Phorochernirrry and Phufobiology, 1976, Vol. 23, pp. 205-208.
Pergamon Press
Prmed ~n Great Britaln
RESEARCH NOTE THE EFFECT OF IRRADIATION WITH ULTRAVIOLET LIGHT ON THE SEDIMENTATION BEHAVIOUR OF PM2 DNA G . VELDHUISEN*,S. BACCHETTI~ and M. J.
VAN DER
VALK*
*Medical Biological Laboratory TNO, Rijswijk Z.H. and ?Laboratory for Molecular Genetics, State University of Leiden, Leiden, The Netherlands (Receiced 19 August 1975; accepted 24 October 1975) INTRODUCTION
7.0, 10 mM /I-mercaptoethanol, 107; ethyleneglycol. Photoreactivating (PR) enzyme was prepared from Streptornyces yriseus by Dr. Eker (Eker et ul., 1975) and was stored in 100 mM NaCI, 10 mM KPi buffer pH 7.4, 307, glycerol (PR buffer).
Denhardt and Kato (1973) have reported that superhelical, closed circular duplex DNA of bacteriophage 6x174 (RFI-DNA) irradiated with ultraviolet light (UV) sediments faster in neutral sucrose gradients than the unirradiated species. The authors ascribed 2500 I this phenomenon to the fact that the pyrimidine :Absorbonce i :Absorbance dimers introduced by the irradiation cause a local unwinding of the superhelical DNA. The main evidence for this conclusion was the observation that exposure of the irradiated DNA to visible light in the presence of photoreactivating enzyme (a treatment known to monomerize the dimers) reduced considerably the amount of the fast sedimenting component. The evidence is not completely convincing, however, because only part of the DNA regained the original sedimentation rate; owing to a nucleolytic activity associated with the photoreactivating enzyme a large Fraction number proportion was converted into more slowly sediment2500 m / ing material. During investigations with PM2 DNA we had noticed that component I, the closed circular duplex form, sediments faster in neutral sucrose gradients after irradiation with UV. In preliminary experiments including photoreactivation, we had not been able, however, to relate the increase in sedimentation rate to the presence of pyrimidine dimers. Therefore, the results of Denhardt and Kato led us to re-examine the phenomenon more closely. The newly obtained results are presented here. They differ from those of Denhardt and Kato in that we definitely do not observe a reversion of the sedimentation coefficient Fraction number Fraction number to the normal value when the irradiated PM2 DNA Figure 1. The effect of UV-irradiation on the scdimenis photoreactivated. tation properties of PM2 DNA in neutral sucrose gra-
rI - - -I I - 7 1 i r jI
dients. 50 pf of a mixture of equal volumes of unlabeled. UV irradiated (500J/m2; 5000ergs/mm2) PM2 DNA (128 pg/m/) and 3H-thymidine labeled unirradiated PM2 DNA MATERIALS AND METHODS (I5 pg/m/) were layered on a sucrose gradient and after D N A . 'H-thymidine labeled PM2 DNA was prepared centrifugation fractionated as described in Methods. The as described by Van der Schans et a/. (1973) with minor broken linc represents the absorbance of the irradiated modifications. DNA was stored in 10 mM Tris pH 8.0, DNA; the histograms represent the radioactivity in the control DNA. Direction of sedimentation is from left to I mM EDTA, 10 mM NaCl (TEN). Enzymes. UV specific endonuclease was prepared from right. I. unirradiated DNA; TI. unirradiated DNA plus PR extracts of Micrococcus luteus as described by Nakayama enzyme; 111. U V irradiated DNA; IV. irradiated DNA plus PR enzyme. Y? a/. (1971) and was stored in 10 m M KPi buffer pH 205
G . VELDHUISI.N.S . BACCHETTI and M. J.
206
44
100
0
100
200 300 400 500 UV Dose(erg/mm2)
Figure 2. Relation between UV-dose and UV-endonucleaseinduced breaks. 3H-thymidine labeled PM2 DNA in TEN (15pg/m/) was exposed to UV with a wavelength of 280 nm from a superhigh-pressure mercury lamp (OSRAM 100 W/2)fitted with a modified Beckman Prism monochromator. The dose rate at sample height was 4.2erg/mm'/s. At intervals 5 p/ aliquots were withdrawn and mixed with 45 p/ TEN; then 40 p/ of I0 m M Tris pH 7.5,1 mM 8-mercaptoethanol, 20% glycerol and 10 II/ of the UV-endonuclease preparation were added. After the final EDTA concentration had been adjusted to 4 mM, the samples were incubated for 10 min at 37°C.The relative amounts of comp. I and 11 and the number of breaks were determined a \ described in Materials and Methods. The broken line represents the expected number of breaks calculated from the number of dimers according to Rahn and Stafford (1974).
VAN IIER
VALK
radioactively labeled, unirradiated P M 2 DNA as an internal marker. The three peaks in the figurc correspond to three forms of double-stranded P M 2 DNA, i.e. from right t o left, superhelical, covalently closed circles (comp. I) which sediment fastest; circular molecules with one or more single-strand breaks (comp. 11);and linear molecules (comp. 111). From a comparison of the position of the absorbance peaks relative t o that of the peaks of the unirradiated, radioactive DNA, it is evident that irradiation with 500 J/m2 (5000 ergs/mm2) leads t o a signilicant increase in the sedimentation rate of comp. I (compare Fig. I , I and 111)which is not changed by a treatment with photoreactivating enzyme and visiblc light (Fig. 1, ITT and IV). When the DNA was exposcd to loo0 J/m' (10,000 ergs/mm') the increase in sedimentation rate was twice as large (data not shown) and also in that case the increase in Sedimentation rate was not changed after photoreactivation. This proportionality between dose of UV irradiation and sedimen~ation rate was also observed by Denhardt and Kato (1973). At lower doses of irradiation 100 J/m2 (1000
U V iirudiufion. DNA samples were exposcd to 500 Jjm' (5000ergslmm') from a low pressure mercury tube (Philips 30 W, T U V ) emitting predominantly at 254 nm. Incubation with PR enzyme. UV irradiated, unlabeled PM2 DNA (128 pg/mr")in TEN was mixed with an equal volume of 3H-thymidine labeled PM2 DNA (1 5 pg/mt). To 100 J of this mixture were added either 50 pc' PR enzyme or 50 J PR buffer. The samples were incubated for 30 min at 37°C underneath two Philips TL W/08 lamps at a distance of 10 cm. Incubation with U V endonuclease. UV irradiated 3Hlabeled PM2 DNA (15 &m/) was mixed with an equal volume of unlabeled unirradiated PM2 DNA (128 pg/m/). After incubation with PR enzyme (with or without light) 50 p/ aliquots were removed to which were added 40 ' p TEN and 10 p/ of a preparation of UV endonuclease. EDTA was added to a final concentration of 4 mM. After incubation a t 37°C during 10 min, 400 p/ cold TEN were added and the amounts of component I and I1 were determined by membrane filtration (Van der Schans et a!., 1973). The number of single strand breaks per molecule was calculated assuming a Poisson distribution. Neutrul .sucrose gradients. Samples to be analyzed were layered on a linear gradient of 3 to 20% sucrose (w/v) in 4 M NaC1, 1 mM EDTA, 10 mM KPi buffer pH 7.0, and centrifuged in an SB 110 rotor (International) for 16 h at 35,000 rpm and 10°C.After centrifugation the gradients were led through a UV-absorptiometer (Van der Schans et al., 1973) and fractions (5 drops) were collected for radioactivity measurements. RESULTS AND DISCUSSION
Figure 1 shows the sedimentation patterns obtained with P M 2 DNA and the effects of U V irradiation and subsequent photoreactivation. I n each gradient the unlabeled test substance was run together with
Figure 3. Electron micrographs of PM2 DNA. DNA molecules were prepared and mounted for electron microscopy using a minor modification of the technique of KleinSchmidt and Zahn (1959).Electron micrographs werc taken with a Philips EM 300 electron microscope. a. Native superhelical PM2 DNA; b. UV-irradiated (500J/m'; 5000 ergs/mm') P M 2 DNA; c. UV-irradiated and photoreactivated PM2 DNA.
Research Note
207
Table 1. The susceptibility of UV-irradiated PM2 DNA to UV-endonuclease before and after photoreactivation. Treatment of the irradiated DNA
PR enzyme
A
-
-
+ + + + B
-
-
+ +
Cornp. II
breaks per
light
UV-endo
%
molecule
+ +
-
20
0.22
+
98
-
+
+ + + + + +
-
25
0.29
+
64
1.0
-
37
0.47
+
98
-
42
0.56
+
75
1.4
29 97
> 4" 0.34
> 4*
> 4"
A) A mixture of UV irradiated 3H-thymidine labeled PM2 DNA and unlabeled, unirradiated PM2 DNA were incubated with PR enzyme followed by an incubation with UV-endonuclease as described in Materials and Methods. B) The conditions were the same as in panel A except that the unlabeled DNA was irradiated with 500 J/mZ (5000 ergs/mmz). This experiment (which was suggested to us by Dr. Denhardt) was included to duplicate the conditions used in Fig. 1. It shows that the presence of an excess cold irradiated DNA has no effect on the activity of the PR enzyme. *A value of 3&40 breaks per molecule should be expected. However with the filter assay the determination of the number of breaks per molecule becomes unreliable for values above -4 breaks/mol (at an average of 4 breaks/mol 1.8% of the DNA is present as comp. I)
ergs/mm2) the distance between the peaks of irradiated and unirradiated DNA becomes too small to be measured reproducibly. Irradiation does not appear to have' a significant effect on the sedimentation of the other two forms of PM2 DNA. The trcatment with photoreactivating enzyme and visible light has no influence on the ratio between comp. I and comp. TI in unirradiated DNA (Fig. I, I and II), but with the irradiated material a slight conversion of comp. I into comp. I1 is seen (Fig. 1, 111 and IV). For the interpretation of these data, knowledge on the efficiency of the photoreactivation is essential. The efficiency was investigated by using a purified endonuclease from Micrococcus luteus which is specific for pyrimidine dimers (Nakayama et al., 1971). Samples of UV-irradiated PM2 DNA were incubated with this enzyme before and after photoreactivation. Conditions of irradiation and photoreactivation were identical to those of Fig. 1. Since this UV-endonuc-
lease introduces one single-strand nick in the DNA near each pyrimidine dimer, comp. I is converted into comp. I1 if it contains one or more dimers. From the extent of this conversion the number of breaks introduced can be calculated. The results, which are given in Table 1, clearly indicate that the irradiated DNA is a much better substrate for the UV-endonuclease than the irradiated and photoreactivated material. Evidently, during the photoreactivation almost all dimers have been removed. This conclusion is warranted only if the endonuclease used is indeed able to introduce a nick next to every pyrimidine dimer. That this assumption is correct was borne out by an experiment in which 3H-labeled PM2 DNA was irradiated with increasing doses of UV and subsequently incubated with the UV endonuclease. From the calibration curve obtained (Fig. 2) it appears that under the conditions used the number of breaks corresponds to the number of dimers present after low doses of UV [according to Rahn and Stafford (1974), one may
208
G . VELDHUISEN,S. BACCHETTI and M.J. VAN
expect 0.7 dimers per 10 Jim2 (100 ergs/mm2) per 6 x 10‘ daltons, the molecular weight of PM2 DNA]. From the data in Table 1 panel A we may conclude that after photoreactivation only 0.7 dimers per molecule are left (subtracting 0.3 breaks not due to dimers). Nevertheless this photoreactivated DNA sediments at the same rate as irradiated and non-photoreactivated DNA which contains (after a dose of 500 J/mz [5000 ergs/mm’]) approximately 35 dimers/ molecule. In their paper Denhardt and Kato (1973) presented electron micrographs in which UV-irradiated superhelical RFT molecules show a more “relaxed” conformation than unirradiated RFI DNA. We also observed that the irradiated superhelical molecules of
DER VALK
PM2 DNA show a “relaxed’ morphology (Fig. 3); this configuration, however, was also seen after photoreactivation. On the basis of our experiments we conclude that indeed exposure to UV enhances the sedimentation rate of superhelical DNA. Our data on photoreactivation, however, indicate that the increase in sedimentation rate and the “relaxed” morphology are not related to the presence of pyrimidine dimers. Consequently, these effects have to be ascribed to some other photoproduct(s) induced by UV. Ack,iowledyernenis-The authors are grateful to Dr. A. P. M. Eker for kindly donating the photoreactivating enzyme, to Dr. R. A. Oosterbaan for the gift of M . luteus UV-endonuclease and to Drs. A. J. van der Eb and F. L. Graham for the electron microscopy.
REFERENCES
Denhardt, D. T., and A. C. Kato (1973) J . h o l . Bid. 77. 479-494. Eker. A. P. M.. and A. M. J. Fichtinper-Schepman (1975) Biochirn. Biophys. Actu 378. 54-63. Kleinschmidt. A. K., and R. K. Zahn (1959) Z . Nutcir:fbrsch.14b. 770-779. Nakayama. H., S. Okubo and Y. Takagi (1971) Biochim. Biophys. Actu 228. 67-82. Rahn, R. O., and R. S. Stafford (1974) Nature 248. 52-54. Schans, G . P. van der. J. F. Bleichrodt and Joh. Blok (1973) Intc‘rri. J . Raditrtiori B i d . 23. 133-150,
Pergamon Press
Prmed ~n Great Britaln
RESEARCH NOTE THE EFFECT OF IRRADIATION WITH ULTRAVIOLET LIGHT ON THE SEDIMENTATION BEHAVIOUR OF PM2 DNA G . VELDHUISEN*,S. BACCHETTI~ and M. J.
VAN DER
VALK*
*Medical Biological Laboratory TNO, Rijswijk Z.H. and ?Laboratory for Molecular Genetics, State University of Leiden, Leiden, The Netherlands (Receiced 19 August 1975; accepted 24 October 1975) INTRODUCTION
7.0, 10 mM /I-mercaptoethanol, 107; ethyleneglycol. Photoreactivating (PR) enzyme was prepared from Streptornyces yriseus by Dr. Eker (Eker et ul., 1975) and was stored in 100 mM NaCI, 10 mM KPi buffer pH 7.4, 307, glycerol (PR buffer).
Denhardt and Kato (1973) have reported that superhelical, closed circular duplex DNA of bacteriophage 6x174 (RFI-DNA) irradiated with ultraviolet light (UV) sediments faster in neutral sucrose gradients than the unirradiated species. The authors ascribed 2500 I this phenomenon to the fact that the pyrimidine :Absorbonce i :Absorbance dimers introduced by the irradiation cause a local unwinding of the superhelical DNA. The main evidence for this conclusion was the observation that exposure of the irradiated DNA to visible light in the presence of photoreactivating enzyme (a treatment known to monomerize the dimers) reduced considerably the amount of the fast sedimenting component. The evidence is not completely convincing, however, because only part of the DNA regained the original sedimentation rate; owing to a nucleolytic activity associated with the photoreactivating enzyme a large Fraction number proportion was converted into more slowly sediment2500 m / ing material. During investigations with PM2 DNA we had noticed that component I, the closed circular duplex form, sediments faster in neutral sucrose gradients after irradiation with UV. In preliminary experiments including photoreactivation, we had not been able, however, to relate the increase in sedimentation rate to the presence of pyrimidine dimers. Therefore, the results of Denhardt and Kato led us to re-examine the phenomenon more closely. The newly obtained results are presented here. They differ from those of Denhardt and Kato in that we definitely do not observe a reversion of the sedimentation coefficient Fraction number Fraction number to the normal value when the irradiated PM2 DNA Figure 1. The effect of UV-irradiation on the scdimenis photoreactivated. tation properties of PM2 DNA in neutral sucrose gra-
rI - - -I I - 7 1 i r jI
dients. 50 pf of a mixture of equal volumes of unlabeled. UV irradiated (500J/m2; 5000ergs/mm2) PM2 DNA (128 pg/m/) and 3H-thymidine labeled unirradiated PM2 DNA MATERIALS AND METHODS (I5 pg/m/) were layered on a sucrose gradient and after D N A . 'H-thymidine labeled PM2 DNA was prepared centrifugation fractionated as described in Methods. The as described by Van der Schans et a/. (1973) with minor broken linc represents the absorbance of the irradiated modifications. DNA was stored in 10 mM Tris pH 8.0, DNA; the histograms represent the radioactivity in the control DNA. Direction of sedimentation is from left to I mM EDTA, 10 mM NaCl (TEN). Enzymes. UV specific endonuclease was prepared from right. I. unirradiated DNA; TI. unirradiated DNA plus PR extracts of Micrococcus luteus as described by Nakayama enzyme; 111. U V irradiated DNA; IV. irradiated DNA plus PR enzyme. Y? a/. (1971) and was stored in 10 m M KPi buffer pH 205
G . VELDHUISI.N.S . BACCHETTI and M. J.
206
44
100
0
100
200 300 400 500 UV Dose(erg/mm2)
Figure 2. Relation between UV-dose and UV-endonucleaseinduced breaks. 3H-thymidine labeled PM2 DNA in TEN (15pg/m/) was exposed to UV with a wavelength of 280 nm from a superhigh-pressure mercury lamp (OSRAM 100 W/2)fitted with a modified Beckman Prism monochromator. The dose rate at sample height was 4.2erg/mm'/s. At intervals 5 p/ aliquots were withdrawn and mixed with 45 p/ TEN; then 40 p/ of I0 m M Tris pH 7.5,1 mM 8-mercaptoethanol, 20% glycerol and 10 II/ of the UV-endonuclease preparation were added. After the final EDTA concentration had been adjusted to 4 mM, the samples were incubated for 10 min at 37°C.The relative amounts of comp. I and 11 and the number of breaks were determined a \ described in Materials and Methods. The broken line represents the expected number of breaks calculated from the number of dimers according to Rahn and Stafford (1974).
VAN IIER
VALK
radioactively labeled, unirradiated P M 2 DNA as an internal marker. The three peaks in the figurc correspond to three forms of double-stranded P M 2 DNA, i.e. from right t o left, superhelical, covalently closed circles (comp. I) which sediment fastest; circular molecules with one or more single-strand breaks (comp. 11);and linear molecules (comp. 111). From a comparison of the position of the absorbance peaks relative t o that of the peaks of the unirradiated, radioactive DNA, it is evident that irradiation with 500 J/m2 (5000 ergs/mm2) leads t o a signilicant increase in the sedimentation rate of comp. I (compare Fig. I , I and 111)which is not changed by a treatment with photoreactivating enzyme and visiblc light (Fig. 1, ITT and IV). When the DNA was exposcd to loo0 J/m' (10,000 ergs/mm') the increase in sedimentation rate was twice as large (data not shown) and also in that case the increase in Sedimentation rate was not changed after photoreactivation. This proportionality between dose of UV irradiation and sedimen~ation rate was also observed by Denhardt and Kato (1973). At lower doses of irradiation 100 J/m2 (1000
U V iirudiufion. DNA samples were exposcd to 500 Jjm' (5000ergslmm') from a low pressure mercury tube (Philips 30 W, T U V ) emitting predominantly at 254 nm. Incubation with PR enzyme. UV irradiated, unlabeled PM2 DNA (128 pg/mr")in TEN was mixed with an equal volume of 3H-thymidine labeled PM2 DNA (1 5 pg/mt). To 100 J of this mixture were added either 50 pc' PR enzyme or 50 J PR buffer. The samples were incubated for 30 min at 37°C underneath two Philips TL W/08 lamps at a distance of 10 cm. Incubation with U V endonuclease. UV irradiated 3Hlabeled PM2 DNA (15 &m/) was mixed with an equal volume of unlabeled unirradiated PM2 DNA (128 pg/m/). After incubation with PR enzyme (with or without light) 50 p/ aliquots were removed to which were added 40 ' p TEN and 10 p/ of a preparation of UV endonuclease. EDTA was added to a final concentration of 4 mM. After incubation a t 37°C during 10 min, 400 p/ cold TEN were added and the amounts of component I and I1 were determined by membrane filtration (Van der Schans et a!., 1973). The number of single strand breaks per molecule was calculated assuming a Poisson distribution. Neutrul .sucrose gradients. Samples to be analyzed were layered on a linear gradient of 3 to 20% sucrose (w/v) in 4 M NaC1, 1 mM EDTA, 10 mM KPi buffer pH 7.0, and centrifuged in an SB 110 rotor (International) for 16 h at 35,000 rpm and 10°C.After centrifugation the gradients were led through a UV-absorptiometer (Van der Schans et al., 1973) and fractions (5 drops) were collected for radioactivity measurements. RESULTS AND DISCUSSION
Figure 1 shows the sedimentation patterns obtained with P M 2 DNA and the effects of U V irradiation and subsequent photoreactivation. I n each gradient the unlabeled test substance was run together with
Figure 3. Electron micrographs of PM2 DNA. DNA molecules were prepared and mounted for electron microscopy using a minor modification of the technique of KleinSchmidt and Zahn (1959).Electron micrographs werc taken with a Philips EM 300 electron microscope. a. Native superhelical PM2 DNA; b. UV-irradiated (500J/m'; 5000 ergs/mm') P M 2 DNA; c. UV-irradiated and photoreactivated PM2 DNA.
Research Note
207
Table 1. The susceptibility of UV-irradiated PM2 DNA to UV-endonuclease before and after photoreactivation. Treatment of the irradiated DNA
PR enzyme
A
-
-
+ + + + B
-
-
+ +
Cornp. II
breaks per
light
UV-endo
%
molecule
+ +
-
20
0.22
+
98
-
+
+ + + + + +
-
25
0.29
+
64
1.0
-
37
0.47
+
98
-
42
0.56
+
75
1.4
29 97
> 4" 0.34
> 4*
> 4"
A) A mixture of UV irradiated 3H-thymidine labeled PM2 DNA and unlabeled, unirradiated PM2 DNA were incubated with PR enzyme followed by an incubation with UV-endonuclease as described in Materials and Methods. B) The conditions were the same as in panel A except that the unlabeled DNA was irradiated with 500 J/mZ (5000 ergs/mmz). This experiment (which was suggested to us by Dr. Denhardt) was included to duplicate the conditions used in Fig. 1. It shows that the presence of an excess cold irradiated DNA has no effect on the activity of the PR enzyme. *A value of 3&40 breaks per molecule should be expected. However with the filter assay the determination of the number of breaks per molecule becomes unreliable for values above -4 breaks/mol (at an average of 4 breaks/mol 1.8% of the DNA is present as comp. I)
ergs/mm2) the distance between the peaks of irradiated and unirradiated DNA becomes too small to be measured reproducibly. Irradiation does not appear to have' a significant effect on the sedimentation of the other two forms of PM2 DNA. The trcatment with photoreactivating enzyme and visible light has no influence on the ratio between comp. I and comp. TI in unirradiated DNA (Fig. I, I and II), but with the irradiated material a slight conversion of comp. I into comp. I1 is seen (Fig. 1, 111 and IV). For the interpretation of these data, knowledge on the efficiency of the photoreactivation is essential. The efficiency was investigated by using a purified endonuclease from Micrococcus luteus which is specific for pyrimidine dimers (Nakayama et al., 1971). Samples of UV-irradiated PM2 DNA were incubated with this enzyme before and after photoreactivation. Conditions of irradiation and photoreactivation were identical to those of Fig. 1. Since this UV-endonuc-
lease introduces one single-strand nick in the DNA near each pyrimidine dimer, comp. I is converted into comp. I1 if it contains one or more dimers. From the extent of this conversion the number of breaks introduced can be calculated. The results, which are given in Table 1, clearly indicate that the irradiated DNA is a much better substrate for the UV-endonuclease than the irradiated and photoreactivated material. Evidently, during the photoreactivation almost all dimers have been removed. This conclusion is warranted only if the endonuclease used is indeed able to introduce a nick next to every pyrimidine dimer. That this assumption is correct was borne out by an experiment in which 3H-labeled PM2 DNA was irradiated with increasing doses of UV and subsequently incubated with the UV endonuclease. From the calibration curve obtained (Fig. 2) it appears that under the conditions used the number of breaks corresponds to the number of dimers present after low doses of UV [according to Rahn and Stafford (1974), one may
208
G . VELDHUISEN,S. BACCHETTI and M.J. VAN
expect 0.7 dimers per 10 Jim2 (100 ergs/mm2) per 6 x 10‘ daltons, the molecular weight of PM2 DNA]. From the data in Table 1 panel A we may conclude that after photoreactivation only 0.7 dimers per molecule are left (subtracting 0.3 breaks not due to dimers). Nevertheless this photoreactivated DNA sediments at the same rate as irradiated and non-photoreactivated DNA which contains (after a dose of 500 J/mz [5000 ergs/mm’]) approximately 35 dimers/ molecule. In their paper Denhardt and Kato (1973) presented electron micrographs in which UV-irradiated superhelical RFT molecules show a more “relaxed” conformation than unirradiated RFI DNA. We also observed that the irradiated superhelical molecules of
DER VALK
PM2 DNA show a “relaxed’ morphology (Fig. 3); this configuration, however, was also seen after photoreactivation. On the basis of our experiments we conclude that indeed exposure to UV enhances the sedimentation rate of superhelical DNA. Our data on photoreactivation, however, indicate that the increase in sedimentation rate and the “relaxed” morphology are not related to the presence of pyrimidine dimers. Consequently, these effects have to be ascribed to some other photoproduct(s) induced by UV. Ack,iowledyernenis-The authors are grateful to Dr. A. P. M. Eker for kindly donating the photoreactivating enzyme, to Dr. R. A. Oosterbaan for the gift of M . luteus UV-endonuclease and to Drs. A. J. van der Eb and F. L. Graham for the electron microscopy.
REFERENCES
Denhardt, D. T., and A. C. Kato (1973) J . h o l . Bid. 77. 479-494. Eker. A. P. M.. and A. M. J. Fichtinper-Schepman (1975) Biochirn. Biophys. Actu 378. 54-63. Kleinschmidt. A. K., and R. K. Zahn (1959) Z . Nutcir:fbrsch.14b. 770-779. Nakayama. H., S. Okubo and Y. Takagi (1971) Biochim. Biophys. Actu 228. 67-82. Rahn, R. O., and R. S. Stafford (1974) Nature 248. 52-54. Schans, G . P. van der. J. F. Bleichrodt and Joh. Blok (1973) Intc‘rri. J . Raditrtiori B i d . 23. 133-150,
