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Biological inactivation of pBR322 plasmid DNA by enzyme- and radiation-induced single-strand damage under various conditions
DNA Repair
ELSEVIER
Mutation Research, DNA Repair 315 (1994) 65-74
Biological inactivation of pBR322 plasmid D N A by enzyme- and radiation-induced single-strand damage under various conditions Yvonne Ventur, Dietrich Schulte-Frohlinde
*
Max-Planek Institut fiir Strahlenchemie, Stiftstrasse 34-36, D-45470Miilheim a.d. Ruhr, Germany
Received 13 August 1993; revision received 4 February 1994; accepted 15 March 1994
Abstract The influence of three different kinds of single-strand breaks (ssb) on the biological activity of plasmid D N A (pBR322) was studied. The single-strand breaks were produced either by y-irradiation (together with base and sugar damage) or by DNase I digestion which introduced ligatable ssb. Non-ligatable ssb - single-strand gaps of three nucleotides in length - were generated in the nicked D N A by exonuclease III treatment. The biological activity ( N / N o) of this damaged D N A was assessed in vivo by transformation of E. coli (CMK) repair wild-type cells. The activity of the enzymes of E. coli was studied in vitro by incubation in a protein extract of E. coli making use of an in vitro assay introduced earlier, which makes it possible to distinguish between enzymatic degradation (dsb formation) and repair of damaged plasmid DNA. The biological activity (D37) of D N A with non-ligatable ssb, as determined by electrotransformation, was about 56% lower than that of D N A with ligatable ssb. The biological activity of enzymatically damaged D N A is greater in calcium-treated cells than in electroporated cells. It is proposed that this is due to a calcium-dependent inhibition of nucleases. In contrast to the enzymatically damaged DNA, with y-radiation-damaged D N A a calcium-dependent increase in survival was not observed. Therefore, calcium-dependent nucleases do not play a role in the repair of damage produced by y-irradiation. The enzyme activity data show that the single-strand damages are either converted into dsb or repaired. A comparison of the efficiency of dsb formation in the extract for two of the single-strand damages is presented. The efficiency depends on the kind of damage and on the presence of cofactors, especially ATP and dNTPs. Key words: Ligatable and non-ligatable single-strand breaks; Transformation of plasmid DNA; Enzymatic doublestrand break formation; E. coli cell extract; Electroporation versus CaC12 transformation
I. Introduction * Corresponding author. Abbreviations: SSD, single-strand damage (sum of strandbreak and non-break damage); ssb, single-strand break; dsb, double-strand break; dsDNA, double-stranded DNA; exo, exonuclease; DNase, deoxyribonuclease; BSA, bovine serum albumin; dNTP, deoxyribonucleoside-5'-triphosphate; oc, open circle; lin, linear; sc, supercoiled.
It has b e e n g e n e r a l l y a c c e p t e d t h a t r e p r o d u c tive cell d e a t h o f E. coli a n d y e a s t is c o r r e l a t e d with o n e u n r e p a i r e d d o u b l e - s t r a n d b r e a k in t h e D N A in E. coli a n d y e a s t ( K a p l a n , 1966; van d e r S c h a n s et al., 1973; Boye a n d Krisch, 1980; F r a n -
0921-8777/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0921-8777(94)00016-Y
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Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65- 74
kenberg et al., 1981; Schulte-Frohlinde, 1987). It has further been proposed that in E. coli a significant proportion of these dsb are not produced directly by radiolysis but arise in the course of enzymatic repair of other lesions in DNA, possibly single-strand breaks (ssb) and base damages (Bonura et al., 1975; Bresler et al., 1984; Schulte-Frohlinde et al., 1991). There is evidence that the biological inactivation of vector D N A is induced predominantly by ssb and non-break damage, e.g. base and sugar damage (Moran and Wallace, 1985; SchulteFrohlinde et al., 1991, 1992; Ventur and SchulteFrohlinde, 1993). A detailed analysis has shown that the damage induced by y-irradiation, at the low scavenger concentration used in the present study, is mainly due to the reaction of randomly distributed O H radicals and the contribution of clustered damages is low (Schulte-Frohlinde et al., 1991, 1992). Conflicting data exist concerning the influence of single-strand breaks. Inactivation efficiencies for ssb range between 2% (Van der Schans et al., 1973, with PM2 and ~bX-174) and 13% (Kow et al., 1991, with ~bX-174). Recently it has been proposed that most of the biological inactivation of damaged plasmid DNA is due to the enzymatic conversion of the single-strand damages (SSD) into lethal damages (dsb) in the cell (Ventur and Schulte-Frohlinde, 1993). Our aim was to study the effect of different types of single-strand damage on the biological activity of transforming plasmid DNA. This activity was determined using two different transformation methods (calcium chloride assay and electroporation). Furthermore, the enzyme activity towards the different kinds of ssb was studied using a recently introduced assay (Ventur, 1992; Ventur and Schulte-Frohlinde, 1993). With this assay the conversion of single-strand damages into dsb is measured following incubation of damaged DNA in a protein extract from E. coli. The assay allows differentiation between degradation (dsb formation) and repair of dsb precursors. The results show that DNase I-produced ligatable ssb are converted into dsb with much smaller yield than y-radiation-induced damages. Furthermore, it is shown that the properties of the extract depend strongly on the presence of
cofactors like Mg 2+, Ca 2+, ATP and nucleoside triphosphates.
2. Materials and methods
Bacterial strains and plasmid DNA Wild-type E. coli CMK (F +, hsdRK+, hsdMK+, lacY, thr, leu, thi, tonA, supE, iambda s) was obtained from Bien et al. (1988). The plasmid pBR322 (Bolivar et al., 1977) was purchased from Boehringer, Mannheim. Enzymes and chemicals DNase I from bovine pancreas (EC 3.1.21.1), exonuclease III (EC 3.1.11.2) ATP, NAD and the dNTPs were all from Boehringer, Mannheim. Gamma-irradiation of plasmid DNA 6°Co y-irradiation of plasmid DNA was performed in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0 according to Sambrook et al., 1989), DNA concentration 10 mg/1, at room temperature and at a dose rate of 45.8 Gy/min. Samples were taken at appropriate times and the plasmid DNA was separated on agarose gels into three fractions: the supercoiled circular form (sc), the open circular form (oc) and the linear form (lin). The gels were stained with ethidium bromide and photographed. The amount of each form was determined by densitometric evaluation on the film. It was assumed that breaks are distributed randomly and that all other prerequisites for using a Poisson distribution are fulfilled. The amount of ssb was calculated from the 'surviving fraction' of the sc form and the amount of dsb from the surviving fraction of the sc + oc form. A linear dose dependence was measured for ssb formation. Above a dose of 300 Gy, where the amount of ssb could not be measured due to the low amount of the sc form, the yield of ssb was calculated by linear extrapolation of the ssb formation at low doses. Unirradiated pBR322 DNA contained 81% supercoiled and 19% open circle form, corresponding to 0.21 ssb, and this was taken into account in the calculation of ssb formation.
Y. Ventur, D. Schulte-Frohlinde/ Mutation Research 315 (1994) 65-74 DNase I digestion DNase I (0.2 n g / ~ g pBR322) digestion was performed in 50 mM Tris-HC1, 10 mM MgSO 4, 0.1 mM D T T and 50 ~ g / m l BSA at p H 7.5 and 37°C (Sambrook et al., 1989). At appropriate times, the reaction was stopped by addition of sodium citrate (final concentration 0.1 M) and the digestion was checked by agarose gel electrophoresis on 0.8% agarose slab gels in T B E buffer (Sambrook et al., 1989). The amount of ssb and dsb formation was determined densitometrically. Ssb formation shows a linear kinetics with time. Ssb values > 3 were calculated by linear extrapolation. Exonuclease III digestion DNase I-damaged D N A ( 2 0 / x g / m l ) was incubated with Exo III (1 U//~g DNA) in 66 mM Tris-HC1, 6.6 mM MgCI2, 1 mM 2-mercaptoethanol at p H 7.6 and 37°C for appropriate times (Sambrook et al., 1989). The reaction was terminated by addition of sodium citrate (final concentration 0.1 M) and heat inactivation for 10 rain at 70°C. The digestion was checked by gel electrophoresis on 0.8% agarose slab gels in T B E buffer. The conditions were such that about three nucleotides were removed from each single-strand break.
67
40). After centrifugation at 48 000 x g for 40 min, the supernatant was collected and the amount of protein was determined colorimetrically using the Bio-Rad Protein Assay. Only the supernatant but not the precipitate was active in producing dsb if Mg 2+ ions were present. This supernatant is called 'extract'. In vitro dsb formation assay Incubation under 'non-repair' conditions was performed with 1 m g / m l protein extract in 25 mM HEPES, 5 mM MgCI2, 80 mM KC1, 2 mM DTT, 50 / z g / m l BSA at 37°C and pH 7.6. This mixture is called 'supplemented extract' under 'non-repair' conditions. After incubation of damaged DNA (pBR322 2 0 / z g / m l ) for 20 min in this supplemented extract, the same volume of icecold sodium citrate solution (0.2 M, pH 7.5) was added to stop the reaction. To provide for 'repair' conditions the following cofactors were added (the final concentrations are indicated): 0.2 mM spermidine, 1.5 mM ATP, 0.5 mM NAD, 1 mM glutathione and 0.1 mM (each) of the four deoxyribonucleotide-5'-triphosphates (Ventur and Schulte-Frohlinde, 1993). The amount of enzyme-induced dsb was determined by densitometric analysis of agarose gels.
3. Results
Transformation (I) Calcium chloride transformation formed according to Bien et al. (1988). (II) Electroporation of E. coli CMK performed according to Hartke and Frohlinde (1991). D N A concentration /xg/l.
was percells was Schultewas 6.25
Preparation of a protein extract from E. coli bacteria Crude cell extract was prepared from E. coli CMK according to Lu et al. (1983) with some modifications. Briefly, bacteria were grown in TBY to O.D.ss 0 0.8-1 and washed twice in extract buffer (25 mM HEPES, 0.1 mM EDTA, pH 7.6). Crude cell extract was prepared by digestion of the bacterial cells with lysozyme followed by treatment with ultrasound (6 x 30 s, sonic power
pBR322 plasmid DNA was y-irradiated in T E (OH scavenger capacity = 3.1 × 107/s) with various doses. The biological inactivation of this damaged D N A was determined by electroporation and the amount of radiation-induced single- and double-strand breaks by densitometric analysis of agarose gels (Fig. 1). The results show that the biological inactivation is lower than the number of ssb per molecule but much larger than the number of dsb per molecule. This is evidence that the biological inactivation of the plasmid DNA is mainly due to single-strand damages (SSD) and only to a minor extent to radiation-induced double-strand breaks (see also Introduction). To assess the inactivation efficiency of yirradiation- as well as enzyme-induced SSD, repair-proficient E. coli (CMK) hosts were trans-
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Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74 7 ,
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0
400
800
1200
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dose [ gray ] Fig. 1. Biological inactivation, and single- and double-strand break formation of y-irradiated pBR322 plasmid D N A as a function of dose. Transformation of E. coli C M K was carried out by electroporation. Concentration of D N A in TE for electroporation 6.25 /xg/l. T h e O H scavenger capacity for irradiation was 3.1 × 107/s, D N A concentration 10 m g / l .
formed by electroporation with pBR322 plasmid D N A containing these lesions. Fig. 2 shows the different survival curves plotted versus the ssb. It can be seen that with all types of lesions survival
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Fig. 2. Survival of transforming pBR322 plasmid D N A with different types of SSD in E. coli C M K wild-type bacteria as a function of ssb. Ligatable ssb were produced by DNase I digestion of the plasmid D N A , non-ligatable ssb are singlestrand gaps of three nucleotides in length which were generated in the nicked D N A by exonuclease III treatment, yirradiation was performed in T E buffer. Transformation was carried out by electroporation.
non-ligatable ssb
T-irradiation
Fig. 3. Comparison of single-strand breaks per molecule per lethal dose from D37 values for electroporation and CaCI 2 transformation of E. coli C M K with damaged plasmid DNA. For irradiation conditions see legend of Fig. 1.
decreases with ssb, but the D 3 7 values are quite different. Ligatable ssb which are produced by DNase I treatment have a low inactivation efficiency. Inactivation of plasmid D N A with nonligatable ssb is 56.1% higher than that for ligatable ssb (ratio of D37 values is 2.3, see Fig. 2). The highest inactivation efficiency - referring to the amount of single-strand breaks - was found for y-irradiated plasmid D N A (Fig. 2). Fig. 3 shows the number of ssb per lethal event for the different types of lesions as measured by the calcium chloride method in comparison to electroporation data. It can be seen that for plasmid D N A containing DNase I-induced ssb the number of ssb per lethal event is much larger than the corresponding ratio for non-ligatable ssb. For y-radiation-induced ssb (together with base and sugar damage) no difference was found (Fig. 3). These data indicate that calcium ions have an inhibitory effect on the biological inactivation of enzymatically damaged plasmid D N A containing single-strand breaks but not on the inactivation of y-irradiated DNA. This inhibitory effect of calcium ions was further investigated using an in vitro assay which makes it possible to distinguish between enzymatic dsb formation and repair of precursors in plasmid D N A (Ventur, 1992; Ventur and Schulte-Frohlinde, 1993). For this assay the damaged
}1.. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65- 74
69
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Fig. 4. Effect of calcium ions on the rate of extract-induced dsb formation. D N a s e I-damaged pBR322 DNA (8.4 ssb per molecule) was incubated for 20 min at 37°C with extract (1 mg protein/ml) without additional supplementation (non-repair conditions), with 10 mM MgCI 2 and increasing concentrations of CaC12. Incubation with HEPES buffer and with extract without additional magnesium served as controls.
plasmid D N A is incubated in a protein extract of E. coli and the enzymatic formation of dsb is measured. Incubation of D N a s e I-damaged plasmid D N A in the extract did not induce any dsb formation if no Mg 2÷ ions were added (extract without Mg) (Fig. 4). Addition of 10 m M MgC12 led to a pronounced increase in dsb formation by the extract. This dsb formation was progressively inhibited by the addition of increasing amounts of Ca 2÷ ions. At 30 m M CaCI 2 the dsb formation was decreased to the same level as the buffer control (Fig. 4). It should be noted that, with other E. coli strains, e.g. ABl157, at incubation times longer than shown in the figures an increasing proportion of the D N A was degraded. We therefore used extracts prepared from strain CMK which is also a repair wild type as is ABl157. We thank Ms Helene Steffen for these measuremeats. The above results support the view that the higher biological activity of D N a s e I-damaged plasmid D N A , using the calcium chloride transformation method in comparison to electroporation, is caused by a calcium-mediated inhibition of enzyme-induced dsb formation. The formation of dsb from single-strand damages was also reduced when the E. coli extract was supplemented
-ATP
controls
-glutsthione
-NAD
Fig. 5. Influence of cofactors on the dsb formation in 3,-irradiated plasmid D N A by incubation in a protein extract of E. coli. The irradiated DNA (503.1 Gy) was incubated in the extract (1 mg protein/ml) with or without supplementation by deoxyribonucleotides for 20 min at 37°C. Complete repair conditions were achieved by addition of all four deoxyribonucleotides (0.1 mM), and of spermidine (0.2 mM), glutathione (1 mM), ATP (1.5 mM) and NAD (0.5 mM). The missing component is marked under each couple of bars. Incubation without nucleotides and cofactors served as a control (buffer).
with certain cofactors. The influence of various cofactors was studied in detail (Figs. 5 and 6). From these investigations it becomes obvious that deoxyribonucleotide-5'-triphosphates ' and A T P are indeed the critical components which must be added to reduce degradation processes. This re-
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Fig. 6. Effect of the ATP concentration on the extract-induced dsb formation. Incubation with all cofactors and nucleotides served as a control.
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Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74 per molecule 6
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Fig. 7, Dsb formaton in y-irradiated plasmid DNA as a function of y-ray dose after incubation for 20 min with E. coli CMK extract under repair and non-repair conditions.
duction of dsb formation could be due to the repair of precursors of dsb or to a suppression of the incision reaction. However, lower dsb formation takes place when the extract is supplemented with just those cofactors which are needed for enzymatic repair (e.g. mainly A T P and deoxyribonucleotide-5'-triphosphates). Therefore, we distinguished two conditions for the usage of the assay. With supplementation of the essential additives, A T P and dNTPs, the condition is designated 'repair condition' and without this supplementation it is designated 'non-repair condition'.
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4. Discussion The present p a p e r deals with the behavior of three different types of single-strand breaks in plasmid D N A towards two different analytical probes. The first probe is a determination of survival by different transformation methods (the CaCI 2 method and electroporation), and the second is m e a s u r e m e n t of the conversion of ssb into dsb by incubating the damaged plasmid D N A in a protein extracts of E. coli. The results are discussed in three sections. Comparison o f biological inactiuation by different types o f ssb
i
_=
We have now used this assay to study differences between D N A with DNase I-induced ssb and radiation-induced ssb (the latter with nonbreak base and sugar damage also present). The comparison was made under repair and under non-repair conditions. Under both conditions the results show that (i) dsb formation is larger per ssb for radiationinduced ssb than for enzyme-induced ssb and that (ii) the dependence on the number of ssb per molecule is different (compare Figs. 7 and 8). Under repair conditions the formation of dsb as a function of the number of DNase I-induced ssb is linear whereas that for y-radiation-induced ssb is curvilinear.
~
2
4 ssb
......... per
6 molecule
8
10
Fig. 8. Dsb formation in DNase I-damaged plasmid DNA after incubation for 20 min with E. coli CMK extract under repair and non-repair conditions.
The low level of inactivation efficiency of DNase I-induced, ligatable ssb for plasmid transformation by electroporation (Fig. 2) corresponds with data from Laipis et al. (1969) and Kow et al. (1991) who used other transformation procedures. The low values are suggested to be due to an efficient, ligation-mediated repair. Repair of dsb virtually does not occur under our conditions because each cell is infected by only a single copy of plasmid D N A (see Bien et al., 1988). To exclude repair by ligation, single-strand gaps (non-ligatable ssb) of three nucleotides in length were generated in the nicked plasmid D N A by exonuclease III treatment (Thomas and Olivera, 1978; Prell and Wackernagel, 1980). Trans-
Z Ventur, D. Schulte-Frohlinde/MutationResearch 315 (1994) 65-74 formation by electroporation with such a D N A led to a 56% lower survival as compared to plasmid D N A with ligatable ssb at D37 with D37 = 21.4 and 9.4 ssb per molecule, respectively (Fig. 2). The D37 values were used because the survival curves are not linear, though of similar shape. These results are in line with those from Kow et al. (1991) insofar as non-ligatable breaks are more difficult to repair than ligatable breaks. The same authors have shown that the difference in survival of the plasmid D N A with ligatable and non-ligatable ssb is a factor of 8-12, whereas we found only a factor of slightly more than 2. A possible explanation is that our non-ligatable breaks are different from theirs and furthermore they used thX174 D N A and we used plasmid pBR322 DNA. Their non-ligatable breaks contain end groups which cannot be used directly for polymerization and have first to be removed before polymerization and ligation can occur and repair can be accomplished. In our case the non-ligatability is due to a gap without unusual end groups. In order to achieve repair only polymerization and ligation has to take place. This is at least one enzymatic step less than in the case of Kow et al. (1991). This may explain the greater difference between the repairability of ligatable and nonligatable breaks in their and our case. It has been shown that several kinds of base damage have the same inactivation efficiency as non-ligatable ssb (Kow et al., 1991). Assuming that this is also valid for radiation-induced base and sugar damage and ssb, it is possible to estimate the contribution of non-break damage to inactivation of y-irradiated plasmid D N A by calculating the ratio of ssb to non-break damage by comparison of the D37 values for non-ligatable enzyme-induced ssb (9.4 ssb/lethal event) with those for y-irradiation-induced damage (3.7 ssb/lethal event). From these values the ratio of ssb to non-break damage was calculated as 1 : 1.54 which means that the contribution of non-break damages to the lethal damage for y-irradiated plasmid D N A is about 60%. This result is consistent with the involvement of base and sugar damages in the biological inactivation of y-irradiated plasmid DNA. This result may be compared with in vitro data
71
from Kohfeld et al. (1988) (1 : 1.8) obtained from y-irradiated A D N A and the use of y endonucleuse for the determination of enzyme-convertible lesions, and those from Strniste and Wallace (1975) (1:2.8) using PM2 D N A y-irradiated in T E and y endonuclease. The biological data may be further compared with the expected chemical yields of ssb and non-break damage. Since the efficiency of production of one ssb in ssDNA (similar in dsDNA) by an O H radical is around 20% (Schulte-Frohlinde et al., 1991), four O H radicals per ssb should produce base and sugar radicals in the DNA. The maximum number of base and sugar non-break-damaged sites per ssb is therefore = 4. If these radicals disappear by disproportionation the minimum number of base and sugar damages per ssb is 2. The values from our transformation measurements and from the in vitro data cited above are in the range of the lower ssb to nonbreak damage ratio. Lower values of 2 per ssb are possible if some kinds of base or sugar damages are better repairable than others or code similar to the original site. It should be mentioned that the distribution of the damage sites in the D N A plays an important role too (SchiJnemann and Schulte-Frohlinde, 1992). The influence of this distribution is not discussed in the present paper. Comparison of CaCl 2 transformation with transformation by electroporation If transformation of plasmid D N A is performed by the calcium chloride method, the inactivation efficiency for ligatable and non-ligatable enzyme-induced ssb is much lower than by electroporation (Fig. 3). One of the main differences between the calcium chloride method and electroporation is the presence of calcium ions during rupture of the membrane of E. coli in the former method. For an explanation we propose that calcium ions enter the E. coli cells during the transformation procedure and that these calcium ions inhibit the action of endo- and exonucleases. Such a calcium-dependent inhibition of certain endoand exonucleases is well known (Rosamond et al., 1979; Williams et al., 1981; Kow, 1989). An inhibitory effect of calcium ions on the enzymatic degradation of enzymatically damaged plasmid
72
Y~ Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74
D N A is shown by the inhibition of dsb formation by calcium ions in an E. coli protein extract (Fig. 4). These results give evidence that enzyme-induced ssb and therefore indeed single-strand damages are converted into lethal damages by enzymatic degradation processes as proposed earlier (Ventur and Schulte-Frohlinde, 1993). Calcium-induced inhibition does not occur with the electroporation method because no calcium ions are present during this procedure. Results from Schulte-Frohlinde et al. (1993) indicated that one main enzyme which is inhibited in E. coli by Ca 2+ is the RecBC enzyme complex, and that this nuclease is involved in the degradation of enzyme-damaged DNA. A calcium ion-dependent inhibition of at least one of the actions of the RecBC enzyme complex was clearly shown to occur with purified enzymes o n phage T7 D N A (Rosamond et al., 1979). With radiation-damaged D N A no difference between the survival measured by the calcium chloride method and electroporation was observed (Fig. 3). It has recently been shown that the purified RecBC enzyme complex has ATPdependent double-strand exonuclease properties with y-irradiated D N A as substrate (Hickson et al., 1985). This may mean that either the RecBC enzyme complex is not inhibited by calcium ions when irradiation-induced damage in D N A is the substrate (as opposed to enzyme,induced damage), or other kinds of degrading enzymes take over in the cell. Many endonuclease activities which can convert radiation-induced base and sugar damages inside the E. coli cell into ssb are not sensitive to calcium inhibition (Kow, 1989). An indication in favor of the possibility that the RecBC enzyme complex might not be sensitive to calcium ions when y-irradiated D N A is the substrate, is that Brcic-Kostic et al. (1991) found that the R e c B C D enzyme complex is involved in the degradation (and repair) of y-irradiated plasmid D N A in E. coli. However, it should be added that the RecBC enzyme complex has several activities which are differently influenced by a change of reaction conditions, especially changes in the ratio of magnesium and calcium ions (Rosamund et al., 1979). These authors have also
reported that purified RecBC enzyme complexes degrade phage DNA, but do not solely produce dsb. However, this result is not at variance with our results since we do not use purified RecBC enzymes but a mixture of all enzymes extractable from E. coli cells. Furthermore, they measured intact and linear D N A but not radiation-damaged DNA. Their results do not indicate that they have measured the effect of incubation times on dsb formation and degradation because they only show results at long incubation times where degradation is complete. We found degradation also at long incubation times. However, a direct comparison of the times of incubation is not meaningful because of the different experimental conditions used. The difference in the properties of a mixture of enzymes, when enzyme-induced and y-irradiation-induced ssb are compared, is clearly due to the chemically different end groups involved in both cases (von Sonntag et al., 1981; von Sonntag, 1987; Obe et al., 1992). Many examples exist which show that a mixture of enzymes react differently with different substrates. One is that a cocktail of four different enzymes capable of degrading undamaged D N A into mononucleotides is not able to do so with y-irradiated D N A (Dizdaroglu et al., 1978).
Incubation in a protein extract o f E. coli With a new in vitro assay a separation of degradation and repair processes which are involved in the enzymatic processing of single-strand damages was achieved (Ventur and SchulteFrohlinde, 1993). By means of this assay we have directly proved that enzyme-, as well as y-irradiation-induced single-strand damages, were converted enzymatically into double-strand breaks by E. coli proteins. In recent experiments our assay was also employed to demonstrate dsb formation from uracil residues in M13 D N A (Schiinemann, 1993). Furthermore, we were able to show that different kinetics are involved in dsb formation under 'repair' and 'non-repair' conditions in the supplemented extracts. The difference between 'repair' and 'non-repair' conditions is due to supplementation by cofactors.
Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74
The data in the present work indicate that an extract from E. coli has a strong degradation activity. This degradation activity is greatly decreased by supplementation with ATP or the four deoxyribonucleotide-5'-triphosphates (Figs. 5 and 6). The fact that the addition of ATP and dNTPs is necessary to inhibit degradation indicates that this inhibition is due to repair. A further proof for the interpretation that the inhibition of dsb formation is due to repair is given by the experimental result of the better survival of T-irradiated plasmid D N A treated with the extract under repair conditions in comparison to the untreated control (Ventur and Schulte-Frohlinde, 1993). If the concentration of ATP is higher than 0.5 mM the addition of dNTPs is not necessary to achieve repair conditions. Addition of supplements to E. coli cell proteins was also necessary in order to achieve mismatch repair (Lu et al., 1983, 1984) and conversion of M13 D N A to its replicative form in vitro (Wickner et al., 1972). Our results also correspond with data from Matson and Bambara (1981) and Masker (1977) who were able to show that excision repair with a dialysed E. coli protein extract is dependent on exogenous dNTPs. This supplementation dependence of the different in vitro systems may be due to the dilution (50-100-fold) of the cellular components by the extract preparation procedure. In the diluted solution the concentration of proteins is high enough to achieve the biological functions but the concentration of cofactors might be too low. The results in the present paper support the proposal made earlier (Ventur and Schulte-Frohlinde, 1993) that in plasmid D N A y-irradiation and enzyme-induced SSD are basically either repaired or enzymatically converted into dsb. The degree of dsb formation depends on the repair capacity of the cell and the extract. The higher yields of dsb per ssb obtained with T-irradiated D N A in comparison with DNase Itreated D N A may have two reasons. First, the majority of the DNase I-produced ssb are repaired by ligation before conversion into dsb occurs. Second, y-irradiated D N A contains, in addition to ssb, base and sugar damage which may contribute to dsb formation.
73
An interesting difference between enzyme-induced ssb and radiation-induced ssb is found in the dependence of the yield of dsb on the number of ssb by incubation in an extract under repair conditions (Figs. 7 and 8). The formation of dsb is linear with enzyme-induced ssb but curvilinear with radiation-induced dsb. This result also points to different mechanisms in the enzymatic processing of the two kinds of damage compared here.
References Bien, M., H. Steffen and D. Schulte-Frohlinde (1988) Repair of the plasmid pBR322 damaged by y irradiation or by restriction endonucleases using different recombinationproficient E. coli strains, Mutation Res., 194, 193-205. Bolivar, F., R.L. Rodriguez, P.J. Greene, M.C. Betlach, H.L. Heyneker and H.W. Boyer (1977) Construction and characterization of new cloning vehicles, II. A multipurpose cloning systems, Gene, 2, 95-113. Bonura, T., K.C. Smith and H.S. Kaplan (1975) Enzymatic induction of DNA double-strand breaks in y-irradiated Escherichia coli K-12, Proc. Natl. Acad. Sci. USA, 72, 4265 -4269. Boye, E. and R.E. Krisch (1980) Induction and repair of double-and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli, Int. J. Radiat. Biol., 37, 119-133. Bresler, S.E., L.A. Noskin and A.V. Suslov (1984) Induction by gamma irradiation of double-strand breaks of Escherichia coli chromosomes and their role in cell lethality, Biophys. J., 45, 749-754. Brcic-Kostic, K., E. Salaj-Smic, N. Marsic, S. Kajic, I. Stojiljkovic and Z. Trgovcevic (1991) Interaction of RecBCD enzyme with DNA damaged by y radiation, Mol. Gen. Genet., 228, 136-142. Dizdaroglu, M., W. Hermes, D. Schulte-Frohlinde and C. von Sonntag (1978) Enzymatic digestion of DNA y-irradiated in aqueous solution. Separation of the digest by ion exchange chromatography, Int. J. Radiat. Biol., 33, 563-569. Frankenberg, D., M. Frankenberg-Schwager, D. Bl6cher and R. Harbich (1981) Evidence for DNA double-strand breaks as the critical lesion in yeast cells irradiated with sparsely or densely ionizing radiation under oxic or anoxic conditions, Radiat. Res,, 88, 524-532. Hartke, A. and D. Schulte-Frohlinde (1991) Survival of M13mpl8 gapped duplex DNA as a function of gap length, Mutation Res., 255, 39-48. Hickson, I.D., C.N. Robson, K.E. Atkinson, L. Hutton and P.T. Emmerson (1985) Reconstitution of RecBC DNase activity from purified _+RecB and RecC proteins, J. Biol. Chem., 260, 1224-1229.
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}1.. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74
Kaplan, H.S. (1966) DNA-strand scission and loss of viability after X irradiation of normal and sensitized bacterial cells, Proc. Natl. Acad. Sci. USA, 55, 1442-1446. Kohfeld, E., H. Bertram and U. Hagen (1988) Action of gamma endonuclease on clustered lesions in irradiated DNA, Radiat. Environ. Biophys., 27, 123-132. Kow, Y.W. (1989) Mechanism of action of Escherichia coli endonuclease III, Biochemistry, 28, 3280-3287. Kow, Y.W., G. Faundez, R.J. Melamede and S.S. Wallace (1991) Processing of model single-strand breaks in &X-174 RF transfecting DNA by Escherichia coli, Radiat. Res., 126, 357-366. Laipis, P.J., B.M. Olivera and A.T. Ganesan (1969) Enzymatic cleavage and repair of transforming DNA, Proc. Natl. Acad. Sci. USA, 62, 289-296. Lu, A.-L., S. Clark and P. Modrich (1983) Methyl-directed repair of DNA base-pair mismatches in vitro, Proc. Natl. Acad. Sci. USA, 80, 4639-4643. Lu, A.-L., K. Welsh, S. Clark, S.-S. Su and P. Modrich (1984) Repair of DNA base-pair mismatches in extracts of Escherichia coli, Cold Spring Harbor Symp. Quant. Biol., 49, 589-596. Masker, W.E. (1977) Deoxyribonucleic acid repair in vitro by extracts of Escherichia coil, J. Bacteriol., 129, 1415-1423. Matson, S.W. and R.A. Bambara (1981) Short deoxyribonucleic acid repair patch length in Escherichia coli is determined by the processive mechanism of deoxyribonucleic acid polymerase I, J. Bacteriol., 146, 275-284. Moran, E. and S.S. Wallace (1985) The role of specific DNA base damages in the X ray-induced inactivation of bacteriophage PM2, Mutation Res., 146, 229-241. Obe, G., C. Johannes and D. Schulte-Frohlinde (1992) DNA double-strand breaks induced by sparsely ionizing radiation and endonucleases as critical lesions for cell death, chromosomal aberrations, mutations and oncogenic transformation, Mutagenesis, 7, 3-12. Prell, A. and W. Wackernagel (1980) Degradation of linear and circular DNA with gaps by the recBC enzyme of Escherichia coli, Eur. J. Biochem., 105, 109-116. Rosamond, J., K.M. Telander and S. Linn (1979) Modulation of the action of the recBC enzyme of Escherichia coli K-12 by Ca 2+, J. Biol. Chem., 254, 8646-8652. Sambrook, J., E.F. Fritsch and T. Maniatis (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Schulte-Frohlinde, D. (1987) DNA strand break formation and biological consequences, Radiat. Res., 115, 158-161. Schulte-Frohlinde, D., E. Bothe, U. Klimczak, D.C. Ludwig, F. Mark, P. Rettberg, S. Schiinemann, Y. Ventur and K.-H. Worm (1991) Radiation-induced damage und biological inactivation of plasmid DNA in water-alcohol mix-
tures: what is the lethal damage?, in: K.H. Chadwick, G. Moschini and M.N. Varma (Eds.), Biophysical Modelling of Radiation Effects, Hilger, New York, pp. 253-260. Schulte-Frohlinde, D., F. Mark and Y. Ventur (1992) Transformation frequency of y-irradiated plasmid DNA and the enzymatic double-strand break formation by incubation in a protein extract of E. coli, Eleventh Symposium on Microdosimetry, 13-18 September 1992, Gatlinburg, TN. Schulte-Frohlinde, D., K.-H. Worm and M. Merz (1993) Double-strand breaks in plasmid DNA and the induction of deletions, Mutation Res., 299, 233-250. Schiinemann, S. (1993) Biologische Aktivit~it uracilierter M13mpl8-DNA in Escherichia coli, Dissertation, Ruhr UniversitS_t Bochum. Schiinemann, S. and D. Schulte-Frohlinde (1992) Survival of phage M13 with uracils on one or both DNA strands, Mol. Gen. Genet., 233, 348-354. Strniste, G.F. and S.S. Wallace (1975) Endonucleolytic incision of X-irradiated deoxyribonucleic acid by extracts of Escherichia coil, Proc. Natl. Acad. Sci. USA, 72, 1997-2001. Thomas, K.R. and B.M. Olivera (1978) Processivity of DNA exonucleases, J. Biol. Chem., 253, 424-429. Van der Schans, G.P., J.F. Bleichrodt and J. Blok (1973) Contribution of various types of damage to inactivation of a biologically-active double-stranded circular DNA by gamma radiation, Int. J. Radiat. Biol., 23, 133-150. Ventur, Y. (1992) EinfluB von Einzelstrangschiiden auf die biologische Aktivit~it transformierender E. coli-DNA, Dissertation, Ruhr Universit~it Bochum. Ventur, Y. and D. Schulte-Frohlinde (1993) Does the enzymatic conversion of DNA single-strand damage into double-strand breaks contribute to biological inactivation of y-irradiated plasmid DNA?, Int. J. Radiat. Biol., 63, 167171. von Sonntag, C. (1987) The Chemical Basis of Radiation Biology, Taylor & Francis, London. von Sonntag, C., U. Hagen, A. Sch6n-Bopp and D. SchulteFrohlinde (1981) Radiation induced strand breaks in DNA: chemical and enzymatic analysis of end-groups and mechanistic aspects, in: J.T. Lett and H. Adler (Eds.), Advances in Radiation Biology, Vol. 9, Academic Press, New York, pp. 109-127. Wickner, W., D. Brutlac, R. Schekman and A. Kornberg (1972) RNA synthesis initiates in vitro conversion of MI3 DNA to its replicative form, Proc. Natl. Acad. Sci. USA, 69, 965-969. Williams, J.G.K., T. Shibata and C.M. Radding (1981) Escherichia coil RecA protein protects single-stranded DNA or gapped duplex DNA from degradation by RecBC DNase, J. Biol. Chem., 256, 7573-7582.
ELSEVIER
Mutation Research, DNA Repair 315 (1994) 65-74
Biological inactivation of pBR322 plasmid D N A by enzyme- and radiation-induced single-strand damage under various conditions Yvonne Ventur, Dietrich Schulte-Frohlinde
*
Max-Planek Institut fiir Strahlenchemie, Stiftstrasse 34-36, D-45470Miilheim a.d. Ruhr, Germany
Received 13 August 1993; revision received 4 February 1994; accepted 15 March 1994
Abstract The influence of three different kinds of single-strand breaks (ssb) on the biological activity of plasmid D N A (pBR322) was studied. The single-strand breaks were produced either by y-irradiation (together with base and sugar damage) or by DNase I digestion which introduced ligatable ssb. Non-ligatable ssb - single-strand gaps of three nucleotides in length - were generated in the nicked D N A by exonuclease III treatment. The biological activity ( N / N o) of this damaged D N A was assessed in vivo by transformation of E. coli (CMK) repair wild-type cells. The activity of the enzymes of E. coli was studied in vitro by incubation in a protein extract of E. coli making use of an in vitro assay introduced earlier, which makes it possible to distinguish between enzymatic degradation (dsb formation) and repair of damaged plasmid DNA. The biological activity (D37) of D N A with non-ligatable ssb, as determined by electrotransformation, was about 56% lower than that of D N A with ligatable ssb. The biological activity of enzymatically damaged D N A is greater in calcium-treated cells than in electroporated cells. It is proposed that this is due to a calcium-dependent inhibition of nucleases. In contrast to the enzymatically damaged DNA, with y-radiation-damaged D N A a calcium-dependent increase in survival was not observed. Therefore, calcium-dependent nucleases do not play a role in the repair of damage produced by y-irradiation. The enzyme activity data show that the single-strand damages are either converted into dsb or repaired. A comparison of the efficiency of dsb formation in the extract for two of the single-strand damages is presented. The efficiency depends on the kind of damage and on the presence of cofactors, especially ATP and dNTPs. Key words: Ligatable and non-ligatable single-strand breaks; Transformation of plasmid DNA; Enzymatic doublestrand break formation; E. coli cell extract; Electroporation versus CaC12 transformation
I. Introduction * Corresponding author. Abbreviations: SSD, single-strand damage (sum of strandbreak and non-break damage); ssb, single-strand break; dsb, double-strand break; dsDNA, double-stranded DNA; exo, exonuclease; DNase, deoxyribonuclease; BSA, bovine serum albumin; dNTP, deoxyribonucleoside-5'-triphosphate; oc, open circle; lin, linear; sc, supercoiled.
It has b e e n g e n e r a l l y a c c e p t e d t h a t r e p r o d u c tive cell d e a t h o f E. coli a n d y e a s t is c o r r e l a t e d with o n e u n r e p a i r e d d o u b l e - s t r a n d b r e a k in t h e D N A in E. coli a n d y e a s t ( K a p l a n , 1966; van d e r S c h a n s et al., 1973; Boye a n d Krisch, 1980; F r a n -
0921-8777/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0921-8777(94)00016-Y
66
Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65- 74
kenberg et al., 1981; Schulte-Frohlinde, 1987). It has further been proposed that in E. coli a significant proportion of these dsb are not produced directly by radiolysis but arise in the course of enzymatic repair of other lesions in DNA, possibly single-strand breaks (ssb) and base damages (Bonura et al., 1975; Bresler et al., 1984; Schulte-Frohlinde et al., 1991). There is evidence that the biological inactivation of vector D N A is induced predominantly by ssb and non-break damage, e.g. base and sugar damage (Moran and Wallace, 1985; SchulteFrohlinde et al., 1991, 1992; Ventur and SchulteFrohlinde, 1993). A detailed analysis has shown that the damage induced by y-irradiation, at the low scavenger concentration used in the present study, is mainly due to the reaction of randomly distributed O H radicals and the contribution of clustered damages is low (Schulte-Frohlinde et al., 1991, 1992). Conflicting data exist concerning the influence of single-strand breaks. Inactivation efficiencies for ssb range between 2% (Van der Schans et al., 1973, with PM2 and ~bX-174) and 13% (Kow et al., 1991, with ~bX-174). Recently it has been proposed that most of the biological inactivation of damaged plasmid DNA is due to the enzymatic conversion of the single-strand damages (SSD) into lethal damages (dsb) in the cell (Ventur and Schulte-Frohlinde, 1993). Our aim was to study the effect of different types of single-strand damage on the biological activity of transforming plasmid DNA. This activity was determined using two different transformation methods (calcium chloride assay and electroporation). Furthermore, the enzyme activity towards the different kinds of ssb was studied using a recently introduced assay (Ventur, 1992; Ventur and Schulte-Frohlinde, 1993). With this assay the conversion of single-strand damages into dsb is measured following incubation of damaged DNA in a protein extract from E. coli. The assay allows differentiation between degradation (dsb formation) and repair of dsb precursors. The results show that DNase I-produced ligatable ssb are converted into dsb with much smaller yield than y-radiation-induced damages. Furthermore, it is shown that the properties of the extract depend strongly on the presence of
cofactors like Mg 2+, Ca 2+, ATP and nucleoside triphosphates.
2. Materials and methods
Bacterial strains and plasmid DNA Wild-type E. coli CMK (F +, hsdRK+, hsdMK+, lacY, thr, leu, thi, tonA, supE, iambda s) was obtained from Bien et al. (1988). The plasmid pBR322 (Bolivar et al., 1977) was purchased from Boehringer, Mannheim. Enzymes and chemicals DNase I from bovine pancreas (EC 3.1.21.1), exonuclease III (EC 3.1.11.2) ATP, NAD and the dNTPs were all from Boehringer, Mannheim. Gamma-irradiation of plasmid DNA 6°Co y-irradiation of plasmid DNA was performed in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0 according to Sambrook et al., 1989), DNA concentration 10 mg/1, at room temperature and at a dose rate of 45.8 Gy/min. Samples were taken at appropriate times and the plasmid DNA was separated on agarose gels into three fractions: the supercoiled circular form (sc), the open circular form (oc) and the linear form (lin). The gels were stained with ethidium bromide and photographed. The amount of each form was determined by densitometric evaluation on the film. It was assumed that breaks are distributed randomly and that all other prerequisites for using a Poisson distribution are fulfilled. The amount of ssb was calculated from the 'surviving fraction' of the sc form and the amount of dsb from the surviving fraction of the sc + oc form. A linear dose dependence was measured for ssb formation. Above a dose of 300 Gy, where the amount of ssb could not be measured due to the low amount of the sc form, the yield of ssb was calculated by linear extrapolation of the ssb formation at low doses. Unirradiated pBR322 DNA contained 81% supercoiled and 19% open circle form, corresponding to 0.21 ssb, and this was taken into account in the calculation of ssb formation.
Y. Ventur, D. Schulte-Frohlinde/ Mutation Research 315 (1994) 65-74 DNase I digestion DNase I (0.2 n g / ~ g pBR322) digestion was performed in 50 mM Tris-HC1, 10 mM MgSO 4, 0.1 mM D T T and 50 ~ g / m l BSA at p H 7.5 and 37°C (Sambrook et al., 1989). At appropriate times, the reaction was stopped by addition of sodium citrate (final concentration 0.1 M) and the digestion was checked by agarose gel electrophoresis on 0.8% agarose slab gels in T B E buffer (Sambrook et al., 1989). The amount of ssb and dsb formation was determined densitometrically. Ssb formation shows a linear kinetics with time. Ssb values > 3 were calculated by linear extrapolation. Exonuclease III digestion DNase I-damaged D N A ( 2 0 / x g / m l ) was incubated with Exo III (1 U//~g DNA) in 66 mM Tris-HC1, 6.6 mM MgCI2, 1 mM 2-mercaptoethanol at p H 7.6 and 37°C for appropriate times (Sambrook et al., 1989). The reaction was terminated by addition of sodium citrate (final concentration 0.1 M) and heat inactivation for 10 rain at 70°C. The digestion was checked by gel electrophoresis on 0.8% agarose slab gels in T B E buffer. The conditions were such that about three nucleotides were removed from each single-strand break.
67
40). After centrifugation at 48 000 x g for 40 min, the supernatant was collected and the amount of protein was determined colorimetrically using the Bio-Rad Protein Assay. Only the supernatant but not the precipitate was active in producing dsb if Mg 2+ ions were present. This supernatant is called 'extract'. In vitro dsb formation assay Incubation under 'non-repair' conditions was performed with 1 m g / m l protein extract in 25 mM HEPES, 5 mM MgCI2, 80 mM KC1, 2 mM DTT, 50 / z g / m l BSA at 37°C and pH 7.6. This mixture is called 'supplemented extract' under 'non-repair' conditions. After incubation of damaged DNA (pBR322 2 0 / z g / m l ) for 20 min in this supplemented extract, the same volume of icecold sodium citrate solution (0.2 M, pH 7.5) was added to stop the reaction. To provide for 'repair' conditions the following cofactors were added (the final concentrations are indicated): 0.2 mM spermidine, 1.5 mM ATP, 0.5 mM NAD, 1 mM glutathione and 0.1 mM (each) of the four deoxyribonucleotide-5'-triphosphates (Ventur and Schulte-Frohlinde, 1993). The amount of enzyme-induced dsb was determined by densitometric analysis of agarose gels.
3. Results
Transformation (I) Calcium chloride transformation formed according to Bien et al. (1988). (II) Electroporation of E. coli CMK performed according to Hartke and Frohlinde (1991). D N A concentration /xg/l.
was percells was Schultewas 6.25
Preparation of a protein extract from E. coli bacteria Crude cell extract was prepared from E. coli CMK according to Lu et al. (1983) with some modifications. Briefly, bacteria were grown in TBY to O.D.ss 0 0.8-1 and washed twice in extract buffer (25 mM HEPES, 0.1 mM EDTA, pH 7.6). Crude cell extract was prepared by digestion of the bacterial cells with lysozyme followed by treatment with ultrasound (6 x 30 s, sonic power
pBR322 plasmid DNA was y-irradiated in T E (OH scavenger capacity = 3.1 × 107/s) with various doses. The biological inactivation of this damaged D N A was determined by electroporation and the amount of radiation-induced single- and double-strand breaks by densitometric analysis of agarose gels (Fig. 1). The results show that the biological inactivation is lower than the number of ssb per molecule but much larger than the number of dsb per molecule. This is evidence that the biological inactivation of the plasmid DNA is mainly due to single-strand damages (SSD) and only to a minor extent to radiation-induced double-strand breaks (see also Introduction). To assess the inactivation efficiency of yirradiation- as well as enzyme-induced SSD, repair-proficient E. coli (CMK) hosts were trans-
68
Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74 7 ,
7
f
100
6
-~5
[ "J
e~ectroooration
[
calcium chloride
transformation
8o]
6 i'
/ biological
o
60 ¸
,o t "~
3
3 ''~
i I ligatable ssb
0
400
800
1200
1600
2000
dose [ gray ] Fig. 1. Biological inactivation, and single- and double-strand break formation of y-irradiated pBR322 plasmid D N A as a function of dose. Transformation of E. coli C M K was carried out by electroporation. Concentration of D N A in TE for electroporation 6.25 /xg/l. T h e O H scavenger capacity for irradiation was 3.1 × 107/s, D N A concentration 10 m g / l .
formed by electroporation with pBR322 plasmid D N A containing these lesions. Fig. 2 shows the different survival curves plotted versus the ssb. It can be seen that with all types of lesions survival
0.1
2
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21.4
Z 0.01 []
Ilgltlble l i b
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'y-Irrldll~d
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10
20
30
40
50
ssb / molecule
Fig. 2. Survival of transforming pBR322 plasmid D N A with different types of SSD in E. coli C M K wild-type bacteria as a function of ssb. Ligatable ssb were produced by DNase I digestion of the plasmid D N A , non-ligatable ssb are singlestrand gaps of three nucleotides in length which were generated in the nicked D N A by exonuclease III treatment, yirradiation was performed in T E buffer. Transformation was carried out by electroporation.
non-ligatable ssb
T-irradiation
Fig. 3. Comparison of single-strand breaks per molecule per lethal dose from D37 values for electroporation and CaCI 2 transformation of E. coli C M K with damaged plasmid DNA. For irradiation conditions see legend of Fig. 1.
decreases with ssb, but the D 3 7 values are quite different. Ligatable ssb which are produced by DNase I treatment have a low inactivation efficiency. Inactivation of plasmid D N A with nonligatable ssb is 56.1% higher than that for ligatable ssb (ratio of D37 values is 2.3, see Fig. 2). The highest inactivation efficiency - referring to the amount of single-strand breaks - was found for y-irradiated plasmid D N A (Fig. 2). Fig. 3 shows the number of ssb per lethal event for the different types of lesions as measured by the calcium chloride method in comparison to electroporation data. It can be seen that for plasmid D N A containing DNase I-induced ssb the number of ssb per lethal event is much larger than the corresponding ratio for non-ligatable ssb. For y-radiation-induced ssb (together with base and sugar damage) no difference was found (Fig. 3). These data indicate that calcium ions have an inhibitory effect on the biological inactivation of enzymatically damaged plasmid D N A containing single-strand breaks but not on the inactivation of y-irradiated DNA. This inhibitory effect of calcium ions was further investigated using an in vitro assay which makes it possible to distinguish between enzymatic dsb formation and repair of precursors in plasmid D N A (Ventur, 1992; Ventur and Schulte-Frohlinde, 1993). For this assay the damaged
}1.. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65- 74
69
3.5 ÷ nucleotide8
0 E ...
2
.~
1.5
- nu¢leotidell
3
2.5
_¢
2.5
_¢
2
0
E 1.5
.Q "0
1
~ 0.5
m
0.5 bufferextract lOmM M control without Mg
1ram
5mM
lOmM
20ram
30ram Ca
0
Fig. 4. Effect of calcium ions on the rate of extract-induced dsb formation. D N a s e I-damaged pBR322 DNA (8.4 ssb per molecule) was incubated for 20 min at 37°C with extract (1 mg protein/ml) without additional supplementation (non-repair conditions), with 10 mM MgCI 2 and increasing concentrations of CaC12. Incubation with HEPES buffer and with extract without additional magnesium served as controls.
plasmid D N A is incubated in a protein extract of E. coli and the enzymatic formation of dsb is measured. Incubation of D N a s e I-damaged plasmid D N A in the extract did not induce any dsb formation if no Mg 2÷ ions were added (extract without Mg) (Fig. 4). Addition of 10 m M MgC12 led to a pronounced increase in dsb formation by the extract. This dsb formation was progressively inhibited by the addition of increasing amounts of Ca 2÷ ions. At 30 m M CaCI 2 the dsb formation was decreased to the same level as the buffer control (Fig. 4). It should be noted that, with other E. coli strains, e.g. ABl157, at incubation times longer than shown in the figures an increasing proportion of the D N A was degraded. We therefore used extracts prepared from strain CMK which is also a repair wild type as is ABl157. We thank Ms Helene Steffen for these measuremeats. The above results support the view that the higher biological activity of D N a s e I-damaged plasmid D N A , using the calcium chloride transformation method in comparison to electroporation, is caused by a calcium-mediated inhibition of enzyme-induced dsb formation. The formation of dsb from single-strand damages was also reduced when the E. coli extract was supplemented
-ATP
controls
-glutsthione
-NAD
Fig. 5. Influence of cofactors on the dsb formation in 3,-irradiated plasmid D N A by incubation in a protein extract of E. coli. The irradiated DNA (503.1 Gy) was incubated in the extract (1 mg protein/ml) with or without supplementation by deoxyribonucleotides for 20 min at 37°C. Complete repair conditions were achieved by addition of all four deoxyribonucleotides (0.1 mM), and of spermidine (0.2 mM), glutathione (1 mM), ATP (1.5 mM) and NAD (0.5 mM). The missing component is marked under each couple of bars. Incubation without nucleotides and cofactors served as a control (buffer).
with certain cofactors. The influence of various cofactors was studied in detail (Figs. 5 and 6). From these investigations it becomes obvious that deoxyribonucleotide-5'-triphosphates ' and A T P are indeed the critical components which must be added to reduce degradation processes. This re-
1.4
1.2
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0.6
0.4
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0.2
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400
600
800
1000
1200
1400
1600
A TP I uM I
Fig. 6. Effect of the ATP concentration on the extract-induced dsb formation. Incubation with all cofactors and nucleotides served as a control.
70
Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74 per molecule 6
ssb
2
5
4
8
10
non-repair condition
~-.Q)EO~324 D
,~
repair condition
1
O.
,
,
200
400
,
dose
,
600 800 [ Gray ]
r
1000
1200
Fig. 7, Dsb formaton in y-irradiated plasmid DNA as a function of y-ray dose after incubation for 20 min with E. coli CMK extract under repair and non-repair conditions.
duction of dsb formation could be due to the repair of precursors of dsb or to a suppression of the incision reaction. However, lower dsb formation takes place when the extract is supplemented with just those cofactors which are needed for enzymatic repair (e.g. mainly A T P and deoxyribonucleotide-5'-triphosphates). Therefore, we distinguished two conditions for the usage of the assay. With supplementation of the essential additives, A T P and dNTPs, the condition is designated 'repair condition' and without this supplementation it is designated 'non-repair condition'.
2.0
~
non-repair condition
tr~
repair condition
1.5
°
~
i 1 0
i
I 0.5
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4. Discussion The present p a p e r deals with the behavior of three different types of single-strand breaks in plasmid D N A towards two different analytical probes. The first probe is a determination of survival by different transformation methods (the CaCI 2 method and electroporation), and the second is m e a s u r e m e n t of the conversion of ssb into dsb by incubating the damaged plasmid D N A in a protein extracts of E. coli. The results are discussed in three sections. Comparison o f biological inactiuation by different types o f ssb
i
_=
We have now used this assay to study differences between D N A with DNase I-induced ssb and radiation-induced ssb (the latter with nonbreak base and sugar damage also present). The comparison was made under repair and under non-repair conditions. Under both conditions the results show that (i) dsb formation is larger per ssb for radiationinduced ssb than for enzyme-induced ssb and that (ii) the dependence on the number of ssb per molecule is different (compare Figs. 7 and 8). Under repair conditions the formation of dsb as a function of the number of DNase I-induced ssb is linear whereas that for y-radiation-induced ssb is curvilinear.
~
2
4 ssb
......... per
6 molecule
8
10
Fig. 8. Dsb formation in DNase I-damaged plasmid DNA after incubation for 20 min with E. coli CMK extract under repair and non-repair conditions.
The low level of inactivation efficiency of DNase I-induced, ligatable ssb for plasmid transformation by electroporation (Fig. 2) corresponds with data from Laipis et al. (1969) and Kow et al. (1991) who used other transformation procedures. The low values are suggested to be due to an efficient, ligation-mediated repair. Repair of dsb virtually does not occur under our conditions because each cell is infected by only a single copy of plasmid D N A (see Bien et al., 1988). To exclude repair by ligation, single-strand gaps (non-ligatable ssb) of three nucleotides in length were generated in the nicked plasmid D N A by exonuclease III treatment (Thomas and Olivera, 1978; Prell and Wackernagel, 1980). Trans-
Z Ventur, D. Schulte-Frohlinde/MutationResearch 315 (1994) 65-74 formation by electroporation with such a D N A led to a 56% lower survival as compared to plasmid D N A with ligatable ssb at D37 with D37 = 21.4 and 9.4 ssb per molecule, respectively (Fig. 2). The D37 values were used because the survival curves are not linear, though of similar shape. These results are in line with those from Kow et al. (1991) insofar as non-ligatable breaks are more difficult to repair than ligatable breaks. The same authors have shown that the difference in survival of the plasmid D N A with ligatable and non-ligatable ssb is a factor of 8-12, whereas we found only a factor of slightly more than 2. A possible explanation is that our non-ligatable breaks are different from theirs and furthermore they used thX174 D N A and we used plasmid pBR322 DNA. Their non-ligatable breaks contain end groups which cannot be used directly for polymerization and have first to be removed before polymerization and ligation can occur and repair can be accomplished. In our case the non-ligatability is due to a gap without unusual end groups. In order to achieve repair only polymerization and ligation has to take place. This is at least one enzymatic step less than in the case of Kow et al. (1991). This may explain the greater difference between the repairability of ligatable and nonligatable breaks in their and our case. It has been shown that several kinds of base damage have the same inactivation efficiency as non-ligatable ssb (Kow et al., 1991). Assuming that this is also valid for radiation-induced base and sugar damage and ssb, it is possible to estimate the contribution of non-break damage to inactivation of y-irradiated plasmid D N A by calculating the ratio of ssb to non-break damage by comparison of the D37 values for non-ligatable enzyme-induced ssb (9.4 ssb/lethal event) with those for y-irradiation-induced damage (3.7 ssb/lethal event). From these values the ratio of ssb to non-break damage was calculated as 1 : 1.54 which means that the contribution of non-break damages to the lethal damage for y-irradiated plasmid D N A is about 60%. This result is consistent with the involvement of base and sugar damages in the biological inactivation of y-irradiated plasmid DNA. This result may be compared with in vitro data
71
from Kohfeld et al. (1988) (1 : 1.8) obtained from y-irradiated A D N A and the use of y endonucleuse for the determination of enzyme-convertible lesions, and those from Strniste and Wallace (1975) (1:2.8) using PM2 D N A y-irradiated in T E and y endonuclease. The biological data may be further compared with the expected chemical yields of ssb and non-break damage. Since the efficiency of production of one ssb in ssDNA (similar in dsDNA) by an O H radical is around 20% (Schulte-Frohlinde et al., 1991), four O H radicals per ssb should produce base and sugar radicals in the DNA. The maximum number of base and sugar non-break-damaged sites per ssb is therefore = 4. If these radicals disappear by disproportionation the minimum number of base and sugar damages per ssb is 2. The values from our transformation measurements and from the in vitro data cited above are in the range of the lower ssb to nonbreak damage ratio. Lower values of 2 per ssb are possible if some kinds of base or sugar damages are better repairable than others or code similar to the original site. It should be mentioned that the distribution of the damage sites in the D N A plays an important role too (SchiJnemann and Schulte-Frohlinde, 1992). The influence of this distribution is not discussed in the present paper. Comparison of CaCl 2 transformation with transformation by electroporation If transformation of plasmid D N A is performed by the calcium chloride method, the inactivation efficiency for ligatable and non-ligatable enzyme-induced ssb is much lower than by electroporation (Fig. 3). One of the main differences between the calcium chloride method and electroporation is the presence of calcium ions during rupture of the membrane of E. coli in the former method. For an explanation we propose that calcium ions enter the E. coli cells during the transformation procedure and that these calcium ions inhibit the action of endo- and exonucleases. Such a calcium-dependent inhibition of certain endoand exonucleases is well known (Rosamond et al., 1979; Williams et al., 1981; Kow, 1989). An inhibitory effect of calcium ions on the enzymatic degradation of enzymatically damaged plasmid
72
Y~ Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74
D N A is shown by the inhibition of dsb formation by calcium ions in an E. coli protein extract (Fig. 4). These results give evidence that enzyme-induced ssb and therefore indeed single-strand damages are converted into lethal damages by enzymatic degradation processes as proposed earlier (Ventur and Schulte-Frohlinde, 1993). Calcium-induced inhibition does not occur with the electroporation method because no calcium ions are present during this procedure. Results from Schulte-Frohlinde et al. (1993) indicated that one main enzyme which is inhibited in E. coli by Ca 2+ is the RecBC enzyme complex, and that this nuclease is involved in the degradation of enzyme-damaged DNA. A calcium ion-dependent inhibition of at least one of the actions of the RecBC enzyme complex was clearly shown to occur with purified enzymes o n phage T7 D N A (Rosamond et al., 1979). With radiation-damaged D N A no difference between the survival measured by the calcium chloride method and electroporation was observed (Fig. 3). It has recently been shown that the purified RecBC enzyme complex has ATPdependent double-strand exonuclease properties with y-irradiated D N A as substrate (Hickson et al., 1985). This may mean that either the RecBC enzyme complex is not inhibited by calcium ions when irradiation-induced damage in D N A is the substrate (as opposed to enzyme,induced damage), or other kinds of degrading enzymes take over in the cell. Many endonuclease activities which can convert radiation-induced base and sugar damages inside the E. coli cell into ssb are not sensitive to calcium inhibition (Kow, 1989). An indication in favor of the possibility that the RecBC enzyme complex might not be sensitive to calcium ions when y-irradiated D N A is the substrate, is that Brcic-Kostic et al. (1991) found that the R e c B C D enzyme complex is involved in the degradation (and repair) of y-irradiated plasmid D N A in E. coli. However, it should be added that the RecBC enzyme complex has several activities which are differently influenced by a change of reaction conditions, especially changes in the ratio of magnesium and calcium ions (Rosamund et al., 1979). These authors have also
reported that purified RecBC enzyme complexes degrade phage DNA, but do not solely produce dsb. However, this result is not at variance with our results since we do not use purified RecBC enzymes but a mixture of all enzymes extractable from E. coli cells. Furthermore, they measured intact and linear D N A but not radiation-damaged DNA. Their results do not indicate that they have measured the effect of incubation times on dsb formation and degradation because they only show results at long incubation times where degradation is complete. We found degradation also at long incubation times. However, a direct comparison of the times of incubation is not meaningful because of the different experimental conditions used. The difference in the properties of a mixture of enzymes, when enzyme-induced and y-irradiation-induced ssb are compared, is clearly due to the chemically different end groups involved in both cases (von Sonntag et al., 1981; von Sonntag, 1987; Obe et al., 1992). Many examples exist which show that a mixture of enzymes react differently with different substrates. One is that a cocktail of four different enzymes capable of degrading undamaged D N A into mononucleotides is not able to do so with y-irradiated D N A (Dizdaroglu et al., 1978).
Incubation in a protein extract o f E. coli With a new in vitro assay a separation of degradation and repair processes which are involved in the enzymatic processing of single-strand damages was achieved (Ventur and SchulteFrohlinde, 1993). By means of this assay we have directly proved that enzyme-, as well as y-irradiation-induced single-strand damages, were converted enzymatically into double-strand breaks by E. coli proteins. In recent experiments our assay was also employed to demonstrate dsb formation from uracil residues in M13 D N A (Schiinemann, 1993). Furthermore, we were able to show that different kinetics are involved in dsb formation under 'repair' and 'non-repair' conditions in the supplemented extracts. The difference between 'repair' and 'non-repair' conditions is due to supplementation by cofactors.
Y. Ventur, D. Schulte-Frohlinde / Mutation Research 315 (1994) 65-74
The data in the present work indicate that an extract from E. coli has a strong degradation activity. This degradation activity is greatly decreased by supplementation with ATP or the four deoxyribonucleotide-5'-triphosphates (Figs. 5 and 6). The fact that the addition of ATP and dNTPs is necessary to inhibit degradation indicates that this inhibition is due to repair. A further proof for the interpretation that the inhibition of dsb formation is due to repair is given by the experimental result of the better survival of T-irradiated plasmid D N A treated with the extract under repair conditions in comparison to the untreated control (Ventur and Schulte-Frohlinde, 1993). If the concentration of ATP is higher than 0.5 mM the addition of dNTPs is not necessary to achieve repair conditions. Addition of supplements to E. coli cell proteins was also necessary in order to achieve mismatch repair (Lu et al., 1983, 1984) and conversion of M13 D N A to its replicative form in vitro (Wickner et al., 1972). Our results also correspond with data from Matson and Bambara (1981) and Masker (1977) who were able to show that excision repair with a dialysed E. coli protein extract is dependent on exogenous dNTPs. This supplementation dependence of the different in vitro systems may be due to the dilution (50-100-fold) of the cellular components by the extract preparation procedure. In the diluted solution the concentration of proteins is high enough to achieve the biological functions but the concentration of cofactors might be too low. The results in the present paper support the proposal made earlier (Ventur and Schulte-Frohlinde, 1993) that in plasmid D N A y-irradiation and enzyme-induced SSD are basically either repaired or enzymatically converted into dsb. The degree of dsb formation depends on the repair capacity of the cell and the extract. The higher yields of dsb per ssb obtained with T-irradiated D N A in comparison with DNase Itreated D N A may have two reasons. First, the majority of the DNase I-produced ssb are repaired by ligation before conversion into dsb occurs. Second, y-irradiated D N A contains, in addition to ssb, base and sugar damage which may contribute to dsb formation.
73
An interesting difference between enzyme-induced ssb and radiation-induced ssb is found in the dependence of the yield of dsb on the number of ssb by incubation in an extract under repair conditions (Figs. 7 and 8). The formation of dsb is linear with enzyme-induced ssb but curvilinear with radiation-induced dsb. This result also points to different mechanisms in the enzymatic processing of the two kinds of damage compared here.
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