INI J Kadurrron Oncology ’ Pergamon Press Ltd

Rio,

, 1979

0 Radiation

0306.3016/791070,-

Ph,, Vol. 5. pp 1055-1060 Printed m the U S A

Sensitivity:

L.

00/O

Facts and Models

RADIOLYSIS

Department

1055/$02

OF DNA AND OTHER BIOPOLYMERSt

S. MYERS, JR., Ph.D.,

and ERNEST KAY,

Ph.D.

of Radiological Sciences and Laboratory of Nuclear Medicine and Radiation University of California, Los Angeles, CA 90024, U.S.A.

Biology,

Studies of radiolysis of biopolymers serve the dual purposes of giving information on (a) chemical mechanisms by which radiation modifies life processes and (b) structure-function relationships in macromolecules. Conditions in living cells are such that both direct and indirect depositions of energy in hiopolymers are possible. Direct effects in chromatin components result in formation of specific radical products, many highly reactive. In irradiated DNA the cationic radical, -Gus+, and the anionic radical, *Thy-, make large contributions to the electron spin resonance (ESR) spectrum. Secondary reactions of the cationic radicals are largely unknown. Indirect effects occur when energy is deposited in water or other components in a solution, and radiolysis products such as e& and -OH react with the biopolymer under investigation. Studies carried out half a century ago show that this process is effective in inactivating enzymes. Conversion of *OH to the less reactive inorganic radical-anion *Bt-- has made it possible to determine the role of tyrosine in functional and structural integrity of several proteinase inhibitors. Both e.,- and -OH react rapidly with DNA, but only *OH initiates reactions which damage DNA. Radiolysis of double-stranded DNA leads to an increase in optical absorption. The *OH is believed to attack the deoxyribose moiety, causing strand breaks and partial denaturation, thus reducing the hypochromic effect. After the DNA is partially denatured, or single-stranded, *OH attacks the bases also. Three kinds of strand breaks have been observed; (1) immediate, (2) those appearing postirradiation, and (3) those appearing on post-irradiation treatment with alkali. Radiolysis of chromatin results in DNA strand breaks, base damage, and protein-DNA cross links. Yields for strand breaks and base damage are lower in chromatin than in purified DNA, and lower still in intact cells. A general hypothesis for radiation damage of chromatin is outlined. Radiolysis,

DNA, Biopolymers.

double-stranded there are significant single-stranded regions at “growing points” and possibly elsewhere. (4) The DNA may be intimately related to membranes at certain locations. (5) Large amounts of water and numerous small molecules and ions are associated with the chromatin. When this in vivo system is exposed to ionizing radiation, energy is deposited in all components, giving many kinds of ions and free radicals. If the energy is deposited in chromatin or a chromatin component, the process is referred to as direct action of radiation; if the energy is deposited in water or other small molecules, with production of free radicals which then react with DNA or chromatin, the process is referred to as indirect action. This distinction is well defined when radiation effects in dry or nearly dry materials are compared with effects in dilute aqueous solution, but it becomes less so in complex systems in which many radiation-induced reactions may be the

In this paper we consider the radiolysis of biopolymers from the point of view of presumed importance in duo. To start, we accept the judgement of biologists that damage of chromatin is the most likely cause of the biological effects of radiation. Consequently, we shall limit our discussion to radiation effects on proteins, DNA, and, to a limited extent, on chromatin itself. The biological system which must be considered is complex and not precisely defined. The structure of chromatin and its relation to other components of the cell are presently subjects of intense investigation. Even so, we can make a few statements which are probably correct: (1) The DNA is in intimate contact with histones and other proteins, including enzymes which monitor integrity of DNA, stimulate repair and replication, etc. (2) The tertiary structure of DNA and its relationship with proteins varies periodically along the chromatin chain. (3) While much of the DNA is tsupported by the U.S. Department of Energy. Reprint requests to: Ernest Kay, Ph.D.

Accepted for publication 1055

21 February

1979.

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same, whether initiated by direct or indirect processes. Both processes probably contribute significantly to the biological effects of radiation. Consequences of absorption of energy directly by DNA or chromatin. The direct process is expected to result in formation of cation-radicals and electrons dissociated from parent molecules. Numerous attempts, mostly utilizing low temperature irradiation of solid compounds and electron spin resonance (ESR) spectroscopy, have been made to identify these products in nucleic acid components and amino acids.20,22 Several general conclusions can be drawn: (1) The initial products tend to be highly reactive even at temperatures of 77°K and lower. Thus, with a few exceptions, the cation-radicals are not observed. Instead, neutral oxidation products are found, formed possibly by deprotonation of the cation radical. Similarly, the dissociated electrons are rarely observed; usually they are captured by a molecule to give an anion-radical which may react with a proton to give a neutral radical-reduction product. (2) The nature of the radicals stabilized in crystals is highly specific. (3) As the irradiated crystals are warmed from 77°K to room temperature, many of the radical products undergo reactions which may be internal rearrangements or radical-molecule interactions. (4) The primary cationic and anionic radicals observed in in crystals are probably the same as those formed viuo by direct action; and the protonation or deprotonation reactions of the ionic-radicals probably also occur in Go. The subsequent secondary reactions in viuo may well be different from secondary processes observed in crystals. Although cation-radicals are rarely observed in irradiated chromatin components, oriented fibers of Li-DNA at 77°K give ESR spectra which indicate that about 25% of the radicals present are guanine cationic radicals (*Gus’).” Thymine anionic radicals, (.Thy-), and unidentified radicals account for the remainder of the spectrum. Strong support for the identification of (=Gua’) has been obtained by comparative ESR studies of photoionized dGMP and DNA.25 While much information is available on the early processes in direct action, the mechanisms leading to final products, and, in many cases, their identity are as yet unknown. Consequences of absorption of energy by water. It is well known that radiolysis of Hz0 gives hydrated free radicals, *OH, in electrons, eaq-, and hydroxyl yields of a little less than 3 PM per 1000 rad; these are, in general, the principal reactants which initiate damage of organic molecules by indirect action. In concentrated solutions the yields may be somewhat‘ different.3 have been The effects of eaq- and -OH on proteins extensively investigated since long before the reac-

July 1979, Volume 5, No. 7

tive species were identified. Over half a century ago, it was observed that indirect action inactivated trypsin, pepsin, and invertase, and converted oxyhemoglobin to methemoglobin.7~8.‘3 The dose-response relation was exponential, and the percentage of damage was found to depend on protein concentration. Subsequently, extensive studies on effects and reaction mechanisms have been carried out with both proteins and model compounds. These are reviewed briefly by Myers.” A major thrust in recent research has been to use radiolytic techniques to study structure-function relationships of proteins by relating radiationinduced changes in biological activity to structural alterations. These studies are facilitated by the conversion of eaqm to *OH by reaction with N20, and of .OH to one of several inorganic radical-anions, for example, .Br2-. .OH + Br-+

OH- + *Br

.Br + Bra -+ .Br2-

Other useful anion radicals are *(CNS)*-, .C03- and .C12-. The advantage of these radicals is that they react more slowly and more specifically than *OH. Thus . Brl reacts with Try, l/2 Cys, Tyr, and His with bimolecular rate constants (k) of 77, 18, 2.0, and 1.5 x 10’ M-‘s-‘, respectively, and with other amino acids with k < 106. The .C12- radical reacts more rapidly, the .C03- and .(CNS),- radicals more slowly choice of attacking than .Br2-. Thus by judicious radical and protein, considerable information can be obtained on the effect of modifying specific amino acid residues on protein conformation and function.‘.* We have been using the radical-anions to elucidate the structure-function relationships of proteinase inhibitors from soybean, lima bean, and bovine pancreas. Enough is known about these proteins so that changes in activity can be related to modification of specific amino acid residues. Circular dichroism spectroscopy was used to determine conformational changes. The Bowman-Birk soybean proteinase inhibitor (BBI) and lima bean proteinase inhibitor (LBI) share several features which are not present in many enzymes: (1) The two proteins are closely related functionally and structurally. The amino acid sequences are almost completely homologous along the entire polypeptide chain,27 and therefore data obtained with these proteins are complementary. (2) Both inhibitors possess two separate independent binding sites, one of which inhibits trypsin, the other chymotrypsin. This feature provides the opportunity to examine 2 parts of the protein separately, and conformational changes can be monitored independently by circular dichroism (damage which changes protein conformation is likely to affect both inhibitory activities simultaneously, while damage which affects only 1 activity may be limited to a specific binding

Radiolysis of DNA and other biopolymers

site). (3) Both proteins contain only a few amino acid residues which are potentially reactive with the inorganic radical-anions. Thus, *(CNS)2- reacts only with Tyr 69 in LB1 and Tyr 69 plus Tyr 55 in BBI. In addition, .Br2- reacts with hystidyl residues; i.e. His 43 in BBI, His 43, His 21, and one or more histidines in the N-terminal segment of LB1.t Radiolysis of BBI in the presence of SCN- and Brshowed a good correlation between the inactivation of the antichymotrypsin activity and the destruction of tyrosyl residues, while the antitrypsin activity remained unchanged under the same conditions.28 The interpretation of these findings is that the integrity of Tyr 55 at the position immediately after the chymotrypsin-binding site, Leu 53-Ser 54, is essential for the chymotrypsin-inhibitory activity. Tyr 69, located in the antitrypsin portion of the inhibitor, is deemed non-essential, as damage to this residue did not lead to functional or conformational changes. Results from the radiolysis of LB1 are consistent with such interpretation.16 In LBI, Tyr 69 is present but Tyr 55 is replaced by isoleucine, an amino acid which does not react appreciably with .Br2- or .(CNS)?-. Radiation damage to the tyrosyl residue in LB1 does not elicit a change in either antiproteinase activity. None of the histydyl residues in the two proteins appear to be essential to either binding or conformational stability. It is of interest to contrast these results with a study using a conventional reagent as a probe. Attempted 0-acetylation of the tyrosyl residues in BBI with N-acetylimidazole suggests that the Tyr 55 adjacent to the antichymotryptic site is relatively inaccessible to the reagent; it can be acetylated in the presence of 6 M guanidine hydrochloride but not in 8 M urea.14 The acetylation of Tyr 55 is accompanied by 60% loss in antichymotryptic activity.” Deacetylation of 0-acetyltyrosines with hydroxylamine restores the activity to the original level. Tyr 69, located in the antitrypsin portion of the inhibitor, is relatively exposed to N-acetylimidazole and can be acetylated without a denaturing agent. The degree of acetylation of Tyr 69 parallels a decrease in antitryptic activity. BBI acetylated at Tyr 69 is fully active toward chymotrypsin and has 30-40% of the antitryptic activity of the native inhibitor. The original level of antitryptic activity is restored upon deacetylation. The loss of antitryptic activity on acetylation of Tyr 69 seems to contradict the evidence of the radiation study. However, *Br2- and acetylation produce different products, and the modification of the same residue in the protein by the two reagents may be expected to have different effects on the functions. tTo simplify the presentation, of the 2 proteins

are aligned

the amino acid sequences and residue

numbers

of LB1

0 L. S. MYERS, JR. and E. KAY

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Acetylation attaches a relatively bulky acetyl group to the hydroxyl group of the tyrosyl side chain, which may sterically hinder the formation of a proper enzyme-inhibitor complex. The derivatives of .Br2and .(CNS)2- attack on tyrosine have not yet been characterized. We anticipate that they will not have large groups attached; more likely, they will be found to be oxidation products formed by electron transfer to the anion-radical. A similar study with LB1 is consistent with the above analysis.1s,‘6 Although Tyr 69 of LB1 is more difficult to acetylate than the corresponding tyrosine in BBI, its acetylation leads to a decline in antitryptic activity but has no effect on antichymotryptic activity. These studies give us some insights into the role of Tyr 55. Since this position is occupied by isoleucine in LB1 without change in antichymotryptic activity, Tyr 55, although adjacent to the chymotrypsin binding site Leu 53-Ser 54, may be regarded as “nonessential”, and presumably does not participate directly in chymotrypsin binding. However, its integrity is essential to maintaining the correct conformation of the chymotrypsin-inhibitory site in BBI. In BBI and LBI, both .Br2- and .(CNS)2- react with Tyr 55 and Tyr 69 readily, though .Br2- shows a higher reactivity. With bovine pancreatic proteinase inhibitor (BPI), however, these two radical anions act very differently.’ BP1 possesses a single binding site, capable of inhibiting both trypsin and chymotrypsin. Only Tyr 10, Tyr 21, Try 23, and Tyr 35 in BP1 are potentially vulnerable to *Brzm and .(CNS),-. However, the .(CNS)2m anion reacts with only 1 of the 4 tyrosyl residues, and the product with one tyrosine damaged by .(CNS)2- retains full activities toward trypsin and chymotrypsin. .Br2-, on the other hand, modifies more than one tyrosine and inactivates both antiproteinase activities. The exact number of tyrosines damaged by .Br2- cannot be quantitated by the spectrophotometric methods used because BP1 exposed to *Brz- precipitated at 150 krad. Only 1 tyrosine can be 0-acetylated by N-acetylimidazole, even in the presence of 6 M guanidine hydrochloride or 8 M urea. The above studies clearly suggest that only 1 of 4 tyrosyl residues in BP1 can be modified easily. Tyr 10 is probably the accessible tyrosine, because X-ray crystallography shows it is exposed and far from the inhibitory site. Of the other tyrosyl residues, one or more, singly or in combination, apparently is (are) essential to the structural stability of the protein. These residues are accessible to some but not to other reagents, presumably because of the relative are then used for both. Therefore the amino of BBI starts with the residue 11.27

acid sequence

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0 Biology 0 Physics

size of the reacting species. Crevices where Tyr 21, Tyr 23, and Tyr 35 are located may permit entry of .Brz-, the smallest of the 3 reagents, but prevent the other 2 from reaching the tyrosyl residues. The above results show that radiation-produced radical anions can be powerful tools in studies of protein structure and function. Their usefulness may be greatly enhanced as we learn more about the mechanisms and products of their reactions. Reactions of water radicals with pure DNA. In and .H (hydrogen atoms) aqueous media, eaq react rapidly with DNA (k(e;,, + DNA) = I .4 x IO’, k(.OH + DNA) = 6 x and k(.H + DNA) = 5 x IO’ 108 ~-ls-l~.Z0.2” Studies with the components of DNA show that all three species react readily with bases or base moieties. With deoxyribose moieties, .OH reacts almost as readily as with the bases, .H reacts more slowly, and ea4- very slowly. Since the yield of .H is low, it will be disregarded in what follows. While e/ reacts with DNA, measurements of the ultraviolet absorption of irradiated DNA in air-free solutions containing .OH scavengers indicate that eaqm does not cause base damage in double-stranded DNA (ds-DNA). Under the conditions noted, the absorbance of a 0.02% DNA solution changes less than 10% at the absorption maximum of DNA for a dose of 106rad, and the shape of the spectrum does not change. Radiolysis of DNA in NzO-saturated solutions permits one to observe the effects of -OH in the absorption of singleabsence of eaq--. The ultraviolet stranded DNA (ss-DNA) at 265 nm decreases in a complex way with increasing radiation dose because of the buildup of light-absorbing products. The decrease is sufficient to show that the base chromophores are attacked by .OH. In solutions saturated with N20, N2 or 02, the absorbance of ds-DNA, in contrast, increases with increasing exposure to low after a maximum absorbance is radiation doses; reached at somewhat less than 40% above the initial value, the absorbance begins to decrease with the same dose-dependence shown by ss-DNA. This result has been interpreted as indicating that in ds-DNA the bases are largely shielded from .OH attack; .OH first attacks mainly the deoxyribose backbone, causing strand breaks and partial denaturation, which expose the bases to .OH attack.3” Although the bases in ds-DNA seem to be shielded from .OH addition, they are not protected from all oxidative attacks. The radical-anion, .Br2-, destroys the base chromophores of intact ds-DNA. This suggests that protection of the bases from .OH results from steric hindrance; if .OH were to add to a double bond in the base, it would have to make room for itself. However, .Brz- can react by electron transfer,

July 1979. Volume 5. No. 7

which apparently is possible if it approaches one of the grooves of ds-DNA. Biological criteria of integrity of DNA are consistent with the chemical studies in indicating that .OH is the principal initiator of DNA damage by indirect action.4,‘2 Mechanisms of base damage and strand breaks. The mechanism of damage of thymine by indirect action is fairly well understood. The .OH adds to the 5,6-double bond, and, if O2 is present (as is usual in uiuo), it adds to the other end of the 5,6-bond to give a hydroxyperoxy radical, which is then reduced by .02- to give the anion of the hydroxyperoxide; this compound is fairly stable. Other derivatives with saturated 5,6-bonds also can be formed, such as thymine glycols. In addition, .OH can abstract .H from the methyl group, to give ultimately 5hydroxymethyluracil. Attack by .OH on the other bases results in the initial formation of base-OH adduct radicals, but details of the secondary reactions, and for the purines the site of addition,‘0 are not as well understood as for thymine. Recent work by Ward and co-workers has begun to clarify the mechanisms leading to strand breaks.‘7,29 The reaction can be initiated by .OH attack either on a base or at one of several positions on the deoxyribose moiety. Three kinds of breaks can be identified by kinetic means; (I) breaks observable immediately after irradiation, (2) breaks which appear slowly over a period of several hours following irradiation, and (3) breaks which occur only on incubation with 0.1 N NaOH. Some of the reactions leading to breaks also result in the release of intact or damaged bases. The ends of the broken strands are believed to be the 3’- and 5’-monophosphate termini. Radiolysis of chromatin and bacteriophages. Irradiation of DNA when it is associated with protein results in DNA damage similar to that observed with isolated DNA, but yields are lower. It is difficult to make quantitative comparisons because different experimental conditions have been used by different workers. In bacteriophage T 1 the yields observed on radiolysis of 0.1 M histidine solutions were in the order, ss-breaks > ds-breaks > DNA-protein crosslinks.s In chromatin the yield of ss-breaks has been reported to be less than the yield of base damage.” Yields are lower in intact cells than in isolated For most of these products, the chromatin. mechanisms of their formation are not established. It has been shown, however, that .OH plays an imin production of crosslinks in portant part chromatin.” Attachment of small molecules to DNA. Many kinds of small molecules have been reported to become attached to DNA or DNA components during

Radiolysis of DNA and other biopolymers

radiolysis. These include alcohols, amino acids, carcinogens, nucleic acid components and several sensitizers to ionizing radiation (N-ethylmaleimide, several nitrofurans, and 2,6,6,6-tetramethyl-4-piperidone-N-oxyl). It is not known whether the attachment or some other reaction such as electron transfer is responsible for the sensitizing effects of the latter compounds (see Myers” for a review). The mechanisms of the attachment reactions are, for the most part, not known. Diffusion distance of .OH in cells. In the vicinity of DNA numerous molecules other than DNA can react with -OH. The probability that a given .OH radical will react with DNA in viuo is therefore low. Correspondingly, the distance which .OH can diffuse before interacting with DNA must be small and its lifetime in uivo short. Several estimates have been made of these parameters. For example, in mouse leukemia cells the diffusion distance has been estimated to be about 60 A for ss-breaks and 93 A for cell killing.24 These values are approximate, but along with earlier estimates,24 they indicate that *OH must be formed close to the target to be effective. Timescale of reactions. The first pulse radiolysis studies of the reaction of *OH with DNA showed that non-radical light-absorbing radicals or, possibly, transient products had a lifetime much longer than the msec period available for observation.23 Brustad et al. have shown that this period may be as long as 40 min in pure DNA solutions. This finding is consistent with the result noted above that the yield of

0 L. S. MYERS,JR. and E. KAY

strand breaks period.6

increases

with

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time

over

a

24-hr

Hypothesis for chromatin radiolysis. The results outlined above make it possible to begin to develop a coherent hypothesis for damage of chromatin by ionizing radiation. Principal points follow: 1. Direct absorption of energy by chromatin can damage bases, the main chain, and the protein. 2. Direct damage of the main chain of DNA probably causes loss of a hydrogen atom followed by strand breakage or reaction with protein or other molecules. 3. At biologically relevant dose levels attack by aOH (indirect action) on ds-DNA damages only the main chain, causing loss of a hydrogen atom, followed by secondary reactions identical to those initiated by direct action as noted above. 4. Attack by *OH on ss-DNA can, in addition, cause base damage. ineffective in 5. Attack by e- or eaq_ is relatively damaging DNA, but may damage proteins. 6. -OH must be formed within 50-+ 25 A of its target in uiuo. 7. Attack on ds-DNA by electron transfer oxidizing agents can damage bases. 8. Sensitization at the earliest stage of radiolysis can be accomplished in principle by: (a) Utilizing electrons to damage DNA. (b) Converting *OH to an oxidizing agent which can react with bases in dsDNA.

REFERENCES G.E., Aldrich, J.E., Bisby, R.H., Cundall, R.B., Redpath, J.L., Willson, R.L.: Selective free radical reactions with proteins and enzymes: reactions of inorganic radical anions with amino acids. Radiat. Res.

1. Adams,

49: 278-289, 1972. 2. Adams, G.E., Redpath, J.L., Bisby, R.H., Cundall, R.B.: The use of free radical probes in the study of mechanisms of enzyme inactivation. Zsr. J. Chem. 10: 1079-1093, 1972. 3. Aldrich, J.E., Lam, K.Y., Shragge, P.C., Hunt, J.W.: Fast electron reactions in concentrated solutions of amino acids and nucleotides. Radiat. Res. 63: 42-52, 1975. 4. Armel, P.R., Strniste, G.F., Wallace, S.S.: Studies on Escherichia coli X-ray endonuclease specificity. Roles of hydroxyl and reducing radicals in the production of DNA lesions. Radiat. Res. 69: 328-338, 1977. 5. Bohne, L., Coquerelle, Th., Hagen, U.: Radiation sensitivity of bacteriophage DNA-II. Breaks and crosslinks after irradiation in viva. Znt. J. Radiat. Biol. 17: 205-215, 1970. 6. Brustad, T., Jones, W.B.G., Wold, E.: Reactions between nitroxyls and radiation-induced long-lived DNAtransients. Znt. J. Radiat. Biol. 24: 3343, 1973.

7. Clark, H., Northrop, J.H.: The inactivation of trypsin by X-rays. J. Gen. Physiol. 9: 87-96, 1925. 8. Fricke, H., Peterson, B.W.: The relation of chemical, colloidal and biological effects of roentgen rays of different wave lengths to the ionization which they produce in air--I. Action of roentgen rays on solutions of oxyhemoglobin in water. Am. J. Roentgenol. 17: 61 l-620, 1927. 9. Gigante, D., Kay, E.: Structure-function relationships of bovine pancreatic proteinase inhibitor. In preparation. 10. Gorin, G., Lehman, C., Mannan, C.A., Raff, L.M., Scheppele, S.E.: Radiolysis of adenine in dilute neutral aqueous solution. J. Phys. Chem. 81: 304-307, 1977. 11. Graslund, A., Ehrenberg, A., Rupprecht, A., Strom, G.: Ionic base radicals in -y-irradiated oriented non-deuterated and fully deuterated DNA. Znt. J. Radiat. Biol. 28: 313-323, 1975. 12. Held, K.D., Synek, R.W., Powers, E.L.: Radiation sensitivity of transforming DNA. Znt. J. Radiat. Biol. 33: 317-324, 1978. 13. Hussey, R.G., Thormpson, W.R.: The effect of radioactive radiations and X-rays on enzymes. J. Gen. Physiol. 5: 647-659, 1923.

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14. Kay, E.: Origins of circular dichroism bands in Bowman-Birk soybean trypsin inhibitor. J. Biol. Chem. 251: 3411-3416 (1976). 15. Kay, E.: Structure-function relationships of proteinase inhibitors-modification by N-acetylimidazole. J. Biol. Chem., Submitted for publication. 16. Kay, E., Gumbiner, B.: Structure-function, relationships of lima bean proteinase inhibitor-modification by selective radical-anions . Brzm and . (CNS)z-. J. Biol. Chem., Submitted for publication. 17. Kay, E., Ward, J.F.: Monophosphate end groups produced in radiation-induced strand breakage in DNA. Radiat. Res. 69: 185-193, 1977. 18. Mee, L.K., Adelstein, S.J.: Radiolysis of chromatin extracted from cultured mammalian cells: Formation of DNA-protein cross links. Radiat. Res. 74: 555, 1978. S.J., Stein, G.: Radiolysis of 19. Mee, L.K., Adelstein, chromatin extracted from cultured mammalian cells: Production of alkali-labile damage in DNA. ht. J. Radiat. Biol. 33: 443-455, 1978. 20. Myers, Jr., L.S.: Radiation chemistry of nucleic acids, proteins, and polysaccharides. In The Radiation Chemed. by Dole, M., New York, istry of Macromolecules, Academic Press, 1973, pp. 323-374. 21. Myers, Jr., L.S.: Ionizing radiation-induced attachment reactions of nucleic acids and their components. In Aging, Carcinogenesis, and Radiation Biology, ed. by Smith, K.C., New York, Plenum, 1976, pp. 261-286. 22. Myers, Jr., L.S.: Free radical damage of nucleic acids and their components: The direct absorption of energy. In Free Radicals in Biology, Vol. IV, ed. by Pryor, W.A., New York, Academic Press, 1979, (in press).

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23. Myers,

Jr., L.S., Hollis, M.L., Theard, L.M.: radiolysis of DNA and related compounds. In Radiation Chemistry, Vol. I, ed. by Gould, R.F. Washington, D.C., American Chemical Society Publications, 1968, pp. 345-367. 24. Roots, R., Okada, S.: Estimation of life times and diffusion distances of radicals involved in X-ray-induced DNA strand breaks of killing of mammalian cells. Radiat. Res. 64, 306-320, 1975. 25. Sevilla, M.D., D’Arcey, J.B., Morehouse, K.M., Engelhardt, M.L.: An ESR study of n-cation radicals in dinucleoside phosphates and DNA, Photochem. PhotoPulse

biol. 29, 37-42, 1979. 26. Shragge, P.C., Michaels,

H.B., Hunt, J.W.: Factors affecting the rate of hydrated electron attack on polynucleotides. Radiat. Res. 47, 598-611, 1971. 27. Stevens, F.C., Wuerz, S., Krahn, J.: Structure-function relationships in lima bean protease inhibitor. In Proteinase Inhibitors, ed. by Fritz, H., Tschesche, H., Greene, L.J., Truscheit, E., Berlin, Springer Verlag, 1974, pp. 344-354. 28. Wandell, J.L., Kay, E.: y-Irradiation of Bowman-Birk soybean proteinase inhibitor. Radiat. Res. 72: 414-426, 1977. 29. Ward, J.F., Kuo,

I.: Strand breaks, base release, and postirradiation changes in DNA -y-irradiated in dilute 02-saturated aqueous solution. Radiat. Res. 66: 485-

498, 1976. 30. Ward, J.F.,

Kuo, I.: Radiation damage to DNA in aqueous solution: A comparison of the response of the single-stranded form with that of the double-stranded form. Radiat. Res. 75: 278-285, 1978.

Radiolysis of DNA and other biopolymers.

INI J Kadurrron Oncology ’ Pergamon Press Ltd Rio, , 1979 0 Radiation 0306.3016/791070,- Ph,, Vol. 5. pp 1055-1060 Printed m the U S A Sensitivi...
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