/ . Biochem. 86, 1469-1478 (1979)
Immunochemical Studies on the Correlation between Conformational Changes of DNA Caused by Ultraviolet Irradiation and Manifestation of Antigenicity Akira WAKIZAKA and Eiji OKUHARA Department of Biochemistry, Akita University School " of Medicine, Hondo, Akita, Akita 010 Received for publication, March 26, 1979
The conformational changes of double-stranded DNA induced during irradiation with ultraviolet, were immunologically investigated. These studies revealed that at least two distinct antigenic sites were induced in the irradiated DNA molecule, giving rise to two different antibodies specific for ultraviolet-irradiated (uv) DNA and thermally denatured DNA-like structure, and these were demonstrable using radioimmunoassay and double diffusion tests. A series of experiments, including melting profile, fluorescence intensity of the ethidium bromide complex and chromatographic behavior on hydroxyapatite, performed on the antigenically active uvDNA indicated that the duplex structure of DNA separated irregularly during irradiation. Furthermore, the data showed that the conformational determinants of uvDNA are located on the exposed single-stranded regions.
It is known that ultraviolet-irradiation of DNA produces several kinds of photochemical products and subsequent conformational changes in the molecule {1-3). Electron microscopic (4) and enzymological studies (5) on irradiated DNA revealed that the double strand of DNA separated during irradiation and locally denatured regions appeared in the molecule. Ultraviolet treatment, on the other hand, makes DNA immunologically active. The production of specific antibodies to uvDNA (6-11) and the usefulness of this antiserum Abbreviations and symbols: uvDNA, ultraviolet-irradiated DNA; dDNA, thermally denatured DNA; EB, ethidium bromide; As(uv), antiserum to uvDNA; Ab(uv), antibody to uvDNA; As(d), antiserum to dDNA; Ab(d), antibody to dDNA; ds-DNA, doublestranded DNA; ss-DNA, single-stranded DNA. Vol. 86, No. 5, 1979
to detect ultraviolet-induced lesions on DNA (8, 9, 11) have been reported by several investigators. Pioneering work on the antigenic determinant of ultraviolet-irradiated DNA, which was thermally denatured before irradiation, was reported by Levine and his associates (6, 7). They demonstrated that the thymine dimers formed during irradiation are the determinant for the DNA. However, little information is available about the antigenic determinant for double-stranded DNA irradiated with ultraviolet. In our previous paper (12) on the antigenic determinant of thermally denatured DNA, we stated that some structural units within the denatured DNA molecule act as the determinant. In addition, we reported that the experimental fact that none of the oligonucleotides showed more than 60% inhibitory activity in the concentration range tested, might
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be thought to reflect the influence of the three dimensional structure on specificity. In similar studies of ours (unpublished) with uvDNA, we noticed that the expression of its antigenicity is susceptible to conformational changes. Therefore, we attempted to investigate the relationship between the manifestation of uvDNA antigenicity and the conformational changes induced, and found that strand-separation of the duplex structure of DNA as such plays a key role in forming the determinant. MATERIALS AND METHODS DNAs—DNA was prepared from lyophilized salmon sperm (oncorhynchus ketd) by the usual method in this laboratory (13). Double-stranded DNA was separated by hydroxyapatite column chromatography (14). [3H]DNA was prepared from mouse embryo labeled in vivo with [6-3H]thymidine by the method described previously (12). Preparation ofuvDNA—3 ml of DNA solution (1 mg/ml of 0 . 1 5 M NaCl) in a petri dish (2.5 cm diameter) was irradiated at 4°C with a commercial germicidal lamp (Toshiba GL-15,15 watt, 254.7 nm emission) at a distance of 5 cm above the surface of the solution with slow and constant stirring. The dose rate of irradiation was 10.3 J/m2/min as determined by dosimetry with a Topcon UVR-254 U.V. Radiometer (Tokyo Kogaku) in the range of 200 to 320 nm. Preparation of Antisera—Rabbit antisera to thermally denatured salmon sperm DNA (dDNA) were prepared by the usual method in our laboratory (75). Rabbit antisera to ultraviolet-irradiated salmon sperm DNA (uvDNA) were prepared in a similar manner; 0.5 mg of DNA dissolved in 1 ml of saline was irradiated at a dose of approximately 2.5xlO 3 J/m 2 with ultraviolet and stirring. The uvDNA solution and an equal weight of methylated bovine serum albumin were mixed. The complex emulsified with an equal volume of Freund's complete adjuvant was injected intramuscularly into rabbits (0.5 mg of uvDNA/animal) at least 6 times at weekly intervals, and blood was drawn one week after the sixth or eighth injection. The remaining procedures have been described in our previous paper (IS). We used the antiserum obtained from rabbit No. 120 in this study. Gamma globulin was obtained from the antiserum
by ammonium sulfate fractionation (16). Antiserum to uvDNA was absorbed with dDNA to remove antibodies to dDNA in the following way; 25 mg of gamma globulin from #R112 antiserum in 0.5 ml of 0.05 M phosphate buffer (pH 7.5) containing 0.15 M NaCl was mixed with 5.0 mg of dDNA in 5.0 ml of the buffer and allowed to stand for 48 to 72 h at 4°C with occasional mixing. The precipitate was spun down at 5,000 rev./min for 20 min and the resulting supernatant was applied to a DEAE-cellulose column (2x2 cm) to remove free dDNA. The antibodies absorbed on the column were eluted with 0.05 M phosphate buffer (pH 6.5). 60 to 70% of the globulin was recovered in the first 30 ml of the eluate. Ouchterlony's double diffusion tests (17) were performed at 4°C for 48 to 72 h in a humid chamber on a glass plate (5x8 cm) laying 0.3% agarose in 0.05 M phosphate buffer (pH 7.5)-0.15 M NaCl containing 0.02% of sodium azide. Radioimmunoassay—[3H]uvDNA bound by anti-uvDNA antibodies was measured by a modification of Farr's assay (12, 18-20). The assay medium contained 1.0 fig of [3H]uvDNA (5,000 dis./min, irradiated at a dose of approx. 2.5 x 10s J/m2) in 0.05 ml of 0.1 M borate buffer (pH 8.5), 0.05 ml of 50-fold diluted anti-uvDNA antisera with the buffer and various amounts of nonradioactive DNA in 0.1 ml of the buffer. When gamma globulin from the antisera absorbed with dDNA was used in the assay, 50 ^g of the globulin in 0.05 ml of the buffer was added in place of the antiserum. The incubation of the mixture was carried out at 4°C for 15 min. After mixing with an equal volume of saturated ammonium sulfate solution at 4°C for 1 h, the precipitate and supernatant fractions were separated by centrifugation (3,000 rev./min for 15 min). 0.05 ml of both fractions was dissolved in 0.5 ml of NCS solubilizer and 10 ml of toluene scintillation cocktail containing 4 g of Omnifluor, and assayed for radioactivity in a Searle Analytic Mark II liquid scintillation counter. The percentage of antigen bound by the antiserum and binding inhibition were calculated as described previously (12). The [3H]dDNAantibody binding was also assayed in the same manner. Melting Profile ofuvDNA—Thermal denaturation curves of uvDNA (75 /*g/ml of 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) were made J. Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA by determination of absorbance at 260 nm with a Hitachi Model 139 spectrophotometer equipped with a Komatsu Solidate SPS-H139 thermoregulator. During the experiment, the temperature of the solution was increased at a constant rate of 1°C per min from room temperature of about 25CC by means of a Komatsu Model KPC-2 automatic temperature programmer. Temperature and absorbance were recorded automatically. The width (47") of the thermal denaturation curve is represented by the temperature difference between 15.9% and 84.1 % of the absorbance rise (21, 22).
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(Hypatite C), from Clarkson (Williamsport, Pa., U.S.A.); agarose (A-45), from Daiichi Kagaku (Tokyo, Japan); ethidium bromide, from Sigma (St. Louis, Mo., U.S.A.). All other chemicals used were of the highest purity commercially available. RESULTS
Antigenic Reactivity of uvDNA—Figure 1 shows the inhibitory activities of DNAs irradiated at different doses on the binding of [3H]uvDNA Fluorometric Determination—The increasing by anti-uvDNA rabbit serum. The inhibitory fluorescence intensity of ethidium bromide (EB) activities of irradiated DNAs were elevated with bound to double-stranded DNA was measured increasing dose of irradiation. Interestingly, DNA 2 according to LePecq (23). A constant volume irradiated at a dose of 10 J/m (about 1 min of (usually 4.0 ml) of DNA solution (2 to 10^g/ml irradiation) also reacted with the antiserum. When of 0.2 M NaCl containing 2 mM EDTA) was mixed the amount of DNA added was 5 //g, the perwith an equal volume of ethidium bromide solution centage of inhibition by the uvDNA irradiated at 2 (20 pg/ml of 0.1 M Tris-HCl buffer, pH 7.5). The doses of 10, 50, 310, 620, and 1,240 J/m was 72, fluorescence intensity of the resulting DNA-EB 80, 83, 90, and 98, respectively. Native DNA complex was measured with a Hitachi MFP-2A fluorescence spectrophotometer. Wavelength of excitation light was 310 nm and emitted fluores100 cence was measured at 580 nm. The intensity of fluorescence of EB solution not containing DNA 80 was subtracted from experimental values. The extent of single-strand formation in uvDNA was calculated from the difference between fluorescence 60 intensity of the uvDNA-EB complex and that of the native DNA-EB complex. Hydroxyapatite Chromatography—Hydroxyapatite column chromatography of uvDNA was performed according to the method of Bernardi (14). A hydroxyapatite column (1.5x20cm), to which was applied 6 ml of saline solution of uvDNA (3 mg), was eluted with 200 ml of phosphate buffer, pH 6.8 (a linear concentration gradient of sodium phosphate, 0.05-0.4 M) at a rate of 20 ml/h and 2 ml fractions were collected. Aliquots of 0.1 ml were used for the radioimmunoassay. Chemicals—[6-3H]Thymidine in aqueous solution (20-30 Ci/mmol) was purchased from The Radiochemical Centre (Amersham, Great Britain); NCSsolubilizer, from Amersham/Searle (Arlington Heights, III., U.S.A.); Omunifluor, from New England Nuclear (Boston Mass., U.S.A.). DEAE cellulose (DE-32) was obtained from Whatman (Springfield Mill, 'Great Britain); hydroxyapatite Vol. 86, No. 5, 1979
0
0.6
1.25
2.5
o
5.0
DNA added, jjg 3
Fig. 1. Inhibition of [ H]uvDNA-antibody binding by DNA irradiated with ultraviolet at different doses. The binding assay medium contains 1.0 fi% of [3H]uvDNA (5,000 dis./min, irradiated at a dose of 2.5XlO3 J/m2), 50 fi\ of a 50-fold diluted anti-uvDNA antiserum, and different amounts of DNA in 0.2 ml of 0.1 M borate buffer (pH 8.5). ( • ) , DNA irradiated with ultraviolet at a dose of 1.2 kJ/m2; ( v ) , at a dose of 6.2x 102 J/m2; ( • ) , at a dose of 3.1 x 102 J/m2; ( A ) , at a dose of 50 J/m2; ( A ) , at a dose of 10J/m a ; (O), unirradiated native DNA; (O), unirradiated dDNA. Results represent the mean of three experiments.
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showed a weak inhibitory activity on the binding when more than 1.25 //g of DNA was added, while dDNA did not show any inhibitory activity. In addition, uvDNA, especially irradiated at a relatively high dose, also inhibited the binding of [3H]dDNA by anti-dDNA rabbit sera, as seen in Fig. 2. The minimum dose of irradiation required for the inhibition of the irradiated DNA on dDNAantibody binding was 6.2 x 102 J/m2. Native DNA did not exhibit any inhibition on this binding. Figure 3 shows the precipitating patterns of anti-uvDNA serum and anti-dDNA serum with
80
w 60
40
20
0.6
1.25 DNA added.
Fig. 2.
2.5
5.0
Fig. 2. Inhibition of [3H]dDNA-antibody binding by DNA irradiated with ultraviolet at different doses. The assay medium contained 1.0/ig of [8H]dDNA (5,000 dis./min), 50 ft] of a 50-fold diluted anti-dDNA antiserum and DNAs (inhibitors). ( • ) , DNA irradiated with ultraviolet at a dose of 9.3 x 102 J/m2. Other symbols are the same as in Fig. 1. Results represent the mean of three experiments.
Fig. 3. Double diffusion tests in agarose gel. Central wells contained 40 fil of the anti-uvDNA antiserum (a) and anti-dDNA antiserum (b).2 The peripheral wells contained 40 //I of each antigen: N, native DNA; 1, uvDNA irradiated at a dose of 50 J/m ; 2, at 2a dose of 1.6 x 10! J/m2; 33, at2 a dose of 3.1 x 102 J/m2; 4, at a dose of 6.2 X 2 2 2 10 J/m ; 5, at a dose of 9.3 x 10 J/m ; 6, at a dose of 1.2x 10 J/m . / . Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA
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Ab(uv)
uvDNA
dDNA
Absorbed Ab(uv)
uvDNA
dDNA
uvDNA
Ab(uv
Ab(d)
Ab(d)
dDNA
uvDNA
Fig. 4. Double diffusion tests of anti-uvDNA serum [Ab(uv)] and anti-dDNA serum [Ab(d)] with uvDNA and dDNA. (a) The reactivities of uvDNA and dDNA with Ab(uv). (b) The reactivities of uvDNA and dDNA with anti-uvDNA gamma globulin absorbed with dDNA [Absorbed Ab(uv)]. (c) The reactivities of uvDNA with antisera, Ab(uv) and Ab(d). (d) The reactivities of dDNA and uvDNA with Ab(d). DNA irradiated at a dose of 2.5 x 103 J/m2 was used as an antigen in double diffusion tests. Vol. 86, No. 5, 1979
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uvDNA irradiated at the different doses. As shown in Fig. 3a, two precipitating lines were formed between anti-uvDNA serum and uvDNAs irradiated at the doses of 3.1 xlO2, 6.2 xlO2, and 1.2xlO3 J/m2. On the other hand, as seen in Fig. 3b, only one precipitating line was observed between anti-dDNA serum and the uvDNA irradiated at a dose of 1.2 x 103 J/m2. lmmunological Characteristics of uvDNA—The
antigenicity of DNA induced by the irradiation was further analyzed by the double diffusion test. Figure 4a shows the reactivities of uvDNA and dDNA to the anti-uvDNA serum. Between uvDNA and Ab(uv), both heavy and weak precipitating lines were formed, suggesting that uvDNA might hold two antigenic sites. The weak line between uvDNA and Ab(uv) was not clearly observed to fuse into one line between Ab(uv) and dDNA, but both weak lines were eliminated by using anti-uvDNA antibodies absorbed with dDNA as seen in Fig. 4b. In order to examine which precipitating line of the two between uvDNA and its antiserum, shown in Figs. 3a and 4a, really connects with the single line between uvDNA and antiserum to dDNA, shown in Fig. 3b, an Ouchterlony's test was performed as depicted in Fig. 4c. The clear explanation of the results is difficult, but it seems likely to be the case that the line formed between uvDNA and Ab(d) crossed over the main precipitating line between uvDNA and Ab(uv) (and thus a double spur was formed) and fused with the second line between them. TABLE I. Melting profiles of uvDNA. Dose 3of UV xlO- J/m= 0 0.3 0.6 1.2 2.4
T_ m
,O/^ (C)
84. 7 ±0.87 82.0+1.80 78. 5± 1.44 71.8 + 0.76 67. 5 ±3. 27
AT,O AT (
7. 3±0.76 11.0 + 0. 50 14.0 + 0.00 18.0±0. 50 26. 3 ±0.30
Total (%) hyperchromism11 39.0 + 0.7 33.4±1.0 32. 3±0. 5 30.0±1.2 26. 5±0.6
Results represent the mean ± S.D. of three different experiments. a AT, the width of the thermal denaturation curves represented by the temperature difference between 15.9 and 84.1% of the absorbance rise (/4260). b Total hyperchromism, percent increase in absorbance at 260 nm resulting from melting divided by the initial absorbance at room temperature.
Hence, uvDNA shares some common determinants with dDNA detectable with the antiserum, Ab(d). Correlation between Antigenicity and Structural Changes—Table I shows changes of Tm and width (J T) of the Tm curve together with changes in total hyperchromism of DNA during irradiation with ultraviolet. As seen in the table, the decrease of Tm and total hyperchromism, and the increase of /IT paralleled the increase of the irradiationdose. These physical properties of uvDNA seem to reflect weakening of the base pairing and partial separation of the duplex of DNA. The relationship between the structural changes and the antigenic manifestation of the DNA during irradiation is summarized in Fig. 5. When the irradiation-dose increased, Tm and fluorescence intensity of the DNA-EB complex decreased along with the increase of the inhibitory activities on the DNA-antibody binding. Inhibitory activity on [3H]uvDNA-Ab(uv) binding was induced with a rather small dose of irradiation, while that on [3H]dDNA-Ab(d) binding appeared "MOO
IOO r
1 90
eo
•. 60
80
20
70
0
0.3
0.6
0.9
1.2
Dose of ultraviolet Irradiation x 103 J/m2
Fig. 5. The relationship of the conformational changes of uvDNA with the inhibitory activities on antigenantibody binding. DNAs irradiated at different doses (as indicated in the abscissa) with ultraviolet were examined with respect to their Tm (V), extent of strandseparation (%) as determined by the fluorometry (O), and inhibitory activities on [3H]uvDNA-Ab(uv) binding ( A ) and on [3H]dDNA-Ab(d) binding ( • ) . 5 fig of uvDNA was used to determine the inhibition on dDNAAb(d) binding, and 2.5 p%, on uvDNA-Ab(uv) binding. Results represent the mean of three or more experiments. J. Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA on the molecule after irradiation with a relatively large dose. Chromatographic Behaviour and Antigenic Reactivity—Figure 6 shows the change of hydroxyapatite chromatographic pattern of DNA during irradiation with ultraviolet. As shown in the figure, a heavy load of native DNA in a preparative scale to the column caused broadening of the elution peak, but the unirradiated nDNA and dDNA were well separated, though some portion of dDNA was eluted at the position of ds-DNA. Irradiation shifted the elution position of DNA from ds-DNA toward ss-DNA according to the intensity of irradiation and the shape of elution peak became narrow like that of dDNA. Figures 7 and 8 show the relationship between chromatographic behaviour of uvDNA and antigenic reactivity. The inhibitory activity of the eluant on [ 3 H]uvDNA-Ab(uv) binding almost coincided with the peak of absorbance at 260 nm as shown in the figure, and a large part of the inhibitory activity still remained in ds-DNA position. On the other hand, the most inhibitable fraction of dDNA-Ab(d) binding was eluted
1475 ss
100 (50) ; < a ,
ds 1
• "0-3 0.5
(§» • j o . 2 • ^o- 1
I 0
s:
I (50)
(b)
-100
• 50 (25) •
0.3
•
0.1
30
0.2
i^o—Q—a 1.0
w Is 40
\
.
i 50 Fraction No.
70
60
Fig. 7. Antigenic reactivity of uvDNA eluted from hydroxyapatite column. 3 mg of uvDNA irradiated at a dose of 6.2xlO 2 J/m 2 (a), and 1.2x10'J/m 2 (b) was applied to a hydroxyapatite column. The chromatographic conditions was the same as in Fig. 6. Inhibitory activity of 0.1 ml of each fraction was assayed in uvDNA-Ab(uv) system ( • ) , and in dDNA-Ab(d) system (O). The inhibition rate to the latter system is indicated in parentheses in the figure. ( ), concentration of phosphate in eluate; ( ), absorbance at 260 nm; (•!», elution position of ds-DNA; ( [ ), that of ss-DNA. Results represent the mean of two assays.
/
f\
/ /
i il 0.5 i /
I
0.3
N
Yi y V i
j
••
•,
. ' /
ii
/
/
/
0.1 •
40
/X\
j/ \
1i 50
'.
0.3 \ ,
I
\ \ \
f
\
-0 . 2
°
0.1 60
70
Fraction Number
Fig. 6. Chromatographic profiles of uvDNA, nDNA, and dDNA on a hydroxyapatite column. 2 to 4 mg of DNAs were loaded on a 1.5 x 20 cm column. The column was eluted with 200 ml of sodium phosphate buffer (pH 6.8) in a linear concentration gradient from 0.05 to 0.4 M. Flow rate, 20 ml/h. 2.1ml fractions were collected and measured for their absorbance at 260 nm. ( ), indicates the absorbance at 260 nm of nDNA (3 mg); ( ), DNA (3 mg) irradiated with 0.6xl0 3 J/m 2 of ultraviolet; ( ), DNA (3.5 mg) irradiated with 1.2 x 103 J/m2; ( ), DNA (3 mg) irradiated with 2.5x 103 J/m2; ( ), unirradiated dDNA (2 mg); (
), molarity of phosphate in eluate.
Vol. 86, No. 5, 1979
g o
50
60
Fraction No.
Fig. 8. Chromatography of thermally and radiationally denatured DNA on a hydroxyapatite column. uvDNA thermally denatured in 0.15 M sodium chloride after irradiation with 2.5x 103 J/m2 ultraviolet (a), and uvDNA thermally denatured in the saline before irradiation at the same dose (b) was chromatographed in the same manner as in Fig. 6. ( • ) , inhibitory activity of 0.1 ml aliquots of each fraction on uvDNA-Ab(uv) binding. ( ), molarity of sodium phosphate; ( ), absorbance at 260 nm; (4J-), elution position of ds-DNA; ( | ) , that for ss-DNA. Results represent the mean of two assays.
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A. WAKIZAKA and E. OKUHARA
slightly earlier than the Ataa peak, typically shown in Fig. 7b. The shift of peak position from ds-DNA to ss-DNA was clearly observed when thermally denatured uvDNA or ultraviolet-irradiated dDNA was chromatographed on the same column, as shown in Fig. 8. DNA thermally denatured after irradiation was eluted at ss-DNA position on the column; serological activity to uvDNA-Ab(uv) system remained and coincided with the Atm peak, as shown in Fig. 8a. Similarly, irradiated DNA thermally denatured before irradiation was also eluted at ss-DNA position with serological activity (Fig. 8b). The shape of Auo peaks of these two cases was different. In the case of Fig. 8a, the peak was rather broad and serological activity was widely distributed around the peak area. However, in the Fig. 8b case, the DNA eluted in a relatively sharper and more compact peak with serological activity eluted slightly earlier than the •^260 peaK.
DISCUSSION Our present studies deal with the relationship between strand separation and the antigenic manifestation of DNA irradiated with ultraviolet. The irradiated DNA reacts with both anti-uvDNA and anti-dDNA antisera. This implies that at least two different antigenic sites are formed in the uvDNA molecule. The experimental data obtained by Ouchterlony's tests revealed that the anti-uvDNA antibodies recognize the unique structure of uvDNA and an additional structure which might be partially identical with that of dDNA. The antigenic structure specific for uvDNA may contain some photoproducts, such as thymine dimer. Levine et al. (6) and Seaman et al. (7) reported that the thymine dimer was most probably the determinant of ultraviolet-irradiated DNA, (thermally denatured before irradiation) on the following evidence: 1) serological activity of uvDNA reflects thymine content of the DNA; 2) antibodies to uvDNA are capable of being inhibited with ultraviolet irradiated thymidine oligonucleotides; 3) the serological activity of the irradiated DNA is decreased by re-irradiation at 235 nm; 4) irradiation of DNA in acetophenone solution with visible light gives a positive serological
response by forming thymine dimer. Their results fully explained the participation of thymine dimer in the antigenic determinant of ultraviolet-irradiated dDNA. However, the antigenic structure induced on the double-stranded DNA by irradiation was not sufficiently investigated. In particular, the relationship between conformational changes caused on the DNA strand by irradiation and formation of antigenic reactivity has not been elucidated. The antigenic determinant literally means "the site of antigen to which an antibody is specifically able to become attached by its combining site" (24). So it may occupy a certain dimensional part larger than single base or nucleotide moiety in DNA, involving the composition of a certain length of nucleotide chain in a certain conformation on the strand. We further investigated the inhibitory activity of pyrimidine oligonucleotides prepared from uvDNA by the known method (25), and found that no considerable extent of inhibition was detectable with those oligonucleotides (data reported preliminarily (26, 21), now under preparation for publication). Nevertheless these oligomers contained an appropriate suitable amount of thymine dimer which remains intact during chemical treatment, as Brunk (28, 29) has demonstrated. From these results we arrived at the idea of the predominancy of certain conformational changes of the strand structure in antigenicity rather than a simple modification of bases. The broadening of the thermal denaturation curve, the decreasing Tm, total hyperchromism and fluorescence intensity of the DNA-EB complex, and chromatographic behaviour on the hydroxyapatite column all give evidence for the formation of single-stranded region on DNA during irradiation. The chromatographic elution profile and the inhibitory activity of the eluate to both antigenantibody systems provides information about the structural changes induced on the strand. The most inhibitorily active fraction of eluate was different for the two antibody systems, Ab(uv) and Ab(d). uvDNA, which was the more inhibitorily active to the former antibody system, eluted slightly later than that for the latter system. This implies the immunochemically different molecular species of DNA or DNAs with denaturated portions of different extents are induced during irradiation and these can be separated by the
/ . Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA
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chromatography. The peak position of A2eo of eluate was shifted towards the elution position of single-stranded DNA during irradiation. But a large part of inhibitory activity to uvDNA-Ab(uv) system still remained close to the ds-DNA position. These findings suggest that a denatured region was formed only locally and to various extents on the DNA with a large native portion remained. The thermal denaturation of uvDNA in saline did not destroy most of uvDNA-specific antigenic reactivity and the denaturated uvDNA was mainly eluted at ss-DNA position on hydroxyapatite column (Fig. 8a). DNA irradiated after thermal denaturation was also eluted at ss-DNA position with serological activity to uvDNA-Ab(uv) system (Fig. 8b). These results suggested that the determinant group for uvDNA-specific antibody is located on the single-stranded conformation of DNA. Therefore, the antigenic structure of ultraviolet-irradiated double-stranded DNA seems to be closely connected with a single-stranded structure induced by irradiation. However, the possibility of participation of interstrand cross linking in the antigenic manifestation of irradiated DNA can not be excluded, since the heated uvDNA was eluted from hydroxyapatite column in a broader peak than irradiated dDNA. This may account for the involvement of interstrand cross links in the antigenic structure of uvDNA.
another way; it is caused by the difference in the physical form and size of the test antigens rather than the actual determinant. dDNA behaves like a small compact structure compared to the uvDNA. As shown in Fig. 1, binding between [ 3 H]uvDNA and Ab(uv) was not inhibited by the addition of cold dDNA. On the contrary, dDNA reacted with antiserum to uvDNA as shown in Fig. 4a. This may be explained as follows: 1) it may be caused by the difference between the immunoassays employed; 2) Fig. 4a and b indicate that two different kinds of antibodies are present in the serum to uvDNA, one specific for uvDNA (Ab(uv)) and the other with some common reactivity to dDNA(Ab(d)). Since Ab(uv) seems very likely to be major antibody populations in the serum, binding between [ 3 H]uvDNA and Ab(uv) may be not inhibited by the addition of cold dDNA. Antibody populations reactive with dDNA are Ab(d) but not Ab(uv) as such.
Radioimmunoassay revealed that the antigenic reactivity for a uvDNA-specific antibody appeared in the molecule with rather smaller doses of irradiation than that for anti-dDNA antibody (Fig. 5). If photoproducts induced on the molecule with ultraviolet cause weakening of base pairing and strand separation, the lag phase for appearance of the antigenic reactivity to Ab(d) on DNA with low irradiation doses (Figs. 2 and 5) may be explained as follows; the radiation-induced denaturated regions may be different immunochemically in structure from the single-stranded region induced by thermal treatment, due to the presence of photoproducts on the site or the smallness in size of the denaturated portion as the antigenic determinant for dDNA antibody. This difference in structure may influence the different reactivity of uvDNA to Ab(d) in radioimmunoassay mentioned above and the Ouchterlony reaction in Figs. 4a and d. The difference in curvatures of precipitating lines in the same figures may be explained in
Vol. 86, No. 5, 1979
Native DNA showed a slight inhibitory activity on uvDNA-Ab(uv) binding as shown in Fig. 1, when a large amount was added to the assay mixture. But it does not seem likely that this inhibition is an immunologically specific one, since the Ouchterlony test shown in Fig. 3a proved no reaction between nDNA and Ab(uv). It must be due to a non-specific DNA-gamma globulin interaction, as we reported previously (50). We conclude from our results that ultravioletirradiation on native DNA induces at least two different antigenic sites which are responsible for the production of two distinct antibodies. And this study seems to indicate that conformational changes of DNA caused by irradiation, especially partial strand-separation in double helical structure of the molecule, greatly contribute to the manifestation of antigenicity. REFERENCES 1. Wacker, A. (1963) in Progress in Nucleic Acid Research (Davidson, J.N. & Cohn, W.E., eds.) Vol. 1, pp. 369-399, Academic Press, New York 2. Setlow, R.B. (1968) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, J.N. & Cohn, W.E., eds.) Vol. 8, pp. 257-295, Academic Press, New York 3. Lomant, A.J. & Fresco, J.R. (1972) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, J.N. & Cohn, W.E., eds.) Vol. 12, pp. 1-27, Academic Press, New York
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4. Stafford, R.S., Allison, D.P., & Rahn, R.O. (1975) Nucleic Acid Res. 2, 143-148 5. Das Gupta, R. & Mitra, S. (1974) Biochim. Biophys. Ada 374, 145-148 6. Levine, L., Seaman, E., Hammerschlag, E., & Van Vunakis, H. (1966) Science 153, 1666-1667 7. Seaman, E., Levine, L., & Van Vunakis, H. (1967) in Nucleic Acids in Immunology (Plescia, O.J. & Braun, W., eds.) pp. 157-164, Springer-Verlag, New York 8. Seaman, E., Van Vunakis, H., & Levine, L. (1972) J. Biol. Chem. 247, 5709-5715 9. Tan, E.M. & Staughton, R.B. (1969) Proc. Natl. Acad. Sci. U.S. 62, 708-714 10. Natali, P.G. & Tan. E.M. (1971) Radiation Res. 46, 506-518 11. Saenco, A.S., Ilyina, T.P., Podgorodnichenko, V.K., & Poverenny, A.M. (1976) Immunochemistry 13, 779-781 12. Wakizaka, A. & Okuhara, E. (1975) Immunochemistry 12, 843-845 13. Okuhara, E. (1970) Analyt. Biochem. 37, 175-178 14. Bernardi, G. (1971) in Methods in Enzymology (Grossman, L. & Moldave, K., eds.) Vol. 21, pp. 95-139, Academic Press, New York 15. Matsumoto, T. & Okuhara, E. (1974) Tohoku J. exp. Med. 113, 245-255 16. Kabat, E.A. (1967) in Experimental Immunology 2nd edn., pp. 760-777, Charles C. Thomas Publisher, Springfield 17. Ouchterlony, O. (1967) in Handbook of Experimental Immunology (Weir, D.M., ed.) 1st edn., pp. 463^J92, Blackwell Scientific Publications, Oxford
A. WAKIZAKA and E. OKUHARA 18. Minden, P. & Fair, R.S. (1967) in Handbook of Experimental Immunology (Weir, D.M., ed.) 1st edn., pp. 655-706, Blackwell Scientific Publications, Oxford 19. Wold, R.T., Young, F.E., Tan, E.M., & Farr, R.S. (1968) Science 161, 806-807 20. Steinberg, A.D., Pincus, T., & Talal, N. (1969) / . Immunol. 102, 788-790 21. Doty, P., Marmur, J., & Sueoka, N. (1959) Proc. Brookhaven Symp. Biol. 12, 1-16 22. Dely, J. (1969) J. Theor. Biol. 22, 89-116 23. LePecq, J-B. (1971) in Methods of Biochemical Analysis (Glick, G., ed.) Vol. 20, pp. 41-86, Interscience Publication, New York 24. Herbert, W.J. & Wilkinson, P.C. (1977) in A Dictionary of Immunology 2nd ed., p. 14, Blackwell Scientific Publications, Oxford 25. Petersen, G.B. & Reeves, J.M. (1966) Biochim. Biophys. Ada 129, 438-440 26. Wakizaka, A. & Okuhara, E. (1976) Seikagaku (in Japanese) 48, 561 (Abstract in Japanese for 48th Annual Meeting of the Japanese Biochemical Society) 27. Wakizaka, A., Kurosaka, K., & Okuhara, E. (1978) Seikagaku 50, 1023 (Abstract in Japanese) 28. Brunk, C.F. (1973) Nature New Biol. 241, 74-76 29. Brunk, C.F. (1975) in Molecular Mechanism for Repair of DNA (Hanawalt, P.C. & Setlow, R.B., eds.) Part A, pp. 61-65, Plenum Press, New York 30. Wakizaka, A. & Okuhara, E. (1979) J. Immunol. Meth. 25, 119-125
/ . Biochem.
Immunochemical Studies on the Correlation between Conformational Changes of DNA Caused by Ultraviolet Irradiation and Manifestation of Antigenicity Akira WAKIZAKA and Eiji OKUHARA Department of Biochemistry, Akita University School " of Medicine, Hondo, Akita, Akita 010 Received for publication, March 26, 1979
The conformational changes of double-stranded DNA induced during irradiation with ultraviolet, were immunologically investigated. These studies revealed that at least two distinct antigenic sites were induced in the irradiated DNA molecule, giving rise to two different antibodies specific for ultraviolet-irradiated (uv) DNA and thermally denatured DNA-like structure, and these were demonstrable using radioimmunoassay and double diffusion tests. A series of experiments, including melting profile, fluorescence intensity of the ethidium bromide complex and chromatographic behavior on hydroxyapatite, performed on the antigenically active uvDNA indicated that the duplex structure of DNA separated irregularly during irradiation. Furthermore, the data showed that the conformational determinants of uvDNA are located on the exposed single-stranded regions.
It is known that ultraviolet-irradiation of DNA produces several kinds of photochemical products and subsequent conformational changes in the molecule {1-3). Electron microscopic (4) and enzymological studies (5) on irradiated DNA revealed that the double strand of DNA separated during irradiation and locally denatured regions appeared in the molecule. Ultraviolet treatment, on the other hand, makes DNA immunologically active. The production of specific antibodies to uvDNA (6-11) and the usefulness of this antiserum Abbreviations and symbols: uvDNA, ultraviolet-irradiated DNA; dDNA, thermally denatured DNA; EB, ethidium bromide; As(uv), antiserum to uvDNA; Ab(uv), antibody to uvDNA; As(d), antiserum to dDNA; Ab(d), antibody to dDNA; ds-DNA, doublestranded DNA; ss-DNA, single-stranded DNA. Vol. 86, No. 5, 1979
to detect ultraviolet-induced lesions on DNA (8, 9, 11) have been reported by several investigators. Pioneering work on the antigenic determinant of ultraviolet-irradiated DNA, which was thermally denatured before irradiation, was reported by Levine and his associates (6, 7). They demonstrated that the thymine dimers formed during irradiation are the determinant for the DNA. However, little information is available about the antigenic determinant for double-stranded DNA irradiated with ultraviolet. In our previous paper (12) on the antigenic determinant of thermally denatured DNA, we stated that some structural units within the denatured DNA molecule act as the determinant. In addition, we reported that the experimental fact that none of the oligonucleotides showed more than 60% inhibitory activity in the concentration range tested, might
1469
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A. WAKIZAKA and E. OKUHARA
be thought to reflect the influence of the three dimensional structure on specificity. In similar studies of ours (unpublished) with uvDNA, we noticed that the expression of its antigenicity is susceptible to conformational changes. Therefore, we attempted to investigate the relationship between the manifestation of uvDNA antigenicity and the conformational changes induced, and found that strand-separation of the duplex structure of DNA as such plays a key role in forming the determinant. MATERIALS AND METHODS DNAs—DNA was prepared from lyophilized salmon sperm (oncorhynchus ketd) by the usual method in this laboratory (13). Double-stranded DNA was separated by hydroxyapatite column chromatography (14). [3H]DNA was prepared from mouse embryo labeled in vivo with [6-3H]thymidine by the method described previously (12). Preparation ofuvDNA—3 ml of DNA solution (1 mg/ml of 0 . 1 5 M NaCl) in a petri dish (2.5 cm diameter) was irradiated at 4°C with a commercial germicidal lamp (Toshiba GL-15,15 watt, 254.7 nm emission) at a distance of 5 cm above the surface of the solution with slow and constant stirring. The dose rate of irradiation was 10.3 J/m2/min as determined by dosimetry with a Topcon UVR-254 U.V. Radiometer (Tokyo Kogaku) in the range of 200 to 320 nm. Preparation of Antisera—Rabbit antisera to thermally denatured salmon sperm DNA (dDNA) were prepared by the usual method in our laboratory (75). Rabbit antisera to ultraviolet-irradiated salmon sperm DNA (uvDNA) were prepared in a similar manner; 0.5 mg of DNA dissolved in 1 ml of saline was irradiated at a dose of approximately 2.5xlO 3 J/m 2 with ultraviolet and stirring. The uvDNA solution and an equal weight of methylated bovine serum albumin were mixed. The complex emulsified with an equal volume of Freund's complete adjuvant was injected intramuscularly into rabbits (0.5 mg of uvDNA/animal) at least 6 times at weekly intervals, and blood was drawn one week after the sixth or eighth injection. The remaining procedures have been described in our previous paper (IS). We used the antiserum obtained from rabbit No. 120 in this study. Gamma globulin was obtained from the antiserum
by ammonium sulfate fractionation (16). Antiserum to uvDNA was absorbed with dDNA to remove antibodies to dDNA in the following way; 25 mg of gamma globulin from #R112 antiserum in 0.5 ml of 0.05 M phosphate buffer (pH 7.5) containing 0.15 M NaCl was mixed with 5.0 mg of dDNA in 5.0 ml of the buffer and allowed to stand for 48 to 72 h at 4°C with occasional mixing. The precipitate was spun down at 5,000 rev./min for 20 min and the resulting supernatant was applied to a DEAE-cellulose column (2x2 cm) to remove free dDNA. The antibodies absorbed on the column were eluted with 0.05 M phosphate buffer (pH 6.5). 60 to 70% of the globulin was recovered in the first 30 ml of the eluate. Ouchterlony's double diffusion tests (17) were performed at 4°C for 48 to 72 h in a humid chamber on a glass plate (5x8 cm) laying 0.3% agarose in 0.05 M phosphate buffer (pH 7.5)-0.15 M NaCl containing 0.02% of sodium azide. Radioimmunoassay—[3H]uvDNA bound by anti-uvDNA antibodies was measured by a modification of Farr's assay (12, 18-20). The assay medium contained 1.0 fig of [3H]uvDNA (5,000 dis./min, irradiated at a dose of approx. 2.5 x 10s J/m2) in 0.05 ml of 0.1 M borate buffer (pH 8.5), 0.05 ml of 50-fold diluted anti-uvDNA antisera with the buffer and various amounts of nonradioactive DNA in 0.1 ml of the buffer. When gamma globulin from the antisera absorbed with dDNA was used in the assay, 50 ^g of the globulin in 0.05 ml of the buffer was added in place of the antiserum. The incubation of the mixture was carried out at 4°C for 15 min. After mixing with an equal volume of saturated ammonium sulfate solution at 4°C for 1 h, the precipitate and supernatant fractions were separated by centrifugation (3,000 rev./min for 15 min). 0.05 ml of both fractions was dissolved in 0.5 ml of NCS solubilizer and 10 ml of toluene scintillation cocktail containing 4 g of Omnifluor, and assayed for radioactivity in a Searle Analytic Mark II liquid scintillation counter. The percentage of antigen bound by the antiserum and binding inhibition were calculated as described previously (12). The [3H]dDNAantibody binding was also assayed in the same manner. Melting Profile ofuvDNA—Thermal denaturation curves of uvDNA (75 /*g/ml of 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) were made J. Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA by determination of absorbance at 260 nm with a Hitachi Model 139 spectrophotometer equipped with a Komatsu Solidate SPS-H139 thermoregulator. During the experiment, the temperature of the solution was increased at a constant rate of 1°C per min from room temperature of about 25CC by means of a Komatsu Model KPC-2 automatic temperature programmer. Temperature and absorbance were recorded automatically. The width (47") of the thermal denaturation curve is represented by the temperature difference between 15.9% and 84.1 % of the absorbance rise (21, 22).
1471
(Hypatite C), from Clarkson (Williamsport, Pa., U.S.A.); agarose (A-45), from Daiichi Kagaku (Tokyo, Japan); ethidium bromide, from Sigma (St. Louis, Mo., U.S.A.). All other chemicals used were of the highest purity commercially available. RESULTS
Antigenic Reactivity of uvDNA—Figure 1 shows the inhibitory activities of DNAs irradiated at different doses on the binding of [3H]uvDNA Fluorometric Determination—The increasing by anti-uvDNA rabbit serum. The inhibitory fluorescence intensity of ethidium bromide (EB) activities of irradiated DNAs were elevated with bound to double-stranded DNA was measured increasing dose of irradiation. Interestingly, DNA 2 according to LePecq (23). A constant volume irradiated at a dose of 10 J/m (about 1 min of (usually 4.0 ml) of DNA solution (2 to 10^g/ml irradiation) also reacted with the antiserum. When of 0.2 M NaCl containing 2 mM EDTA) was mixed the amount of DNA added was 5 //g, the perwith an equal volume of ethidium bromide solution centage of inhibition by the uvDNA irradiated at 2 (20 pg/ml of 0.1 M Tris-HCl buffer, pH 7.5). The doses of 10, 50, 310, 620, and 1,240 J/m was 72, fluorescence intensity of the resulting DNA-EB 80, 83, 90, and 98, respectively. Native DNA complex was measured with a Hitachi MFP-2A fluorescence spectrophotometer. Wavelength of excitation light was 310 nm and emitted fluores100 cence was measured at 580 nm. The intensity of fluorescence of EB solution not containing DNA 80 was subtracted from experimental values. The extent of single-strand formation in uvDNA was calculated from the difference between fluorescence 60 intensity of the uvDNA-EB complex and that of the native DNA-EB complex. Hydroxyapatite Chromatography—Hydroxyapatite column chromatography of uvDNA was performed according to the method of Bernardi (14). A hydroxyapatite column (1.5x20cm), to which was applied 6 ml of saline solution of uvDNA (3 mg), was eluted with 200 ml of phosphate buffer, pH 6.8 (a linear concentration gradient of sodium phosphate, 0.05-0.4 M) at a rate of 20 ml/h and 2 ml fractions were collected. Aliquots of 0.1 ml were used for the radioimmunoassay. Chemicals—[6-3H]Thymidine in aqueous solution (20-30 Ci/mmol) was purchased from The Radiochemical Centre (Amersham, Great Britain); NCSsolubilizer, from Amersham/Searle (Arlington Heights, III., U.S.A.); Omunifluor, from New England Nuclear (Boston Mass., U.S.A.). DEAE cellulose (DE-32) was obtained from Whatman (Springfield Mill, 'Great Britain); hydroxyapatite Vol. 86, No. 5, 1979
0
0.6
1.25
2.5
o
5.0
DNA added, jjg 3
Fig. 1. Inhibition of [ H]uvDNA-antibody binding by DNA irradiated with ultraviolet at different doses. The binding assay medium contains 1.0 fi% of [3H]uvDNA (5,000 dis./min, irradiated at a dose of 2.5XlO3 J/m2), 50 fi\ of a 50-fold diluted anti-uvDNA antiserum, and different amounts of DNA in 0.2 ml of 0.1 M borate buffer (pH 8.5). ( • ) , DNA irradiated with ultraviolet at a dose of 1.2 kJ/m2; ( v ) , at a dose of 6.2x 102 J/m2; ( • ) , at a dose of 3.1 x 102 J/m2; ( A ) , at a dose of 50 J/m2; ( A ) , at a dose of 10J/m a ; (O), unirradiated native DNA; (O), unirradiated dDNA. Results represent the mean of three experiments.
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A. WAKIZAKA and E. OKUHARA
showed a weak inhibitory activity on the binding when more than 1.25 //g of DNA was added, while dDNA did not show any inhibitory activity. In addition, uvDNA, especially irradiated at a relatively high dose, also inhibited the binding of [3H]dDNA by anti-dDNA rabbit sera, as seen in Fig. 2. The minimum dose of irradiation required for the inhibition of the irradiated DNA on dDNAantibody binding was 6.2 x 102 J/m2. Native DNA did not exhibit any inhibition on this binding. Figure 3 shows the precipitating patterns of anti-uvDNA serum and anti-dDNA serum with
80
w 60
40
20
0.6
1.25 DNA added.
Fig. 2.
2.5
5.0
Fig. 2. Inhibition of [3H]dDNA-antibody binding by DNA irradiated with ultraviolet at different doses. The assay medium contained 1.0/ig of [8H]dDNA (5,000 dis./min), 50 ft] of a 50-fold diluted anti-dDNA antiserum and DNAs (inhibitors). ( • ) , DNA irradiated with ultraviolet at a dose of 9.3 x 102 J/m2. Other symbols are the same as in Fig. 1. Results represent the mean of three experiments.
Fig. 3. Double diffusion tests in agarose gel. Central wells contained 40 fil of the anti-uvDNA antiserum (a) and anti-dDNA antiserum (b).2 The peripheral wells contained 40 //I of each antigen: N, native DNA; 1, uvDNA irradiated at a dose of 50 J/m ; 2, at 2a dose of 1.6 x 10! J/m2; 33, at2 a dose of 3.1 x 102 J/m2; 4, at a dose of 6.2 X 2 2 2 10 J/m ; 5, at a dose of 9.3 x 10 J/m ; 6, at a dose of 1.2x 10 J/m . / . Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA
1433
Ab(uv)
uvDNA
dDNA
Absorbed Ab(uv)
uvDNA
dDNA
uvDNA
Ab(uv
Ab(d)
Ab(d)
dDNA
uvDNA
Fig. 4. Double diffusion tests of anti-uvDNA serum [Ab(uv)] and anti-dDNA serum [Ab(d)] with uvDNA and dDNA. (a) The reactivities of uvDNA and dDNA with Ab(uv). (b) The reactivities of uvDNA and dDNA with anti-uvDNA gamma globulin absorbed with dDNA [Absorbed Ab(uv)]. (c) The reactivities of uvDNA with antisera, Ab(uv) and Ab(d). (d) The reactivities of dDNA and uvDNA with Ab(d). DNA irradiated at a dose of 2.5 x 103 J/m2 was used as an antigen in double diffusion tests. Vol. 86, No. 5, 1979
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A. WAKIZAKA and E. OKUHARA
uvDNA irradiated at the different doses. As shown in Fig. 3a, two precipitating lines were formed between anti-uvDNA serum and uvDNAs irradiated at the doses of 3.1 xlO2, 6.2 xlO2, and 1.2xlO3 J/m2. On the other hand, as seen in Fig. 3b, only one precipitating line was observed between anti-dDNA serum and the uvDNA irradiated at a dose of 1.2 x 103 J/m2. lmmunological Characteristics of uvDNA—The
antigenicity of DNA induced by the irradiation was further analyzed by the double diffusion test. Figure 4a shows the reactivities of uvDNA and dDNA to the anti-uvDNA serum. Between uvDNA and Ab(uv), both heavy and weak precipitating lines were formed, suggesting that uvDNA might hold two antigenic sites. The weak line between uvDNA and Ab(uv) was not clearly observed to fuse into one line between Ab(uv) and dDNA, but both weak lines were eliminated by using anti-uvDNA antibodies absorbed with dDNA as seen in Fig. 4b. In order to examine which precipitating line of the two between uvDNA and its antiserum, shown in Figs. 3a and 4a, really connects with the single line between uvDNA and antiserum to dDNA, shown in Fig. 3b, an Ouchterlony's test was performed as depicted in Fig. 4c. The clear explanation of the results is difficult, but it seems likely to be the case that the line formed between uvDNA and Ab(d) crossed over the main precipitating line between uvDNA and Ab(uv) (and thus a double spur was formed) and fused with the second line between them. TABLE I. Melting profiles of uvDNA. Dose 3of UV xlO- J/m= 0 0.3 0.6 1.2 2.4
T_ m
,O/^ (C)
84. 7 ±0.87 82.0+1.80 78. 5± 1.44 71.8 + 0.76 67. 5 ±3. 27
AT,O AT (
7. 3±0.76 11.0 + 0. 50 14.0 + 0.00 18.0±0. 50 26. 3 ±0.30
Total (%) hyperchromism11 39.0 + 0.7 33.4±1.0 32. 3±0. 5 30.0±1.2 26. 5±0.6
Results represent the mean ± S.D. of three different experiments. a AT, the width of the thermal denaturation curves represented by the temperature difference between 15.9 and 84.1% of the absorbance rise (/4260). b Total hyperchromism, percent increase in absorbance at 260 nm resulting from melting divided by the initial absorbance at room temperature.
Hence, uvDNA shares some common determinants with dDNA detectable with the antiserum, Ab(d). Correlation between Antigenicity and Structural Changes—Table I shows changes of Tm and width (J T) of the Tm curve together with changes in total hyperchromism of DNA during irradiation with ultraviolet. As seen in the table, the decrease of Tm and total hyperchromism, and the increase of /IT paralleled the increase of the irradiationdose. These physical properties of uvDNA seem to reflect weakening of the base pairing and partial separation of the duplex of DNA. The relationship between the structural changes and the antigenic manifestation of the DNA during irradiation is summarized in Fig. 5. When the irradiation-dose increased, Tm and fluorescence intensity of the DNA-EB complex decreased along with the increase of the inhibitory activities on the DNA-antibody binding. Inhibitory activity on [3H]uvDNA-Ab(uv) binding was induced with a rather small dose of irradiation, while that on [3H]dDNA-Ab(d) binding appeared "MOO
IOO r
1 90
eo
•. 60
80
20
70
0
0.3
0.6
0.9
1.2
Dose of ultraviolet Irradiation x 103 J/m2
Fig. 5. The relationship of the conformational changes of uvDNA with the inhibitory activities on antigenantibody binding. DNAs irradiated at different doses (as indicated in the abscissa) with ultraviolet were examined with respect to their Tm (V), extent of strandseparation (%) as determined by the fluorometry (O), and inhibitory activities on [3H]uvDNA-Ab(uv) binding ( A ) and on [3H]dDNA-Ab(d) binding ( • ) . 5 fig of uvDNA was used to determine the inhibition on dDNAAb(d) binding, and 2.5 p%, on uvDNA-Ab(uv) binding. Results represent the mean of three or more experiments. J. Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA on the molecule after irradiation with a relatively large dose. Chromatographic Behaviour and Antigenic Reactivity—Figure 6 shows the change of hydroxyapatite chromatographic pattern of DNA during irradiation with ultraviolet. As shown in the figure, a heavy load of native DNA in a preparative scale to the column caused broadening of the elution peak, but the unirradiated nDNA and dDNA were well separated, though some portion of dDNA was eluted at the position of ds-DNA. Irradiation shifted the elution position of DNA from ds-DNA toward ss-DNA according to the intensity of irradiation and the shape of elution peak became narrow like that of dDNA. Figures 7 and 8 show the relationship between chromatographic behaviour of uvDNA and antigenic reactivity. The inhibitory activity of the eluant on [ 3 H]uvDNA-Ab(uv) binding almost coincided with the peak of absorbance at 260 nm as shown in the figure, and a large part of the inhibitory activity still remained in ds-DNA position. On the other hand, the most inhibitable fraction of dDNA-Ab(d) binding was eluted
1475 ss
100 (50) ; < a ,
ds 1
• "0-3 0.5
(§» • j o . 2 • ^o- 1
I 0
s:
I (50)
(b)
-100
• 50 (25) •
0.3
•
0.1
30
0.2
i^o—Q—a 1.0
w Is 40
\
.
i 50 Fraction No.
70
60
Fig. 7. Antigenic reactivity of uvDNA eluted from hydroxyapatite column. 3 mg of uvDNA irradiated at a dose of 6.2xlO 2 J/m 2 (a), and 1.2x10'J/m 2 (b) was applied to a hydroxyapatite column. The chromatographic conditions was the same as in Fig. 6. Inhibitory activity of 0.1 ml of each fraction was assayed in uvDNA-Ab(uv) system ( • ) , and in dDNA-Ab(d) system (O). The inhibition rate to the latter system is indicated in parentheses in the figure. ( ), concentration of phosphate in eluate; ( ), absorbance at 260 nm; (•!», elution position of ds-DNA; ( [ ), that of ss-DNA. Results represent the mean of two assays.
/
f\
/ /
i il 0.5 i /
I
0.3
N
Yi y V i
j
••
•,
. ' /
ii
/
/
/
0.1 •
40
/X\
j/ \
1i 50
'.
0.3 \ ,
I
\ \ \
f
\
-0 . 2
°
0.1 60
70
Fraction Number
Fig. 6. Chromatographic profiles of uvDNA, nDNA, and dDNA on a hydroxyapatite column. 2 to 4 mg of DNAs were loaded on a 1.5 x 20 cm column. The column was eluted with 200 ml of sodium phosphate buffer (pH 6.8) in a linear concentration gradient from 0.05 to 0.4 M. Flow rate, 20 ml/h. 2.1ml fractions were collected and measured for their absorbance at 260 nm. ( ), indicates the absorbance at 260 nm of nDNA (3 mg); ( ), DNA (3 mg) irradiated with 0.6xl0 3 J/m 2 of ultraviolet; ( ), DNA (3.5 mg) irradiated with 1.2 x 103 J/m2; ( ), DNA (3 mg) irradiated with 2.5x 103 J/m2; ( ), unirradiated dDNA (2 mg); (
), molarity of phosphate in eluate.
Vol. 86, No. 5, 1979
g o
50
60
Fraction No.
Fig. 8. Chromatography of thermally and radiationally denatured DNA on a hydroxyapatite column. uvDNA thermally denatured in 0.15 M sodium chloride after irradiation with 2.5x 103 J/m2 ultraviolet (a), and uvDNA thermally denatured in the saline before irradiation at the same dose (b) was chromatographed in the same manner as in Fig. 6. ( • ) , inhibitory activity of 0.1 ml aliquots of each fraction on uvDNA-Ab(uv) binding. ( ), molarity of sodium phosphate; ( ), absorbance at 260 nm; (4J-), elution position of ds-DNA; ( | ) , that for ss-DNA. Results represent the mean of two assays.
1476
A. WAKIZAKA and E. OKUHARA
slightly earlier than the Ataa peak, typically shown in Fig. 7b. The shift of peak position from ds-DNA to ss-DNA was clearly observed when thermally denatured uvDNA or ultraviolet-irradiated dDNA was chromatographed on the same column, as shown in Fig. 8. DNA thermally denatured after irradiation was eluted at ss-DNA position on the column; serological activity to uvDNA-Ab(uv) system remained and coincided with the Atm peak, as shown in Fig. 8a. Similarly, irradiated DNA thermally denatured before irradiation was also eluted at ss-DNA position with serological activity (Fig. 8b). The shape of Auo peaks of these two cases was different. In the case of Fig. 8a, the peak was rather broad and serological activity was widely distributed around the peak area. However, in the Fig. 8b case, the DNA eluted in a relatively sharper and more compact peak with serological activity eluted slightly earlier than the •^260 peaK.
DISCUSSION Our present studies deal with the relationship between strand separation and the antigenic manifestation of DNA irradiated with ultraviolet. The irradiated DNA reacts with both anti-uvDNA and anti-dDNA antisera. This implies that at least two different antigenic sites are formed in the uvDNA molecule. The experimental data obtained by Ouchterlony's tests revealed that the anti-uvDNA antibodies recognize the unique structure of uvDNA and an additional structure which might be partially identical with that of dDNA. The antigenic structure specific for uvDNA may contain some photoproducts, such as thymine dimer. Levine et al. (6) and Seaman et al. (7) reported that the thymine dimer was most probably the determinant of ultraviolet-irradiated DNA, (thermally denatured before irradiation) on the following evidence: 1) serological activity of uvDNA reflects thymine content of the DNA; 2) antibodies to uvDNA are capable of being inhibited with ultraviolet irradiated thymidine oligonucleotides; 3) the serological activity of the irradiated DNA is decreased by re-irradiation at 235 nm; 4) irradiation of DNA in acetophenone solution with visible light gives a positive serological
response by forming thymine dimer. Their results fully explained the participation of thymine dimer in the antigenic determinant of ultraviolet-irradiated dDNA. However, the antigenic structure induced on the double-stranded DNA by irradiation was not sufficiently investigated. In particular, the relationship between conformational changes caused on the DNA strand by irradiation and formation of antigenic reactivity has not been elucidated. The antigenic determinant literally means "the site of antigen to which an antibody is specifically able to become attached by its combining site" (24). So it may occupy a certain dimensional part larger than single base or nucleotide moiety in DNA, involving the composition of a certain length of nucleotide chain in a certain conformation on the strand. We further investigated the inhibitory activity of pyrimidine oligonucleotides prepared from uvDNA by the known method (25), and found that no considerable extent of inhibition was detectable with those oligonucleotides (data reported preliminarily (26, 21), now under preparation for publication). Nevertheless these oligomers contained an appropriate suitable amount of thymine dimer which remains intact during chemical treatment, as Brunk (28, 29) has demonstrated. From these results we arrived at the idea of the predominancy of certain conformational changes of the strand structure in antigenicity rather than a simple modification of bases. The broadening of the thermal denaturation curve, the decreasing Tm, total hyperchromism and fluorescence intensity of the DNA-EB complex, and chromatographic behaviour on the hydroxyapatite column all give evidence for the formation of single-stranded region on DNA during irradiation. The chromatographic elution profile and the inhibitory activity of the eluate to both antigenantibody systems provides information about the structural changes induced on the strand. The most inhibitorily active fraction of eluate was different for the two antibody systems, Ab(uv) and Ab(d). uvDNA, which was the more inhibitorily active to the former antibody system, eluted slightly later than that for the latter system. This implies the immunochemically different molecular species of DNA or DNAs with denaturated portions of different extents are induced during irradiation and these can be separated by the
/ . Biochem.
CONFORMATIONAL DETERMINANT OF uvDNA
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chromatography. The peak position of A2eo of eluate was shifted towards the elution position of single-stranded DNA during irradiation. But a large part of inhibitory activity to uvDNA-Ab(uv) system still remained close to the ds-DNA position. These findings suggest that a denatured region was formed only locally and to various extents on the DNA with a large native portion remained. The thermal denaturation of uvDNA in saline did not destroy most of uvDNA-specific antigenic reactivity and the denaturated uvDNA was mainly eluted at ss-DNA position on hydroxyapatite column (Fig. 8a). DNA irradiated after thermal denaturation was also eluted at ss-DNA position with serological activity to uvDNA-Ab(uv) system (Fig. 8b). These results suggested that the determinant group for uvDNA-specific antibody is located on the single-stranded conformation of DNA. Therefore, the antigenic structure of ultraviolet-irradiated double-stranded DNA seems to be closely connected with a single-stranded structure induced by irradiation. However, the possibility of participation of interstrand cross linking in the antigenic manifestation of irradiated DNA can not be excluded, since the heated uvDNA was eluted from hydroxyapatite column in a broader peak than irradiated dDNA. This may account for the involvement of interstrand cross links in the antigenic structure of uvDNA.
another way; it is caused by the difference in the physical form and size of the test antigens rather than the actual determinant. dDNA behaves like a small compact structure compared to the uvDNA. As shown in Fig. 1, binding between [ 3 H]uvDNA and Ab(uv) was not inhibited by the addition of cold dDNA. On the contrary, dDNA reacted with antiserum to uvDNA as shown in Fig. 4a. This may be explained as follows: 1) it may be caused by the difference between the immunoassays employed; 2) Fig. 4a and b indicate that two different kinds of antibodies are present in the serum to uvDNA, one specific for uvDNA (Ab(uv)) and the other with some common reactivity to dDNA(Ab(d)). Since Ab(uv) seems very likely to be major antibody populations in the serum, binding between [ 3 H]uvDNA and Ab(uv) may be not inhibited by the addition of cold dDNA. Antibody populations reactive with dDNA are Ab(d) but not Ab(uv) as such.
Radioimmunoassay revealed that the antigenic reactivity for a uvDNA-specific antibody appeared in the molecule with rather smaller doses of irradiation than that for anti-dDNA antibody (Fig. 5). If photoproducts induced on the molecule with ultraviolet cause weakening of base pairing and strand separation, the lag phase for appearance of the antigenic reactivity to Ab(d) on DNA with low irradiation doses (Figs. 2 and 5) may be explained as follows; the radiation-induced denaturated regions may be different immunochemically in structure from the single-stranded region induced by thermal treatment, due to the presence of photoproducts on the site or the smallness in size of the denaturated portion as the antigenic determinant for dDNA antibody. This difference in structure may influence the different reactivity of uvDNA to Ab(d) in radioimmunoassay mentioned above and the Ouchterlony reaction in Figs. 4a and d. The difference in curvatures of precipitating lines in the same figures may be explained in
Vol. 86, No. 5, 1979
Native DNA showed a slight inhibitory activity on uvDNA-Ab(uv) binding as shown in Fig. 1, when a large amount was added to the assay mixture. But it does not seem likely that this inhibition is an immunologically specific one, since the Ouchterlony test shown in Fig. 3a proved no reaction between nDNA and Ab(uv). It must be due to a non-specific DNA-gamma globulin interaction, as we reported previously (50). We conclude from our results that ultravioletirradiation on native DNA induces at least two different antigenic sites which are responsible for the production of two distinct antibodies. And this study seems to indicate that conformational changes of DNA caused by irradiation, especially partial strand-separation in double helical structure of the molecule, greatly contribute to the manifestation of antigenicity. REFERENCES 1. Wacker, A. (1963) in Progress in Nucleic Acid Research (Davidson, J.N. & Cohn, W.E., eds.) Vol. 1, pp. 369-399, Academic Press, New York 2. Setlow, R.B. (1968) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, J.N. & Cohn, W.E., eds.) Vol. 8, pp. 257-295, Academic Press, New York 3. Lomant, A.J. & Fresco, J.R. (1972) in Progress in Nucleic Acid Research and Molecular Biology (Davidson, J.N. & Cohn, W.E., eds.) Vol. 12, pp. 1-27, Academic Press, New York
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