Volume 3 no.4 April 1976
Nucleic Acids Research
On the use of ultraviolet light to study protein-DNA crosslinking.
Julio E . Celis*+ , Michelle Fink and Keld Kaltofto * MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, E ngland, UK
Received 6 February 1976 A BSTRACT Irradiation of Ehrlich ascites chromatin with ultraviolet light (u. v.) leads to protein-DNA crosslinking as determined by CsCl isopycnic ultracentrifugation and SDS-polyacrylamide gel electrophoresis. At the most 4.5% of the chromatin proteins labelled with (14C)-lysine and (14C)-arginire can be criosslinked to DNA at u.v. doses between 3. 6 x 104 to 10. 8 x 10 ergs/mm'. We find however that the crosslinking reaction is hindered by protein breakage induced by u.v. light of wave length of less than 2950 A. Our results point out that caution must be used in the interpretation of studies on protein-nucleic acid interactions using u.v. light.
INTRODUCTION Research on the structure of chromatin needs crosslinking agents that will allow the determination of which proteins are in close proximity to the DNA. It was first noted by Smith that ultraviolet light (u.v.) irradiation induced DNA-to-protein crosslinking in bacteria. This observation has been further confirmed using mammalian cells by several laboratories2-4 There are at least two ways in which u.v. induced crosslinking between protein and DNA could be used to approach our problem; (1) irradiation of cells or chromatin with u.v. light and (2) irradiation of BUdR treated cells or chromatin (BUdR-substituted DNA) with u.v. light of about 3130 A5 8. In this article we describe experiments in which we have irradiated Ehrlich ascites chromatin in vitro with u.v. light. We show that although a fraction of the chromatin proteins (4.5%) can be crosslinked to the DNA this reac tion is hindered by protein breakage induced by u.v. irradiation 9-10. The experiments concerning irradiation of BUdR treated cells and chromatin will be a matter of a further communication. METHODS Preparation of chromatin. Chromatin from Ehrlich ascites mouse 11 tumor cells was prepared essentially as described by Ilyin et al. . The cells were broken with 0. 3% Triton X-100 in 0. 25 M sucrose buffer con-
C Information Retrieval Umited 1 Falconberg Court London W1 V 5FG England
1065
Nucleic Acids Research taining 50 mM Tris-HCl, pH 8. 0, 10 mM MgCl2 and 10 mM dithiothreitol. The nuclei were recovered from the lysed cells by pelleting at 600 xg. for 10 min. and washed three times in the 0.25 M sucrose buffer. Chromatin was ext racted by homogenizing the nuclei continously for 1 min. in 75 mM NaCl, 25 mM EDTA, pH 8.0, 2 mM 2-mercaptoethanol, 10 mM NaHSO3. The suspension was pelleted at 3. 000 xg. for 10 min. and the extraction was repeated twice. The final pellet was resuspended in 5 mM Tris-HCl pH 8.00, 2 mM 2 mercaptoethanol, 5 mM NaHSO3 and layered over 25 ml of 1. 7 M sucrose in the same buffer. The chromatin was pelleted by centrifuging in a Beckman SW 25 rotor at 22. 000 rpm at 4 0C for 3 hrs. The chromatin gel was allowed to swell overnight in twice deionized H20 at 0 0C. u.v. irradiation of chromatin. Chromatin at concentration of 5 A 260/ml (15 ml in a 10 cm petri-dish) was irradiated with constant shaking at 40C with a Mercury arc lamp uvs-100, 125 Watt, Hanovia made inSlough, England. The energy output of the lamp was determined with a Blak-Ray u.v. meter model J-225. Samples withdrawn at different times were lyophylized 12 and resuspended in SDS-dissociation buffer SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel slabs containing 17% acrylamide were prepared as described by Laemmli12 and run at 40C and 20 mA for 15 hrs. The slab gels were stained with 0. 1% (w/v) coomassie brilliant blue in methanol/acetic acid/water (40: 7: 53) and destained with the same solvent.
RESULTS AND DISCUSSION Chromatin exposed to increasing doses of u.v. light radiation was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis as described in Methods. We assumed that proteins crosslinked to DNA will not enter the gel; thus, a total or partial disappearance of a polypeptide band from the running gel followed by a concomittant appearance of protein staining material at the top of the gel is taken as a sign of proteinDNA crosslinking. Although this assay is not specific (it cannot distinguish between protein-protein and protein-DNA crosslinking) it allowed a rapid screening of many samples. It is clear from the gels shown in fig. 1 that at u.v. doses between 3. 6 to 10. 8 x 104 ergs/mm2 some chromosomal proteins, mainly the non-histone proteins, disappeared from the gel (partially or totally) with a simultaneous appearance of protein staining material at the top of the gel. Irradiation with even higher u.v. doses leads to a reduction of the intensities of protein bands corresponding to f., f3 f2b and f2a2 respectively, without accumulation of crosslinked material at the top of the gel or appearance of new bands in the running gel. 1066
Nucleic Acids Research B --
Origin
A
Origin -
cl
C
l
f
f 2 b_
W::~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. .....:
f3
+
0
36
72 108
216 432
ergs/ mm2 x 1O3 ~~~~~~~~~~~~
u.v dose f3 b
Figure 1. SDS-polyacrylamide gel electrophoresis of chromatin irradiated with u. v. light. 30 pug of protein was applied to each gel slot. A more extensive analysis of the irradiated samples of chromatin (double labelled with (methyl 3H) -thymidine and ~14C) -lysine and (14C )-arginine by means of CsCl isopycnic ultracentrifugation is shown in fig. 2. Irradiation with a u. v. dose of 3. 8 x 1 leads to splitting of the dine counts in two peaks with densities of 1. 68 and 1 52 respectively
04ergs/mm2
g/cm3
(3H)-thymi-
(fig. 2B). Analysis of the C-protein counts showed that 4% of the radioactivity was pre sent unde r the DNA peak of density 1. 52 g/ cm3 while no counts 3 could be detected under the peak of density 1. 68 g/cm Irradiation with a -_fig. 2C) results in an increase in higher u. v. dose (1 0.8 x the size of the peak of density 1g 52 a substantial increase in the amount of C-protein present under the peak (4.5%). At present we do not know what causes the DNA to shift to a density of 1.52 g/cm 3 .in such a fashion. First the amount of protein under the peak does not correspond with the expected protein content of a nucleoprotein of density of 1. 52 g/cm3p13 and s of density secondly one would expect intermediate species between the .
4ergs/mm2 glcm3without
1.68 and
1.o52 g/cm 3respectively. Further experiments
will be necessary
to clarify these points.
1067
Nucleic Acids Research
0
0
,x
X 9
ECL
E
C)
I
Fraction Number
Figure 2. CsCl isopycnic ult{acentrifugation of Y4v. irradiated chromatin double labelled with(methyl- H)-thymidine and ( C)-lysine and (14C)-arginine. Mice carrying E)rlich ascites tumor were injected intraperitoneally with 1 mF of (methyl- H)-thymidine (Amersham 43 Ci!mmol) and 500 pCi each of ( C)-lysine (Amersham 270 mCi/mmol)and (14C)-arginine (Amersham 270 mCi/mmol). Samples after irradiation were centrifuged in CsCl at 40. 000 rpm for 72 hrs. in the SW 50. 1 rotor at 100C. A) controlchromatin B) chromatin irradiated with a u.v. dose of 3.8 x 104 ergs,/mm and C) chromatin irradiated with a u.v. dose of 10. 8 x 104 ergs/mm .
Since the amount of crosslinked protein did not vary significantly within the range of u.v. irradiation analyzed and considering that proteins were disappearing from the SDS-polyacrylamide gels without accumulation of staining material at the top of the gel we believe that our results could be best explained as follows: a small fraction of the chromosomal proteins does in 9-10 fact crosslink to the DNA but this reaction is hindered by protein breakage Since it was possible that u.v. actually induced denaturation of the proteins 14, 15, 10 which could then be degraded by a proteolytic system present in the chromatin we analyzed the effect of u.v. radiation on chromatin proteins isolated under denaturating conditions. The result of such an experiment is shown in fig. 3A.
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Nucleic Acids Research B
A
*-i*Il* w__
u-Origin
-----
fl
0 7 14 70
uNv
dose
0
ergs/mm2
4.8 9.6 x
10-3
Figure 3. Irradiation of chromatin proteins and BSA. Proteins were dissociated from chromatin with 1% SDS, 2% 2 mercaptoethanol and 1 M NaCl. The viscous suspension was centrifuged at 15-200C for 7 hrs. at 47.000 rpm in the Ti 50 rotor. The supernatant was saved and the pellet was extracted once more with the same solution. The supernatant fractions were pooled and dialyzed extensively against twice deonized water. The proteins were dried by lyophylization and were resuspended in 10 mM Tris-HCl pH 8. 0. Proteins at a concentration of 250 ug/ml in 10 mM Tris-HCl pH 8. 0 were irradiated at 40C and analyzed in SDS-polyacrylamide gels as described in Methods. A) chromatin proteins. Only the non-histone protein region and histone f1 are shown. Approximately the same amount of protein (30 pg) was added to each gel. B) BSA. The band moving slower than BSA corresponds to an impurity. 30 ,ug of protein was added to each gel.
Only the non-histone protein region and f1 histone are shown. Again we obtained the same results as when whole chromatin was irradiated. Further irradiation leads to disappearance of the histones such as when whole chromatin is irradiated (results not shown). At present we do not know whether the proteins not entering the gel correspond to protein-protein or to protein-DNA crosslinking. The latter is possible since small amounts of low molecular weight DNA could have contaminated the protein preparation. We have not examined these possibilities any further. It thus seems that chromosomal proteins do break down as a result of u.v. irradiation and the sensitivity towards irradiation probably depends on the amino acid composition of the protein10 (based on the different amino acid composition between nonhistone and histone proteins). Finally, the effect of u.v. is not only confined to chromosomal proteins. As shown in fig. 2B irradiation of bovine serum albumin (BSA) leads to the production of polypeptide fragments9. These frag-
1069
Nucleic Acids Research ments could be seen even at low doses of irradiation (1.2 x 103 ergs/mm2 results not shownl. We could not detect protein breakage or crosslinking under conditions in which a pyrex cover was placed on top of the sample even results not up to quite large doses of irradiation (7.5 x 105 ergs/mm2 shown). Only u.v. of a wave-length higher than 2950 A is transmitted under this condition. Our results then raise many questions about the use of u.v. of less than 2950 A as a probe to study protein-nucleic acid interaction. Even though polypeptide breakage does not take place at low u.v. doses it is possible that amino acid conversion and modification could take place 9, 17 18 -
ACKNOWLEDGEMENTS We thank the Cambridge MRC Chromatin Group for discussion. We also thank Professor B.F.C. Clark and Dr. J.D. Smith for comments and reading the manuscript.
+Present address:
Division of Biostructural Chemistry, Institute of Chemistry, Aarhus University, 8000 Aarhus C, Denmark. °Division of Biostructural Chemistry, Institute of Chemistry, Aarhus University, 8000 Aarhus C Denmark.
REFERENCES 1. Smith, K.C. (1962) Biochem.Biophys.Res.Commun. a, 157-163. 2. Alexander, P., and Moroson, H. (1962) Nature, Lond 194, 882-883. 3. Habazin, V., and Han, A. (1970) Int.Radiat.Biol. 17, 569575. 4. Han, A., Korbelik, M., and Ban, J. (1975) Int.J.Radiat.Biol. 27, 63-74. 5. Smets,L.A., and Cornelis,J.J. (1971) Int.Radiat.Biol. 19, 445-457. 6. Han,,A. (1973) Studia Biophys. 36-37, 127-137. 7. Hutchinson, F. (1973) Quarterly Reviews of Biophys. 6, 201-246. 8. Weintraub, H. (1973) Cold Spring Harbor Symp.Quant.Biol.38, 247-256. 9. Rideal, E.K., and Roberts, R. (1951) Proc.Roy.Soc. 205A, 391-405. 10. Smith, K.C. and Hanawalt, P.C. (1969) in Molecular Photobiology pp 85-95. Academic Press, New York. 11. Ilyin, Yu.V., Varshawsky, A.J., Mickelsaar, -U.N., and Georgiev, G.P. (1971) Eur.J.Biochem. 22, 235-245. 12. Laemmli, U.K. (1970) Nature2n7, 680-685. 13. Ilyin, V.Y., Varshavskii, A.J., and Georgiev, G.P. (1970) Mol.Biol. USSR 4, 821-830. 14. McLaren, A.D. and Hidalgo-Salvatierra, 0. (1964) Photochem. Photobiol. 3, 349-353. 15. Augenstein, L., and Riley, P. (1964) Photochem. Photobiol. 3, 353-367. 16. Vecchio, G., Dose, K. and Salvatore, G. (1968) European J. Biochem. 5, 422-428.
1070
Nucleic Acids Research 17. 18.
Aktipis, S., and Iammartino, A.J. (1972) Biochem.Biophys.Acta, 278, 239-242. Schultz, R.M., Jammartino, A.J., andAktipis, S. (1975) Biochem. Biophys.Acta 386, 120-128.
1071
Nucleic Acids Research
On the use of ultraviolet light to study protein-DNA crosslinking.
Julio E . Celis*+ , Michelle Fink and Keld Kaltofto * MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, E ngland, UK
Received 6 February 1976 A BSTRACT Irradiation of Ehrlich ascites chromatin with ultraviolet light (u. v.) leads to protein-DNA crosslinking as determined by CsCl isopycnic ultracentrifugation and SDS-polyacrylamide gel electrophoresis. At the most 4.5% of the chromatin proteins labelled with (14C)-lysine and (14C)-arginire can be criosslinked to DNA at u.v. doses between 3. 6 x 104 to 10. 8 x 10 ergs/mm'. We find however that the crosslinking reaction is hindered by protein breakage induced by u.v. light of wave length of less than 2950 A. Our results point out that caution must be used in the interpretation of studies on protein-nucleic acid interactions using u.v. light.
INTRODUCTION Research on the structure of chromatin needs crosslinking agents that will allow the determination of which proteins are in close proximity to the DNA. It was first noted by Smith that ultraviolet light (u.v.) irradiation induced DNA-to-protein crosslinking in bacteria. This observation has been further confirmed using mammalian cells by several laboratories2-4 There are at least two ways in which u.v. induced crosslinking between protein and DNA could be used to approach our problem; (1) irradiation of cells or chromatin with u.v. light and (2) irradiation of BUdR treated cells or chromatin (BUdR-substituted DNA) with u.v. light of about 3130 A5 8. In this article we describe experiments in which we have irradiated Ehrlich ascites chromatin in vitro with u.v. light. We show that although a fraction of the chromatin proteins (4.5%) can be crosslinked to the DNA this reac tion is hindered by protein breakage induced by u.v. irradiation 9-10. The experiments concerning irradiation of BUdR treated cells and chromatin will be a matter of a further communication. METHODS Preparation of chromatin. Chromatin from Ehrlich ascites mouse 11 tumor cells was prepared essentially as described by Ilyin et al. . The cells were broken with 0. 3% Triton X-100 in 0. 25 M sucrose buffer con-
C Information Retrieval Umited 1 Falconberg Court London W1 V 5FG England
1065
Nucleic Acids Research taining 50 mM Tris-HCl, pH 8. 0, 10 mM MgCl2 and 10 mM dithiothreitol. The nuclei were recovered from the lysed cells by pelleting at 600 xg. for 10 min. and washed three times in the 0.25 M sucrose buffer. Chromatin was ext racted by homogenizing the nuclei continously for 1 min. in 75 mM NaCl, 25 mM EDTA, pH 8.0, 2 mM 2-mercaptoethanol, 10 mM NaHSO3. The suspension was pelleted at 3. 000 xg. for 10 min. and the extraction was repeated twice. The final pellet was resuspended in 5 mM Tris-HCl pH 8.00, 2 mM 2 mercaptoethanol, 5 mM NaHSO3 and layered over 25 ml of 1. 7 M sucrose in the same buffer. The chromatin was pelleted by centrifuging in a Beckman SW 25 rotor at 22. 000 rpm at 4 0C for 3 hrs. The chromatin gel was allowed to swell overnight in twice deionized H20 at 0 0C. u.v. irradiation of chromatin. Chromatin at concentration of 5 A 260/ml (15 ml in a 10 cm petri-dish) was irradiated with constant shaking at 40C with a Mercury arc lamp uvs-100, 125 Watt, Hanovia made inSlough, England. The energy output of the lamp was determined with a Blak-Ray u.v. meter model J-225. Samples withdrawn at different times were lyophylized 12 and resuspended in SDS-dissociation buffer SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel slabs containing 17% acrylamide were prepared as described by Laemmli12 and run at 40C and 20 mA for 15 hrs. The slab gels were stained with 0. 1% (w/v) coomassie brilliant blue in methanol/acetic acid/water (40: 7: 53) and destained with the same solvent.
RESULTS AND DISCUSSION Chromatin exposed to increasing doses of u.v. light radiation was analyzed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis as described in Methods. We assumed that proteins crosslinked to DNA will not enter the gel; thus, a total or partial disappearance of a polypeptide band from the running gel followed by a concomittant appearance of protein staining material at the top of the gel is taken as a sign of proteinDNA crosslinking. Although this assay is not specific (it cannot distinguish between protein-protein and protein-DNA crosslinking) it allowed a rapid screening of many samples. It is clear from the gels shown in fig. 1 that at u.v. doses between 3. 6 to 10. 8 x 104 ergs/mm2 some chromosomal proteins, mainly the non-histone proteins, disappeared from the gel (partially or totally) with a simultaneous appearance of protein staining material at the top of the gel. Irradiation with even higher u.v. doses leads to a reduction of the intensities of protein bands corresponding to f., f3 f2b and f2a2 respectively, without accumulation of crosslinked material at the top of the gel or appearance of new bands in the running gel. 1066
Nucleic Acids Research B --
Origin
A
Origin -
cl
C
l
f
f 2 b_
W::~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. .....:
f3
+
0
36
72 108
216 432
ergs/ mm2 x 1O3 ~~~~~~~~~~~~
u.v dose f3 b
Figure 1. SDS-polyacrylamide gel electrophoresis of chromatin irradiated with u. v. light. 30 pug of protein was applied to each gel slot. A more extensive analysis of the irradiated samples of chromatin (double labelled with (methyl 3H) -thymidine and ~14C) -lysine and (14C )-arginine by means of CsCl isopycnic ultracentrifugation is shown in fig. 2. Irradiation with a u. v. dose of 3. 8 x 1 leads to splitting of the dine counts in two peaks with densities of 1. 68 and 1 52 respectively
04ergs/mm2
g/cm3
(3H)-thymi-
(fig. 2B). Analysis of the C-protein counts showed that 4% of the radioactivity was pre sent unde r the DNA peak of density 1. 52 g/ cm3 while no counts 3 could be detected under the peak of density 1. 68 g/cm Irradiation with a -_fig. 2C) results in an increase in higher u. v. dose (1 0.8 x the size of the peak of density 1g 52 a substantial increase in the amount of C-protein present under the peak (4.5%). At present we do not know what causes the DNA to shift to a density of 1.52 g/cm 3 .in such a fashion. First the amount of protein under the peak does not correspond with the expected protein content of a nucleoprotein of density of 1. 52 g/cm3p13 and s of density secondly one would expect intermediate species between the .
4ergs/mm2 glcm3without
1.68 and
1.o52 g/cm 3respectively. Further experiments
will be necessary
to clarify these points.
1067
Nucleic Acids Research
0
0
,x
X 9
ECL
E
C)
I
Fraction Number
Figure 2. CsCl isopycnic ult{acentrifugation of Y4v. irradiated chromatin double labelled with(methyl- H)-thymidine and ( C)-lysine and (14C)-arginine. Mice carrying E)rlich ascites tumor were injected intraperitoneally with 1 mF of (methyl- H)-thymidine (Amersham 43 Ci!mmol) and 500 pCi each of ( C)-lysine (Amersham 270 mCi/mmol)and (14C)-arginine (Amersham 270 mCi/mmol). Samples after irradiation were centrifuged in CsCl at 40. 000 rpm for 72 hrs. in the SW 50. 1 rotor at 100C. A) controlchromatin B) chromatin irradiated with a u.v. dose of 3.8 x 104 ergs,/mm and C) chromatin irradiated with a u.v. dose of 10. 8 x 104 ergs/mm .
Since the amount of crosslinked protein did not vary significantly within the range of u.v. irradiation analyzed and considering that proteins were disappearing from the SDS-polyacrylamide gels without accumulation of staining material at the top of the gel we believe that our results could be best explained as follows: a small fraction of the chromosomal proteins does in 9-10 fact crosslink to the DNA but this reaction is hindered by protein breakage Since it was possible that u.v. actually induced denaturation of the proteins 14, 15, 10 which could then be degraded by a proteolytic system present in the chromatin we analyzed the effect of u.v. radiation on chromatin proteins isolated under denaturating conditions. The result of such an experiment is shown in fig. 3A.
1068
Nucleic Acids Research B
A
*-i*Il* w__
u-Origin
-----
fl
0 7 14 70
uNv
dose
0
ergs/mm2
4.8 9.6 x
10-3
Figure 3. Irradiation of chromatin proteins and BSA. Proteins were dissociated from chromatin with 1% SDS, 2% 2 mercaptoethanol and 1 M NaCl. The viscous suspension was centrifuged at 15-200C for 7 hrs. at 47.000 rpm in the Ti 50 rotor. The supernatant was saved and the pellet was extracted once more with the same solution. The supernatant fractions were pooled and dialyzed extensively against twice deonized water. The proteins were dried by lyophylization and were resuspended in 10 mM Tris-HCl pH 8. 0. Proteins at a concentration of 250 ug/ml in 10 mM Tris-HCl pH 8. 0 were irradiated at 40C and analyzed in SDS-polyacrylamide gels as described in Methods. A) chromatin proteins. Only the non-histone protein region and histone f1 are shown. Approximately the same amount of protein (30 pg) was added to each gel. B) BSA. The band moving slower than BSA corresponds to an impurity. 30 ,ug of protein was added to each gel.
Only the non-histone protein region and f1 histone are shown. Again we obtained the same results as when whole chromatin was irradiated. Further irradiation leads to disappearance of the histones such as when whole chromatin is irradiated (results not shown). At present we do not know whether the proteins not entering the gel correspond to protein-protein or to protein-DNA crosslinking. The latter is possible since small amounts of low molecular weight DNA could have contaminated the protein preparation. We have not examined these possibilities any further. It thus seems that chromosomal proteins do break down as a result of u.v. irradiation and the sensitivity towards irradiation probably depends on the amino acid composition of the protein10 (based on the different amino acid composition between nonhistone and histone proteins). Finally, the effect of u.v. is not only confined to chromosomal proteins. As shown in fig. 2B irradiation of bovine serum albumin (BSA) leads to the production of polypeptide fragments9. These frag-
1069
Nucleic Acids Research ments could be seen even at low doses of irradiation (1.2 x 103 ergs/mm2 results not shownl. We could not detect protein breakage or crosslinking under conditions in which a pyrex cover was placed on top of the sample even results not up to quite large doses of irradiation (7.5 x 105 ergs/mm2 shown). Only u.v. of a wave-length higher than 2950 A is transmitted under this condition. Our results then raise many questions about the use of u.v. of less than 2950 A as a probe to study protein-nucleic acid interaction. Even though polypeptide breakage does not take place at low u.v. doses it is possible that amino acid conversion and modification could take place 9, 17 18 -
ACKNOWLEDGEMENTS We thank the Cambridge MRC Chromatin Group for discussion. We also thank Professor B.F.C. Clark and Dr. J.D. Smith for comments and reading the manuscript.
+Present address:
Division of Biostructural Chemistry, Institute of Chemistry, Aarhus University, 8000 Aarhus C, Denmark. °Division of Biostructural Chemistry, Institute of Chemistry, Aarhus University, 8000 Aarhus C Denmark.
REFERENCES 1. Smith, K.C. (1962) Biochem.Biophys.Res.Commun. a, 157-163. 2. Alexander, P., and Moroson, H. (1962) Nature, Lond 194, 882-883. 3. Habazin, V., and Han, A. (1970) Int.Radiat.Biol. 17, 569575. 4. Han, A., Korbelik, M., and Ban, J. (1975) Int.J.Radiat.Biol. 27, 63-74. 5. Smets,L.A., and Cornelis,J.J. (1971) Int.Radiat.Biol. 19, 445-457. 6. Han,,A. (1973) Studia Biophys. 36-37, 127-137. 7. Hutchinson, F. (1973) Quarterly Reviews of Biophys. 6, 201-246. 8. Weintraub, H. (1973) Cold Spring Harbor Symp.Quant.Biol.38, 247-256. 9. Rideal, E.K., and Roberts, R. (1951) Proc.Roy.Soc. 205A, 391-405. 10. Smith, K.C. and Hanawalt, P.C. (1969) in Molecular Photobiology pp 85-95. Academic Press, New York. 11. Ilyin, Yu.V., Varshawsky, A.J., Mickelsaar, -U.N., and Georgiev, G.P. (1971) Eur.J.Biochem. 22, 235-245. 12. Laemmli, U.K. (1970) Nature2n7, 680-685. 13. Ilyin, V.Y., Varshavskii, A.J., and Georgiev, G.P. (1970) Mol.Biol. USSR 4, 821-830. 14. McLaren, A.D. and Hidalgo-Salvatierra, 0. (1964) Photochem. Photobiol. 3, 349-353. 15. Augenstein, L., and Riley, P. (1964) Photochem. Photobiol. 3, 353-367. 16. Vecchio, G., Dose, K. and Salvatore, G. (1968) European J. Biochem. 5, 422-428.
1070
Nucleic Acids Research 17. 18.
Aktipis, S., and Iammartino, A.J. (1972) Biochem.Biophys.Acta, 278, 239-242. Schultz, R.M., Jammartino, A.J., andAktipis, S. (1975) Biochem. Biophys.Acta 386, 120-128.
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