific DNA sequence sites were identified by their nucleotide distance from the 5'-end of 95 by o cDNA .

429

(1 .6 x 100 cm ; IBF) and were eluted with buffer that included 50 mm Tris pH 7 . 8, 5 mm DTT, 1 mm EDTA, 5% glycerol, 500 mm NaCl overnight at 4°C . Before the Ultrogel column, DNA was removed from cell lysates by eluting through 1 x 2 . 5 cm DEAE Sepharose columns (Pharmacia) using 300 mm NaCl, 50 mm Tris, pH 7 . 8, 5mM DTT, 1 mm EDTA, and 5% glycerol .

2.5. Partial purification by column chromatography

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2 .6. 8-Oxoguanine duplex DNA substrate Superose 12 columns . Lysates (200 µl) were loaded onto a Pharmacia Superose 12 HR 10/30 pre-packed column and eluted with buffer that included 100 mm NaCl, 50mM Tris pH 7 . 8, 2 mm DTT, 1 mm EDTA, 5% glycerol at room temperature . AcA-54 Ultrogel columns . In some experiments lysates were loaded onto an Ultrogel AcA-54 column UNIRRADIATED CELL LYSATE

8-Oxoguanine synthetic 23-mer and its complementary DNA (prepared by R . Rieger and F . Johnson following published procedures of Bodepudi et al . 1991) and FPG protein (Boiteux et al. 1987, 8oxoguanine DNA glycosylase) were gifts from A . Grollman, State University of N .Y . at Stony Brook IRRADIATED CELL LYSATE 5 Gy

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Figure 1 . Digestion of irradiated 95 by a DNA by Superose 12 fractions . Lysates from 8 x 10 7 unirradiated CV-1 cells and from 8 x 10 7 cells which had received 5 Gy 12h earlier were prepared by sonication . Protein (7 mg) was recovered from the unirradiated cell sonicate and 5 . 5 mg from the sonicate of the irradiated cells . 5'-32 P-labelled 95 by substrate a DNA which had received 400 Gy in 10 mm KI (100 ng containing 1 x 10 5 cpm) were incubated with a 10µl aliquot of each Superose 12 fraction for 30 min at 37 ° C in 100 ul of enzyme reaction buffer . Alternate fractions from the column eluate from lysate of unirradiated CV-1 cells are numbered C16-C32 . The corresponding fractions from lysate of CV-1 cells harvested 12 h after exposure to 5 Gy are numbered R16-R32 . After the incubation, each reaction mixture was extracted with phenol and precipitated with ethanol . Each gel lane received DNA containing the following : lanes C16-C32 received : 1 . 6, 3 . 2, 3 . 4, 2 . 7, 3 . 0, 3 . 0, 2 . 3, 4 . 2 and 2 . 9x 10 4 cpm, respectively ; lanes R16-R32 received : 0 . 7, 3 . 7, 2 . 5, 2 . 7, 2 . 5, 3 . 1, 3 . 4, 3.0 and 2 . 9cpmx 10 4 , respectively. The first lane (2 x 10 4 cpm) and the three lanes to the right of lane R32 contain DNA which received 400 Gy in 10 mm KI, (1, 2 and 5 x 104 cpm, respectively) . Lane 2 and the right hand lane and a middle lane contain Maxam and Gilbert standards (G .std .), showing the location of guanines in the sequence . The 20% acrylamide gel autoradiogram is from a 17-day exposure . The upper panels show the concentration of protein in each fraction tested . Most of the radiolabel placed on the gel remained at the origins at the top and are not shown .



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1 2 3 4 5 6 7 G la 1P 2P 3P 4P 5P 6P 7P G 1b G22-

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Figure 2 . Lysates from irradiated CV-1 cells release more fragments of intermediate mobility from irradiated 5'- 32 P-end labelled 95 by a DNA substrate than lysates from unirradiated cells . Incubations with lysates from fractions 26 from irradiated and unirradiated cells of the experiment of Figure 1 were done at 37 ° C for 30 min in enzyme reaction buffer . Sequence gel lanes 1, I a and 1 b : irradiated DNA substrate incubated without lysate fraction : 8 . 5, 3 . 0 and 4. 5 x 10 4 cpm of labelled DNA . Lanes 2-4 : irradiated DNA substrate incubated with 3 µg of protein from column fraction 26 from unirradiated cells, incubated 30, 60 or 90 min . Lanes 2-4 received DNA containing 6 . 9, 8 . 7, 8 . 6 x 10 4 cpm, respectively . Lanes 5-7 the same treatment as in lanes 2-4 except incubations were with 3µg of protein from column fraction 26 from irradiated cells . Lanes 5-7 each received DNA containing 1 x 10 5 cpm . The autoradiogram was exposed for 5 days. Lane G is a reference lane showing location of fragments ending at G's (Maxam and Gilbert) . DNA of lanes 1P-7P received treatments identical to the DNA of lanes 1-7, but were heated for 20 min at 90 ° C in 1 M piperidine before being placed on the gel . These lanes received 1 . 6, 2 . 9, 3 . 1, 2 . 8, 2 . 1, 2 . 5, 2 . 8 x 10 4 cpm of DNA, respectively . After piperidine treatment all intermediate mobility bands were converted to fragments with mobility of 3'-phosphoryl or to distinctly different slowly migrating species .

(Tchou et al . 1991) . The oligonucleotide (sequence # 13, Tchou et al . 1991) was labelled at the 5'-end, as described . The complementary oligonucleotide was referred to there as sequence # 14) . The sequence of the 8-oxodG containing oligodoxynucleotide is : 5'-CTCTCCCTTCG*CTCCTTTCCTCT-3' .

3 . Results 3.1 . Enhanced excision repair activity in lysates of CV-1 cells 12 h after 5 Gy

To isolate enzymes which might account for the enhanced level of redoxyendonuclease activity found

in cells after radiation, CV-1 cells were given 5 Gy 12 h before harvesting . Lysates from unirradiated and irradiated cells were prepared and assayed as described in Materials and methods . 5'- 32 P-end-labelled 95 by cDNA which had received 400 Gy in 10 mm KI was used as a substrate for the lysates . K I quenched radiation-induced free radicals, thereby reducing the background level of DNA strand breaks . Base damage was then unmasked by endonuclease III of E . coli or similar enzymes in cell lysates, as detected by the release of fragments of rapidly migrating DNA (Doetsch et al. 1987, Bases et al . 1990) . The different mobilities of fragments at each nucleotide position in the sequence provide clues to the nature of the 3'-terniini (Haukanes et al. 1988, Bases et al. 1990) . Figure 1 shows assays of cell lysate fractions from



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Radiation-enhanced excision repair

Superose 12 gel eluates . The lower numbered fractions contain the larger proteins, which are eluted earlier . Precise determinations of the size of the excision enzymes will be provided in Figures 4 and 6 . Fractions 26 and 28 of lysates from irradiated cells released more fragments of intermediate mobility than corresponding fractions from unirradiated cells . To confirm this, lysate fractions 26 from irradiated and unirradiated cells were compared in 30, 60 and 90-min incubations (Figure 2) . Gel autoradiograms of DNA fragments at sequence locations T-14 -G- 18 from the 5'-termini in Figure 2 were scanned ; the densitometer values were normalized for the cpm of DNA placed in each gel lane . From these scans we estimated that fragments with the mobility of DNA with 3'-phosphoryl ends in lanes 5-7 (released by the lysates from irradiated cells) were 2 . 4, 1 . 4 and 1 . 1-fold more intense than corresponding autoradiographic bands

in lanes 2-4 (released by the lysates from unirradiated cells) . This 1 . 6-fold increase is to be compared with the more than three-fold increases found when lysates from irradiated and unirradiated CV-1 and Krebs II mouse cells in other studies were compared (Figures 5, 6 and 8) . In Figure 2 the autoradiograms from fragments of intermediate mobility were 2 . 9, 1 .4 and 2 . 5 times more intense than corresponding autoradiograms in lanes 2-4 . This 2 . 3-fold increase is to be compared with 3-4-fold increases found in the experiments of Figures 3 and 6 and in other experiments not shown. Heat and piperidine treatment of aliquots of DNA which had received these digestions (lanes 1 P-7P) caused certain intermediate mobility fractions to disappear, e .g. retarded A-12, G-13, T-15, while releasing other retarded species, e .g . T-14 . After heat and piperidine treatment, lanes 1P-7P exhibited more intense radiographic bands due to fl-

RELEASE OF INTERMEDIATE MOBILITY FRAGMENTS BY : LYSATES FROM IRRADIATED CV-1 CELLS 4

LYSATES FROM UNIRRADIATED CV-1 CELLS

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SUPEROSE 12 FRACTION NUMBER Figure 3 . Enhanced endonuclease activity in irradiated monkey CV-1 cells. 5 x 10' untreated cells were harvested from ten 100-mm dishes and from 10 dishes which had received 5 Gy 12 h earlier . Sonicates were prepared and the cell lysates were concentrated three-fold by use of Amicon 10 filters . Five milligrams of each preparation were fractionated on Superose 12 columns . After discarding the void volumes, 0. 5 ml fractions were collected . Aliquots (10 µl) of certain corresponding fractions shown in Figure 3A and B were assayed by measuring their ability to release intermediate mobility fragments from 5'- 32 P-end labelled a DNA given 400 Gy in 10 mm KI . The digested DNA was analysed on a 20% sequencing gel . The data plotted correspond with the densitometer values obtained in an autoradiogram of DNA fragments whose mobility was intermediate between G-13 and T-14 (i .e . retarded G-13) : •, values from lysates of irradiated cells ; 0, values from lysates

of unirradiated cells ; values obtained at fragments intermediate between T-14 and T-15 (i .e . retarded T-14) : A, released by lysates from irradiated cells ; A, released by lysates from unirradiated cells .



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RETARDED A-12 FRAGMENTS (Intermediate mobility)

A LYSATES FROM UNIRRADIATED CV-1 CELLS

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Radiation-enhanced excision repair elimination and strand scission at sites of damaged bases . These results indicated that the Superose 12 fraction 26 from irradiated cells was up to 2 . 9-fold more active in releasing intermediate mobility fragments from radiation damaged DNA . This was confirmed in a similar experiment (Figure 3) . Superose 12 fractions from irradiated and unirradiated CV-1 cells were compared . The amounts of DNA of intermediate mobility released at the sequence site of guanine, 13 nucleotides from the 5'-end, were determined autoradiographically . Fractions from irradiated cells (Figure 3A) were approximately threefold more active in releasing intermediate fragments at sequence position G-13 than fractions from unirradiated cells (Figure 3B) . They were nearly twofold more active in releasing intermediate mobility fragments at thymine, 14 nucloetides from the 5'-end . EDTA resistance of the endonuclease activity allowed it to be determined without interference from exonucleases and other enzymes which require divalent cations . When 3 mm Mg + 2 was included in the enzyme digestion buffer, results similar to those of Figure 1 were obtained, but they were more difficult to interpret (not shown) . 3.2. ED TA-resistant endonuclease activity from irradiated and unirradiated cells in 60-70 kDa proteins Cell lysates from 7 x 10 8 CV-1 cells which had been given 5 Gy 12 h before harvesting were fractionated by size on a 100-cm Ultrogel AcA-54 column . Lysates from an equal number of unirradiated cells were processed identically . Figures 4A and B show that most of the endonuclease activity

433

was present in lysate fractions corresponding to 60-70 kDa . Excision repair activity from lysates of irradiated CV-1 cells exited from the Ultrogel column as 60 kDa proteins while the active enzymes from unirradiated cells were 70 kDa in size . With mouse Krebs II cell lysates a similar difference was found (Figure 6) . The differences in size are small but they appear significant. They are not yet understood . A minor amount of endonuclease activity was recovered from irradiated cells in fractions corresponding to 35 kDa . The enhancement of enzyme activity by radiation was only N 30% above control values in this experiment, in contrast to the three-fold or more enhancement in the separate studies of Figures 1, 3 and 6 . The causes of variability in enhancement are not yet understood . Figure 4A and B clearly demonstrate the size of the active proteins as 60-70 kDa . EDTA resistance is common to these enzymes and to endonuclease III of E. coli. Estimates of the quantity of DNA of each size class which were released from irradiated 95 by ocDNA substrate by lysates were made by correlating gel autoradiographic density with corresponding radiolabel . The linear relationship between radiolabel and autoradiographic density made the estimates feasible . We estimated that the retarded DNA fragments at A-12 and T-17 corresponded to 1 . 0 x 10 -3 pmol (or 8 x 10 -5 pmol ends) and 0 . 47 x 10 -3 pmol (or 4 x 10 -5 pmol ends) respectively . 3.3. Concentration and characterization of radiationenhanced endonuclease activity Active fractions from AcA-54 filtration of lysates from the irradiated and unirradiated cells of the

Figure 4 . Endonuclease activity in irradiated and unirradiated CV-1 cells is associated with 60 kDa protein . Sonicates were prepared from a total of 7 x 10 8 CV-1 cells grown in 135 100-mm Petri dishes. The cells had been given 5 Gy 12 h before harvest . Unirradiated cells from 135 dishes were also harvested, sonicated and otherwise processed identically . Nucleic acids were removed from the sonicates using DEAE sepharose columns . After this step, 26 mg of each sonicate from unirradiated or irradiated cells were applied to a 1 .6 x 100 cm Ultrogel AcA-54 column . Fractions were eluted with buffer containing 50 mm Tris pH 7 . 8, 5 mm DTT, I mm EDTA, 5% glycerol and 500 mm NaCl . After the void volume of 60 ml was discarded, 60 fractions of 2 . 3 ml each were collected ; protein determinations were done and enzyme activity was tested on 32 P-95 by aDNA irradiated in 10 mm KI at 400 Gy . Densitometer scans were done and density units were obtained for intermediate mobility fragments (Figure 4A at A- 12 and Figure 4B at T- 17) released by fractions R34, 36, 38, 40, 42 ( A), and C34, 36, 38, 40 and 42 (A) . It was estimated that 1 . 25 x 10 -3 pmol of retarded A- 12 (Figure 4A) and 0 . 44 x 10 -3 pmol of retarded T-17 (Figure 4B) were released . (A) lysate from irradiated CV-1 cells ; (A) lysate from unirradiated CV-1 cells . A linear relationship between radiolabel in the gel and autoradiographic density for this experiment was obtained ; e .g. 30 min exposure of gel lanes with 3000 or 1500 cpm gave corresponding densities of 32 . 2 and 16. 7 units . The substrate for each gel lane was 2 x 10 5 cpm, i .e . 0 . 1 pg of DNA . The AcA-54 column was calibrated by eluting bovine serum albumin (-66 kDa), carbonic anhydrase (- 30 kDa) and cytochrome C (- 12 . 4 kDa) . We estimated from the calibration curve (not shown) that fractions 34, 36, 38, 40, 42 and 48 contain proteins of 77, 70, 60 . 5, 55, 50 and 36 kDa, respectively .

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experiment of Figure 4A and B were concentrated and their endonuclease activities were compared (Figure 5) . Their endonuclease activities were measured by release of intermediate mobility fragments . Intermediate fragments produced by irradiated cell lysates were 4 . 0-fold greater at A-12 and 4 . 6-fold greater at T-17 than fragments released by unirradiated cell lysates . In Figure 5, compare lane 8 (irradiated DNA digested with lysate fraction 40 from unirradiated cells) with lane 10 (irradiated DNA digested with lysate fraction 40 from irradiated cells) . Since we achieved an eight-fold protein concentration by Amicon concentration of fractions 40, we might have expected an eight-fold increase in its endonuclease activity. In fact, it was increased 4 . 0-

4.6-fold, suggesting some loss of activity during purification . We cannot readily account for the 4 . 0 and 4 . 6-fold higher specific activity of excision activity in Amicon concentrated Ultrogel fractions of cell lysates from irradiated cells compared with preparations from unirradiated cells . Overall, the Ultrogel fractions were only - 30% more active (Figure 4) . This result could reflect differences in elution profiles from the same column . It may reflect (as we believe) a decrease in the actual size of cellular excision endonucleases in lysates from irradiated cells so that same-numbered fractions from the preparations do not correspond . It may also indicate enhancement (or inhibition) of enzyme activities by

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-G23 -G22 -C21 -A20 -A19 -G18 -T17 -T16 -T15 -T14 -G13 -A12 Figure 5 . Enhanced endonuclease activity in 60 kDa proteins from irradiated CV-1 cells . AcA-54 Ultrogel fractions 38 and 40 from irradiated or unirradiated cells of the experiment of Figure 4 were assayed in endonuclease digestion buffer . 5'- 32P-end-labelled 95 by a DNA was used as substrate . The Maxam and Gilbert G standard and the radiation-induced doublets produced by irradiating the DNA with 100 Gy in H20 are shown in the two lanes at the right with the base sequence . The figure is from a 7-day exposure of the autoradiogram. Lanes 1-16 each received 2 x 10 5 cpm of labelled DNA . To prepare irradiated substrate, 95 by a DNA received 400 Gy in 10 mm KI . Irradiated substrate was used for the reactions of lanes 3, 4, 6, 8, 10, 12, 14, 16 . The digestions were done with the lysate fractions from Figure 4 : lane 3, no enzyme ; lane 4, 4µg concentrated from fraction 38 (unirradiated cells) ; lane 6, 4pg concentrated from fraction 38 (irradiated cells) ; lane 8, 12 pg fraction concentrated from fraction 40 (unirradiated cells) ; lane 10, 12 pg concentrated from fraction 40 (irradiated cells) ; lane 12, 0 . 8 pg unconcentrated fraction 40 (unirradiated cells) ; lane 14, 1 . 5 pg unconcentrated fraction 40 (irradiated cells) ; lane 16, 200 ng endonuclease III of E . coli. Unirradiated substrate was used for the reactions of lanes 1, 2, 5, 7, 9, 11, 13 and 15 . Digestions were done with lysate fractions from Figure 4 : lane 1, no enzyme ; lane 2, 4µg concentrated fraction 38 (unirradiated cells) ; lane 5, 4pg concentrated fraction 38 (irradiated cells) ; lane 7, 12 µg concentrated fraction 40 (unirradiated cells) ; lane 9, 12 pg concentrated fraction 40 (irradiated cells) ; lane 11, 0 . 8µg unconcentrated fraction 40 (unirradiated cells) ; lane 13, 1 . 5 µg unconcentrated fraction 40 (irradiated cells) ; lane 15, 200 ng endonuclease III of E. coli.



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M • M M T and A -+ C substitution (Cheng et al. 1992) . Its presence in double-stranded DNA is recognized by the FPG protein (8-oxoguanine DNA glycosylase), which excises the damaged base and by an associated endonuclease activity which incises the strand (Tchou et al . 1991) . To determine the endonuclease specificity of the cell lysates described here, we measured their ability to recognize and endonucleolytically cleave a 23 nt long synthetic substrate with a single 8-oxyguanine 11 bases from the 5'-end . Without further purification of cell lysates we could not determine if the results obtained were due to several enzymes or to multiple activities of individual enzymes . Figure 7A and B demonstrate release of incised products from 5'- 32P-end labelled 8-oxoguanine containing duplexes by the lysate fractions previously studied in the experiment of Figure 3 . The amount of substrate which was converted is expressed as a fraction of the original substrate ; it is shown in Figure 7B as the number of pmols released during a 10-min incubation . Lysates from irradiated and unirradiated cells were nearly equal in activity with this substrate, unlike results in Figures 1 and 3-5 . The DNA products' electrophoretic mobilities were slightly less than that of the product released by authentic FPG protein, seen in Figure 7A . The cell lysate fractions released > 25-fold more product from the synthetic substrate than from irradiated ocDNA, comparing results of Figures 7 and 4 . Cell lysate activity was destroyed by heating for 2 min at 90°C . Aspecific activity was checked by testing lysate fractions from irradiated and unirradiated cells on unirradiated DNA, in the experiment of Figure 5 . A background was found, some of which is likely due to self irradiation of the DNA substrate from its 5'- 32P-end label . For the 8-oxoguanine containing oligodeoxynucleotide duplex we found only one major and two

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minor products . Aspecific effects might have been expected by scissions at sequence sites containing unmodified G but for FPG protein this has been excluded by (Tchou et al . 1991) . We found lytic activity only at 8-oxoG when cell lysate fractions were tested . 3.6. 60 kDa lysate fractions from irradiated Krebs II mouse ascites tumour cells recognize 8-oxodG and are more active than lysates from unirradiated cells

The AcA-54 cell lysate fractions from cells of irradiated mice described in Figure 6 were examined using the 8-oxoguanine containing synthetic oligodeoxynucleotide described earlier (Figure 8A) . Lysates from irradiated cells were 2-6-fold more active. This data is demonstrated quantitatively in Figure 8B. The major product released by the mouse cell lysate fractions co-migrated with the fragments released by FPG protein, i.e. fragments with 3'phosphoryl ends . Fragments of intermediate mobility were less abundant with lysate fractions from the mouse cells than with lysates from CV-1 cells . 4. Discussion Our results are the first to demonstrate enhanced DNA excision repair of ionizing radiation damage induced in animal cells by ionizing radiation . The enzymes described here were only purified 25-50fold by gel filtration and are likely to contain a spectrum of excision and repair enzymes (Robins et al . 1991) . The 60-70 kDa EDTA resistant endonuclease activity found in monkey CV-1 cells and Krebs II mouse ascites tumour cells is similar to the 60 kDa activity isolated from human CEM-Cl lymphoblasts (Lee et al. 1987) . However, the scission products released by the human enzyme were consistent with species that contain a 3'-terminal phosphoryl group, while the partially purified lysates described in this report released DNA fragments with the mobilities of fragments that contain 3'-terminal base-free sugars as well as fragments with 3'-phosphoryl ter-

Figure 8(b) . Quantitation of DNA released by digestion with the Ultrogel mouse lysate fractions of Figure 8(a) . The amount of lysate product that comigrated with the FPG protein product (lane PFG on Figure 8(a)) was correlated with the fraction tested . An estimate of the number of pmols released was made by comparing the autoradiographic density corresponding to 10 nt long fragments (lanes 9-14 of Figure 8 (a)) after 6 h of exposure . Each of these densities was related to the densities obtained from its corresponding undigested 23-mer DNA substrate, determined by 2 min of exposure of the same autoradiogram . Aliquots from cells of irradiated mice were approximately three-fold higher in protein concentration ; the values obtained with lysates from the unirradiated cells were normalized, represented by the thin line and open circles .

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R . Bases et al .

mini . Further purification of these lysates is needed to learn if the fractions contain multiple enzymes . A calf thymus Mg t+ dependent 37kDa lymphoblast enzyme has been described which leaves a 3'hydroxyl group (Sanderson et al. 1989) . An EDTAresistant - 30 kDa calf thymus repair endonuclease which incised damaged DNA both 3' and 5' to the AP site left a nucleoside free site flanked by 3' and 5' terminal phosphate groups (Doetsch et al . 1986) . The lysates from monkey CV-1 cells recognized 8oxoguanines. While characterization of the products will be instructive, so far it seems unlikely from results of Figure 7 that 8-oxoguanine is the sole substrate for the enzyme activity which was enhanced by pre-irradiation as shown in Figure 1-3 . The lysates we have described appear to contain enzymes unlike those described by others, but further purification will be needed before critical comparisons can be made. Lysates from Krebs II mouse cells also recognised 8-oxoguanine as a substrate . They released fragments with 3'-phosphoryl ends and exhibited more than three-fold enhancement of activity when they were pre-irradiated . Release of DNA fragments of intermediate mobility was less than with monkey CV-1 cells . Recently, an endonuclease activity from human polymorphonuclear leukocytes was found which removes 8-oxoguanine from a synthetic ds DNA substrate (Chung et al. 1991) . This activity was dependent upon divalent cations and it incised DNA simultaneously at phosphodiester bonds 5' and 3' to 8-oxoguanine, liberating fragments with free 3'hydroxyl groups or 5'-phosphoryl groups . Cheng et al . 1992, reported that 8-oxoguanine is mutagenic in certain systems ; it causes G -+ T or A ---> C substitution . The enhancement of endonuclease activity in cells by pre-irradiation was anticipated from results obtained with pre-irradiated CV-1 cells which had received 300 Gy and were then incubated for 1 h (Bases et al. 1990) . Intermediate mobility fragments and enhanced abundance of fragments with 3'phosphoryl termini were noted in pre-irradiated cells . Enhanced abundance of cDNA fragments with 3'-phosphoryl termini by lysate fractions from irradiated cells was also a regular finding . The assay system described here was 100-fold more sensitive than the usual plasmid nicking assays . However, it does require 3-4 days for the autoradiographic assays . It was close in sensitivity to the plasmid repair synthesis techniques of Wood et al. (1988) and Robins et al. (1991) . Non-enzymatic protein co-factors such as stress proteins may enhance enzyme activity (Gething and Sambrook 1992) ; single strand DNA binding pro-

teins have recently been described as cofactors in repair (Coverly et al. 1991) . The enhancement of excision endonuclease described here might be due to increased abundance of stress protein cofactors, rather than enhanced amounts of the repair enzymes . The arrest of cells in G-2 and slowing of their progression through S-phase are well-known effects of ionizing radiation . Twelve hours incubation after 5 Gy might be sufficient to account for cell cycle disturbances (increases) in levels of repair enzymes . Studies to evaluate this are now in progress. Laval has shown that a few Gy increased the level of 0 6-methylguanine-DNA-methyl transferase 5-6fold in tumour cells (Laval 1990, Habraken and Laval 1991) . Laval's results may be relevant to joint radiotherapy/chemotherapy protocols in clinical regimens because pre-radiation might actually confound the joint treatments . On the other hand, protraction in clinical radiation therapy has been found empirically to be beneficial for clinical outcomes . Our results may provide insights at the molecular level into the mechanisms of DNA repair which accompany split dose therapy .

Acknowledgements We thank Dr Arthur P. Grollman and J . Tchou, Department of Pharmacological Sciences, State University of New York at Stony Brook, NY 11794 for a generous gift of 8-oxoguanine DNA glycosylase and for advice and encouragement . We are grateful to Mr Robert Rieger and Dr Francis Johnson for synthesizing the oligonucleotides . This work was supported in part by CA36492 from NIH and Core Support Cancer Research Center NIH-NCI-P30 CA-13330 and the Rome Sisters Foundation . W.A .F. was supported by NIH grant CA52025 .

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