- Email: [email protected]
Degradation of DNA by cysteine
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Degradation HERBERT Department
146, 483-487
of DNA
S. ROSENKRANZ2
of Microbiology,
AND
(1971)
by Cysteine’ SAMUEL
ROSENKRANZ
College of Physicians and Surgeons, New York, New York 1005%
Received
June 10, 1971; accepted
July
Columbia
University,
6, 1971
Exposure of DNA solutions to cysteine resulted in degradation of the polydeoxynucleotide. This reaction was inhibited by Naz EDTA and by sodium citrate. Methylcyste-ine, mercaptoethanol, serine, and homocysteine did not exhibit this degradative effect. Preincubation of cysteine resulted in a more rapid rate of degradation thus suggesting that a product formed during incubation was responsible for the observed effects.
Recent studies in our laboratory have revealed that L-cysteine, a naturally occurring amino acid, possessed antibacterial properties (1). This activity was made evident when bacteria were exposed to levels of L-cysteine of the order of 0.005 M. In view of the possible significance of this finding on our understanding of regulatory mechanisms, the basis of this biologic activity was investigated. The present report deals with the effects of L-oysteine and related substances on the properties of purified DNA. MATERIALS
AND
tration of 5%. After standing in the cold for 30 min, the insoluble residues were collected on the nitrocellulose discs and thoroughly washed with 5% trichloroacetic acid. The filter discs were dried, and the retained radioactivity was determined in a liquid-scintillation counter in 10 ml of omnifluor (4 g/liter of toluene). Physical chemical analysis. Sedimentation coefficients were determined in a Spinco Model E ultracentrifuge equipped with an ultraviolet optical system. The percentage of material sedimenting was determined as described previously (3). RESULTS
METHODS
Exposure of various DNA preparations to L-cysteine result,ed in degradation which was detectable by decreases in sedimentation coefficients (Table I) and by the formation of acid-soluble products (Table II). This depolymerization was dependent upon temperature and duration of exposure (Tables I-III). It was not due to contaminating nucleases present in the various DNA preparations as incubation of DNA in the absence of cysteine and its congeners did not result in degradation (Tables I and II). The possibility that cysteine activates cryptic deoxyribonucleases is also unlikely as the cysteine-induced degradation occurred even when heat-denatured DNA was examined (Table IV). Such a treatment is expected t,o abolish enzyme activit,y . The reaction between DNA and cysteine
Nucleic acids. Calf thymus DNA was purchased either from Nutritional Biochemicals Corp. or from CalBiochem. These preparations had sedimentation coeflicients of 12 and 21 S, respectively. Escherichia coli DNA was prepared by the procedure of Marmur (2: from E. coli C600. The bacteriophage DNA was isolated from Klebsiells phage E-l currently under investigation in this laboratory. 3H-Labeled DNA derived from chicken embryos was prepared as described previously (3). Stability oj DNA. Solutions of radioactive DNA were incubated at 37 or 56”. At intervals portions from each solution were removed and trichloroacetic acid was added to a final conceni This investigation was supported by a grant from the Damon Runyon Memorial Fund for Cancer Research. 2 Research Career Development Awardee of the U.S. Public Health Service (2-K3-GM29,024). 483
484
ROSENKRANZ
AND TABLE
EFFECT Erpt.
I
OF CYSTEINE
AND
DNA specimen
OTHER
COMPOUNDS Temperature (“C)
Calf
t,hymus
56
Calf
thymus
37
III
Bacteriophage
56
IV
E. coli
37
II
ROSENKRANZ I ON SEDIMENTATION Time (hr)
0 46 46 0 65 14.3 41 65 14.3 41 65 65 0 98 98 98 0 98 98 98
BEHAVIOR
OF DNA”
Additions
Sedimentation coefficient (S)
None None L-cysteine None None L-cysteine L-cysteine L-cysteine L-cysteine + EDTA L-cysteine + EDTA L-cysteine + EDTA EDTA None None L-cysteine S-Methyl-L-cysteine None None L-cysteine L-Serine
12.0 12.4 7.9 12.2 12.4 10.4 10.4 0 12.3 13.0 11.3 12.3 38.2 37.2 0 39.2 30.2 29.9 19.1 31.4
were incubated in the dark at the temperatures (1Sodium deoxynucleates (1 mg/ml 0.015 M N&l) indicated. At intervals portions of the reaction mixtures were removed diluted with 0.15 M NaCl and analyzed in the analytical ultracentrifuge. (Occasionally prior to ultracentrifugal analyses the DNA specimens were first precipitated with ethanol. This additional procedure did not affect the results). The final concentration of L-cysteine (L-cysteine hydrochloride.HsO; Mann Research Laboratories), S-methvl-L-cvsteine (Sigma Chemical Co.) and L-serine (Mann Research Laboratories) was 0.005 M; ” ” that of Na*EDTA was 2 mg/ml.
was influenced by the pH of the solvent. The smallest effect was near neutrality (Table III). This degradation was observed when three different preparations of L-cysteine, as well as the D- and m-isomers (Table II) were used. The material responsible for this degradation was dialyzable and resisted autoclaving-thus eliminating the possibility that it was due to bacterial contaminants or to a nuclease present in various batches of L-cysteine. Solubilization of the DNA was prevented by NazEDTA and by sodium citrate. No significant degradation was observed when DNA was exposed to methylcysteine (Tables serine, and I and II), mercaptoethanol, homocysteine (Table II) ; cysteamine caused some depolymerization at elevated temperatures while 2-aminoethylisothiouronium bromide was moderately active (Table II). Preincubation of cysteine at 56” increased its reactivity toward DNA as evidenced by
the more rapid rate of depolymerization exhibited by such a preincubated solution (Table V). DISCUSSION
The present findings of an effect of cysteine on isolated DNA may provide a possible chemical basis for the observed antibacterial action of this amino acid. It should be mentioned, however, that the in vivo effects are exhibited mainly by the L-isomer (1) while the present study indicates that the L- as well as the n-form degrades DNA. This discrepancy may be a reflection of the fact that the natural (L-) isomer has easier access into the cell. Current studies in this Laboratory are concerned with an elucidation of the chemical nature of the reaction between DNA and cysteine. However, in view of the results obtained with preincubated cysteine (Table V), it may be predicted that the degradation of DNA is
DEGRADATION
OF DNA TABLE
EFFECT Tern erature k)
Expt.
I
37
II
56
III
37
IV
37
V
37
VI
56
VII
37 56
OF CYSTEINE
AND
OTHER
II
COMPOUNDS
OF DNAa
ON DEGRADATION Radioactivity
Additions
n-Cysteine, Prep. No. 1 n-Cysteine, prep. No. 2 n-Cysteine, prep. No. 3 n-Cysteine n-Cysteine, autoclaved S-Methyl-n-cysteine Homocysteine n-Cysteine + EDTA EDTA n-Cysteine S-Methyl-t-cysteine L-Serine n-Cysteine-autoclaved Homocysteine Mercaptoethanol n-Cysteine L-Cysteine + citrate Citrate L-Cysteine, dialyzed AET* Cysteamine L-Cysteine n-Cysteine + citrate Citrate L-Cysteine, dialyzed AET” Cysteamine o-Cysteine nn-Cysteine o-Cysteine on-Cysteine
485
BY CYSTEINE
20
43
73 66 60 16 12 97 93 90 109
73 57 58 13 11 100 98 89 100 49 99 100 65 100 100 64 96 100 100 82 99 9 92 99 90 41 89
11 89 97 87 62 90
retained (% of Control, hr) 68
76
93
56 46 22
46 34 19
49 100 100 29 100 100
40 93 62 19 92 82 37 97 100 100 70 100
65 69
44 44
29 19
into a Two-milliliter portions of 3H-DNA (37 pg/ml of 0.015 M NaCl, 970 cpm/Gg) were distributed tubes containing premeasured amounts of cysteine or its congeners (final concentration 0.005 M), the final concentration of NazEDTA was 2 mg/ml and that of sodium citrate was 0.005 M. The reaction mixtures were incubated in the dark and at intervals replicate 0.1.ml portions were withdrawn and radioactivity remaining acid-precipitable was determined. The source of the chemicals used has been indicated elsewhere (1). Unless otherwise indicated, cysteine Prep. No. 1 (1) was used. b AET, 2-aminoethylisothiouronium bromide, HCl.
mediated by a product which is formed when cysteine is incubated at elevated temperatures. It should be pointed out that this action of cysteine, a, naturally occurring substance, on DNA is quite unique. Thus, many agents which degrade DNA have been recognized. Usually, however, these substances are mutagens. For example, X-rays, peroxides, and alkylating agents degrade DNA presumably through a mechanism involving depurina-
tion followed by chain-scission (4-12); exposure of DNA to uv light leads to a diminution in molecular weight, the mechanism of which is not fully understood (13, 14) and hydroxylamine and its derivatives also depolymerize DNA (15), probably through the formation of peroxides and free radicals (7, 16). This generation of peroxides and free radicals appears to be catalyzed by trace metals (7). In view of the present results, which indicate that the degradative effect
486
ROSENKRANZ
AND
ROSENKRANZ
TABLE EFFECT
OF
pH
ON
THE
III
PROPERTIES
DNA
OF
EXPOSED
TO
Sedimentation Time
(hr)
Buffer
CYSTEINEY behavior
DKA
and pH Sedimentation coefhcient (S)
DNA
+ cyst&e
Sedimentation coefficient (S)
VO Sedimenting
% Sedimenting -
24 48 72 0 24 48 72 24 48 72 24 48 72
Acetate, pH 4.6 Acetate, pH 4.6 Acetate, pH, 4.6 Phosphate, pH 7.0 Phosphate, pH 7.0 Phosphate, pH 7.0 Phosphate, pH 7.0 Tris, pH 8.0 Tris, pH 8.0 Tris, pH 8.0 Phosphate, pH 12 Phosphate, pH 12 Phosphate, pH 12
11.4 7.8 6.4 21.3 21.4 20.3 20.3 22.7 20.1 20.3
100 100 100 100 106 100 100 100 100 100
22.2
100
a Calf thymus DNA solutions (1 mg/ml of 0.01 M buffer in 0.015 L-cysteine at 56’. At intervals samnles were removed, diluted in 0.15 cients were determined. TABLE DEGRADATION
OF
IV BY
EFFECT
Radioactivity cpm/ml
18.4 16.0 14.1 16.4 15.5 12.5 16.5 10.8 10.1
100 loo 100 100 100 100 106 100 100
NaCl) were exposed to 0.005 M NaCl, and sedimentation coeffi-
2740 382
106 13.9
2756 342
100 12.4
of cysteine is inhibited by chelating agents, it is conceivable that the generation of free radicals or peroxides is also the basis of the action of cysteine. REFERENCES H. S., CARR, H. S., AND ZYROFF, J., J. Bacterial. 102, 672 (1970). 2. M.4RMUR, J., J. Mol. Biol. 3, 208 (1961). 3. JACOBS, S. J., A.ND ROSENKRANZ, H. S., Cancer Res. 30, 1084 (1970).
V
PREINCUB~TED
retain&
CYSTEINE
24 48
Control
21.4 20.3
ON
THE
OF (2.4~~ THYMUS DN& Sedimentation
Time (hr)
% Total
a One.milliliter portions of 3H-DNA were incubated with 0.005 M L-cysteine at 56” for 66 hr at which time radioactivity remaining acidinsoluble was determined. Thermally denatured DNA was prepared by heating DNA at 100” for 10 min and then immersing the specimens into an ice bath.
ROSENKRANZ,
OF
PROPERTIES
Additions
Native DNA Native DNA + 0.005 M Lcysteine Heat-denatured DNA Heat-denatured DNA+ 0.005 M L-cysteine
56 36 0
TABLE DNA
DEBT-DENBTURED L-CYSTEINE’
1.
M M
4.0 2.6 0
DNA + “fresh” cysteine
18.4 16.0
coefficients DNA + preincubated cyst&e
13.9 12.1
a L-Cysteine (0.05 M) in 0.01 M phosphate buffer, pH 7, was incubated for 24 hr at 56” whereupon it was diluted 1:lO upon addition to DNA. In parallel another specimen of DNA was supplemented with a fresh solution of L-cysteine. 4. 5.
6.
7. 8. 9.
J. A. V., Radiat. Res. Suppl. 1, 403 (1959). WI:ISS, J. J., in Progress in Nlicleic Acid Research and Molecular Biology Vol. 3 (J. N. Davidson and W. E. Cohn, eds.), Academic Press Inc., New York, N.Y. 1964, p. 103. FREESE, E. E., in “Molecular Genetics,” Part I (J. H. Taylor, ed.), p. 207. Academic Press, New York, 1963. FREESE, E., AND FREk:sIC, E. B., Radiat. Res. Suppl. 6, 97 (1966). FREIFGLDkx, D., Radial. Res. &kppl. 6, 80 (1966). LUZZATI, D., SCHWIUTZ, H., BACH, M. L., AND CHEVALIER, M. R., J. Chim. Phys. 66, 1021 (1961). BUTLER,
DEGRADATION
OF DNA
10. SCHWEITZ, II., Biopolymers 8, 101 (1969). 11. LAWLF,Y, P. D., in Progress in Nucleic Acid Research and Molecular Biology. Vol. 5 (J. N. Davidson and W. E. Cohn, eds.), Academic Press, Inc., New York, N.Y. 1966, p. 8!). 12. ROSENKRANZ, H. S., ROSENKRANZ, S., AND SCHMIDT, R. M., Biochim. Biophys. Acta 196, 262 (1969). 13. MARMUR, J., ANDERSON, W. F., MATTHEWS, L., BERNS, K., GAJEWSKA, E., LANE, I).,
BY CYSTEINE
487
AND DOTY, P., J. Cell. Comp. Physiol. Suppl. 1, 68, 33 (1961). 14. MOROSON, H., AND ALEXANDER, P., Radiat. Res. 14, 29 (1961). 15. BENDICH, A., AND ROSENKRANZ, H. S. in Progress in Nucleic Acid Research, Vol. 1, (J. N. Davidson and W. E. Cohn, eds.), Academic Press Inc., New York, N.Y. 1963, p. 219. 16. FREESE, E., FREESE, E. B., AND GRAHAM, S., Biochim. Biophys. Acta 133, 17 (1966).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Degradation HERBERT Department
146, 483-487
of DNA
S. ROSENKRANZ2
of Microbiology,
AND
(1971)
by Cysteine’ SAMUEL
ROSENKRANZ
College of Physicians and Surgeons, New York, New York 1005%
Received
June 10, 1971; accepted
July
Columbia
University,
6, 1971
Exposure of DNA solutions to cysteine resulted in degradation of the polydeoxynucleotide. This reaction was inhibited by Naz EDTA and by sodium citrate. Methylcyste-ine, mercaptoethanol, serine, and homocysteine did not exhibit this degradative effect. Preincubation of cysteine resulted in a more rapid rate of degradation thus suggesting that a product formed during incubation was responsible for the observed effects.
Recent studies in our laboratory have revealed that L-cysteine, a naturally occurring amino acid, possessed antibacterial properties (1). This activity was made evident when bacteria were exposed to levels of L-cysteine of the order of 0.005 M. In view of the possible significance of this finding on our understanding of regulatory mechanisms, the basis of this biologic activity was investigated. The present report deals with the effects of L-oysteine and related substances on the properties of purified DNA. MATERIALS
AND
tration of 5%. After standing in the cold for 30 min, the insoluble residues were collected on the nitrocellulose discs and thoroughly washed with 5% trichloroacetic acid. The filter discs were dried, and the retained radioactivity was determined in a liquid-scintillation counter in 10 ml of omnifluor (4 g/liter of toluene). Physical chemical analysis. Sedimentation coefficients were determined in a Spinco Model E ultracentrifuge equipped with an ultraviolet optical system. The percentage of material sedimenting was determined as described previously (3). RESULTS
METHODS
Exposure of various DNA preparations to L-cysteine result,ed in degradation which was detectable by decreases in sedimentation coefficients (Table I) and by the formation of acid-soluble products (Table II). This depolymerization was dependent upon temperature and duration of exposure (Tables I-III). It was not due to contaminating nucleases present in the various DNA preparations as incubation of DNA in the absence of cysteine and its congeners did not result in degradation (Tables I and II). The possibility that cysteine activates cryptic deoxyribonucleases is also unlikely as the cysteine-induced degradation occurred even when heat-denatured DNA was examined (Table IV). Such a treatment is expected t,o abolish enzyme activit,y . The reaction between DNA and cysteine
Nucleic acids. Calf thymus DNA was purchased either from Nutritional Biochemicals Corp. or from CalBiochem. These preparations had sedimentation coeflicients of 12 and 21 S, respectively. Escherichia coli DNA was prepared by the procedure of Marmur (2: from E. coli C600. The bacteriophage DNA was isolated from Klebsiells phage E-l currently under investigation in this laboratory. 3H-Labeled DNA derived from chicken embryos was prepared as described previously (3). Stability oj DNA. Solutions of radioactive DNA were incubated at 37 or 56”. At intervals portions from each solution were removed and trichloroacetic acid was added to a final conceni This investigation was supported by a grant from the Damon Runyon Memorial Fund for Cancer Research. 2 Research Career Development Awardee of the U.S. Public Health Service (2-K3-GM29,024). 483
484
ROSENKRANZ
AND TABLE
EFFECT Erpt.
I
OF CYSTEINE
AND
DNA specimen
OTHER
COMPOUNDS Temperature (“C)
Calf
t,hymus
56
Calf
thymus
37
III
Bacteriophage
56
IV
E. coli
37
II
ROSENKRANZ I ON SEDIMENTATION Time (hr)
0 46 46 0 65 14.3 41 65 14.3 41 65 65 0 98 98 98 0 98 98 98
BEHAVIOR
OF DNA”
Additions
Sedimentation coefficient (S)
None None L-cysteine None None L-cysteine L-cysteine L-cysteine L-cysteine + EDTA L-cysteine + EDTA L-cysteine + EDTA EDTA None None L-cysteine S-Methyl-L-cysteine None None L-cysteine L-Serine
12.0 12.4 7.9 12.2 12.4 10.4 10.4 0 12.3 13.0 11.3 12.3 38.2 37.2 0 39.2 30.2 29.9 19.1 31.4
were incubated in the dark at the temperatures (1Sodium deoxynucleates (1 mg/ml 0.015 M N&l) indicated. At intervals portions of the reaction mixtures were removed diluted with 0.15 M NaCl and analyzed in the analytical ultracentrifuge. (Occasionally prior to ultracentrifugal analyses the DNA specimens were first precipitated with ethanol. This additional procedure did not affect the results). The final concentration of L-cysteine (L-cysteine hydrochloride.HsO; Mann Research Laboratories), S-methvl-L-cvsteine (Sigma Chemical Co.) and L-serine (Mann Research Laboratories) was 0.005 M; ” ” that of Na*EDTA was 2 mg/ml.
was influenced by the pH of the solvent. The smallest effect was near neutrality (Table III). This degradation was observed when three different preparations of L-cysteine, as well as the D- and m-isomers (Table II) were used. The material responsible for this degradation was dialyzable and resisted autoclaving-thus eliminating the possibility that it was due to bacterial contaminants or to a nuclease present in various batches of L-cysteine. Solubilization of the DNA was prevented by NazEDTA and by sodium citrate. No significant degradation was observed when DNA was exposed to methylcysteine (Tables serine, and I and II), mercaptoethanol, homocysteine (Table II) ; cysteamine caused some depolymerization at elevated temperatures while 2-aminoethylisothiouronium bromide was moderately active (Table II). Preincubation of cysteine at 56” increased its reactivity toward DNA as evidenced by
the more rapid rate of depolymerization exhibited by such a preincubated solution (Table V). DISCUSSION
The present findings of an effect of cysteine on isolated DNA may provide a possible chemical basis for the observed antibacterial action of this amino acid. It should be mentioned, however, that the in vivo effects are exhibited mainly by the L-isomer (1) while the present study indicates that the L- as well as the n-form degrades DNA. This discrepancy may be a reflection of the fact that the natural (L-) isomer has easier access into the cell. Current studies in this Laboratory are concerned with an elucidation of the chemical nature of the reaction between DNA and cysteine. However, in view of the results obtained with preincubated cysteine (Table V), it may be predicted that the degradation of DNA is
DEGRADATION
OF DNA TABLE
EFFECT Tern erature k)
Expt.
I
37
II
56
III
37
IV
37
V
37
VI
56
VII
37 56
OF CYSTEINE
AND
OTHER
II
COMPOUNDS
OF DNAa
ON DEGRADATION Radioactivity
Additions
n-Cysteine, Prep. No. 1 n-Cysteine, prep. No. 2 n-Cysteine, prep. No. 3 n-Cysteine n-Cysteine, autoclaved S-Methyl-n-cysteine Homocysteine n-Cysteine + EDTA EDTA n-Cysteine S-Methyl-t-cysteine L-Serine n-Cysteine-autoclaved Homocysteine Mercaptoethanol n-Cysteine L-Cysteine + citrate Citrate L-Cysteine, dialyzed AET* Cysteamine L-Cysteine n-Cysteine + citrate Citrate L-Cysteine, dialyzed AET” Cysteamine o-Cysteine nn-Cysteine o-Cysteine on-Cysteine
485
BY CYSTEINE
20
43
73 66 60 16 12 97 93 90 109
73 57 58 13 11 100 98 89 100 49 99 100 65 100 100 64 96 100 100 82 99 9 92 99 90 41 89
11 89 97 87 62 90
retained (% of Control, hr) 68
76
93
56 46 22
46 34 19
49 100 100 29 100 100
40 93 62 19 92 82 37 97 100 100 70 100
65 69
44 44
29 19
into a Two-milliliter portions of 3H-DNA (37 pg/ml of 0.015 M NaCl, 970 cpm/Gg) were distributed tubes containing premeasured amounts of cysteine or its congeners (final concentration 0.005 M), the final concentration of NazEDTA was 2 mg/ml and that of sodium citrate was 0.005 M. The reaction mixtures were incubated in the dark and at intervals replicate 0.1.ml portions were withdrawn and radioactivity remaining acid-precipitable was determined. The source of the chemicals used has been indicated elsewhere (1). Unless otherwise indicated, cysteine Prep. No. 1 (1) was used. b AET, 2-aminoethylisothiouronium bromide, HCl.
mediated by a product which is formed when cysteine is incubated at elevated temperatures. It should be pointed out that this action of cysteine, a, naturally occurring substance, on DNA is quite unique. Thus, many agents which degrade DNA have been recognized. Usually, however, these substances are mutagens. For example, X-rays, peroxides, and alkylating agents degrade DNA presumably through a mechanism involving depurina-
tion followed by chain-scission (4-12); exposure of DNA to uv light leads to a diminution in molecular weight, the mechanism of which is not fully understood (13, 14) and hydroxylamine and its derivatives also depolymerize DNA (15), probably through the formation of peroxides and free radicals (7, 16). This generation of peroxides and free radicals appears to be catalyzed by trace metals (7). In view of the present results, which indicate that the degradative effect
486
ROSENKRANZ
AND
ROSENKRANZ
TABLE EFFECT
OF
pH
ON
THE
III
PROPERTIES
DNA
OF
EXPOSED
TO
Sedimentation Time
(hr)
Buffer
CYSTEINEY behavior
DKA
and pH Sedimentation coefhcient (S)
DNA
+ cyst&e
Sedimentation coefficient (S)
VO Sedimenting
% Sedimenting -
24 48 72 0 24 48 72 24 48 72 24 48 72
Acetate, pH 4.6 Acetate, pH 4.6 Acetate, pH, 4.6 Phosphate, pH 7.0 Phosphate, pH 7.0 Phosphate, pH 7.0 Phosphate, pH 7.0 Tris, pH 8.0 Tris, pH 8.0 Tris, pH 8.0 Phosphate, pH 12 Phosphate, pH 12 Phosphate, pH 12
11.4 7.8 6.4 21.3 21.4 20.3 20.3 22.7 20.1 20.3
100 100 100 100 106 100 100 100 100 100
22.2
100
a Calf thymus DNA solutions (1 mg/ml of 0.01 M buffer in 0.015 L-cysteine at 56’. At intervals samnles were removed, diluted in 0.15 cients were determined. TABLE DEGRADATION
OF
IV BY
EFFECT
Radioactivity cpm/ml
18.4 16.0 14.1 16.4 15.5 12.5 16.5 10.8 10.1
100 loo 100 100 100 100 106 100 100
NaCl) were exposed to 0.005 M NaCl, and sedimentation coeffi-
2740 382
106 13.9
2756 342
100 12.4
of cysteine is inhibited by chelating agents, it is conceivable that the generation of free radicals or peroxides is also the basis of the action of cysteine. REFERENCES H. S., CARR, H. S., AND ZYROFF, J., J. Bacterial. 102, 672 (1970). 2. M.4RMUR, J., J. Mol. Biol. 3, 208 (1961). 3. JACOBS, S. J., A.ND ROSENKRANZ, H. S., Cancer Res. 30, 1084 (1970).
V
PREINCUB~TED
retain&
CYSTEINE
24 48
Control
21.4 20.3
ON
THE
OF (2.4~~ THYMUS DN& Sedimentation
Time (hr)
% Total
a One.milliliter portions of 3H-DNA were incubated with 0.005 M L-cysteine at 56” for 66 hr at which time radioactivity remaining acidinsoluble was determined. Thermally denatured DNA was prepared by heating DNA at 100” for 10 min and then immersing the specimens into an ice bath.
ROSENKRANZ,
OF
PROPERTIES
Additions
Native DNA Native DNA + 0.005 M Lcysteine Heat-denatured DNA Heat-denatured DNA+ 0.005 M L-cysteine
56 36 0
TABLE DNA
DEBT-DENBTURED L-CYSTEINE’
1.
M M
4.0 2.6 0
DNA + “fresh” cysteine
18.4 16.0
coefficients DNA + preincubated cyst&e
13.9 12.1
a L-Cysteine (0.05 M) in 0.01 M phosphate buffer, pH 7, was incubated for 24 hr at 56” whereupon it was diluted 1:lO upon addition to DNA. In parallel another specimen of DNA was supplemented with a fresh solution of L-cysteine. 4. 5.
6.
7. 8. 9.
J. A. V., Radiat. Res. Suppl. 1, 403 (1959). WI:ISS, J. J., in Progress in Nlicleic Acid Research and Molecular Biology Vol. 3 (J. N. Davidson and W. E. Cohn, eds.), Academic Press Inc., New York, N.Y. 1964, p. 103. FREESE, E. E., in “Molecular Genetics,” Part I (J. H. Taylor, ed.), p. 207. Academic Press, New York, 1963. FREESE, E., AND FREk:sIC, E. B., Radiat. Res. Suppl. 6, 97 (1966). FREIFGLDkx, D., Radial. Res. &kppl. 6, 80 (1966). LUZZATI, D., SCHWIUTZ, H., BACH, M. L., AND CHEVALIER, M. R., J. Chim. Phys. 66, 1021 (1961). BUTLER,
DEGRADATION
OF DNA
10. SCHWEITZ, II., Biopolymers 8, 101 (1969). 11. LAWLF,Y, P. D., in Progress in Nucleic Acid Research and Molecular Biology. Vol. 5 (J. N. Davidson and W. E. Cohn, eds.), Academic Press, Inc., New York, N.Y. 1966, p. 8!). 12. ROSENKRANZ, H. S., ROSENKRANZ, S., AND SCHMIDT, R. M., Biochim. Biophys. Acta 196, 262 (1969). 13. MARMUR, J., ANDERSON, W. F., MATTHEWS, L., BERNS, K., GAJEWSKA, E., LANE, I).,
BY CYSTEINE
487
AND DOTY, P., J. Cell. Comp. Physiol. Suppl. 1, 68, 33 (1961). 14. MOROSON, H., AND ALEXANDER, P., Radiat. Res. 14, 29 (1961). 15. BENDICH, A., AND ROSENKRANZ, H. S. in Progress in Nucleic Acid Research, Vol. 1, (J. N. Davidson and W. E. Cohn, eds.), Academic Press Inc., New York, N.Y. 1963, p. 219. 16. FREESE, E., FREESE, E. B., AND GRAHAM, S., Biochim. Biophys. Acta 133, 17 (1966).