KENNETH H. KINGDON
1710
Indirect Inactivation of Deoxyribonucleic Acid
by Kenneth H. Kingdon 1174 Phoeniz Avenue, Schenectady, h’ew York 1.9308
(Received October 9, 1966)
This paper explores the concept that radiation -inactivation of a DNA molecule is caused by the generation of a single pair of H radicals in a sugar, which migrate through the adjacent bases by a two-dimensional random walk, caused by excitation of the electron atmosphere of the molecule by the radiation which generated the H radicals. The chance is estimated that this single pair of H radicals shall react with both members of an adjacent base pair, thus leading to the formation of a covalent cross-link between the bases and inactivation of the entire molecule. This chance is estimated to be 0.12, and from this and the experimental G value of 2.0 for producing the H radicals, the D3, for virus DNA is found to be 7.5 X 1012/(molecular weight) rads, in fair agreement with experiment. A molecule of diploid cell DNA has much lower sensitivity to radiation than a molecule of equal mass of virus DNA. Part of this is due to the duplication of genes, but it is suggested that another factor is the existence of a three-dimensional secondary structure in the diploid DNA formed by H bonds with adsorbed water, in which the pair of H radicals is much more widely dispersed by a three-dimensional random walk and therefore is less effective for cross-linking the bases. An initial effect of radiation on a diploid cell is to break some of these water H bonds, giving isolated pieces of virus-like twin DNA helix, which are more sensitive to radiation. The production of isolated twin helix DNA by the radiation is estimated, and it is shown that a log-log plot of the fraction of diploid cells surviving at dose D, against cosh (D/D37),should be linear, with a slope of -2.3. This is in agreement with experiment.
I. Introduction One process by which radiation is known to inactivate DNA is by the formation of a covalent’ cross-link between the two bases in a pair of nucleotides.’ Such cross-links may inactivate the DNA biologically by preventing complete separation of the strands of a twin helix. In other experiments a cross-link may serve as an anchor to locate the partially separated strands of a twin helix, giving the “reversible DNA” studied by Geiduschek.2 These cross-links presumably result from chemical interactions of radicals formed in the two bases. However, all bases are resonating ring structures and are therefore quite insensitive to radiation, so the possibility should be considered that other radicals are produced by the radiation elsewhere and that these radicals migrate to the bases and put them in a chemically reactive condition. Although indirect inactivation is usually thought to be due to radicals formed from water, which diffuse The Journal of Physical Chemistry
into the molecule to be inactivated, it is true that for ionizing radiations some organic substances such as n-octane, cyclohexane, alanine, and glutamic acid have radical yields greater than that for ~ a t e r . ~ , ~ It seems clear, therefore, that nonaqueous sources of radicals, such as other components of DNA, may be important. Biemann and TvIcClo~key~~~ have demonstrated from the mass spectra of nucleosides that H (1) J. Marmur and L. Grossman, Proc. Natl. Acad. Sci. U . S., 47, 778 (1961). (2) E. P. Geiduschek, ibid., 47, 950 (1961). (3) L. Bouby, A. Chapiro, M. Magat, E. Migirdicyan, A. Prevot-
Bernas, L. Reinisch, and J. Sebban, Proc. Intern. Conf. Peaceful Uses At. Energy, Geneva, 1966, 7, 526 (1956). (4) T. Henriksen, T. Sanner, and A. Pihl, Radiation Res., 18, 147 (1963). (5) K. Biemann, “Mass Spectrometry,” McGraw-Hill Book Co., Inc., New York, N. Y . , 1962, p 351. (6) K. Biemann and J. A. McCloskey, J . Am. Chem. SOC.,84, 2005 (1962).
INDIRECT INACTIVATION OF DEOXYRIBONUCLEIC ACID
1711
atoms are produced in the sugar and migrate to the base. The very great intensity of the (base H) 2H) peaks shows that this is a very and (base probable process and illustrates the stability of the bases against ionizing radiations, as compared with the sugars. A very large DNA molecule is formed from only six kinds of small molecules-the four bases, the sugar, and the phosphate. The bonds between phosphorus and oxygen are known to be very strong and therefore radiation resistant; and the work of Biemann and McCloskeyG demonstrates that the bases are much more radiation resistant than the sugar. Accordingly, we shall assume that the G of 2.0 for DNA, as found by Lett, Parkins, Alexander, and Ormerod,’ is due entirely to H atoms liberated from the sugar and shall attempt to show that the migration of these H atoms to the bases can account quantitatively for the inactivation of virus twin helix DNA by ionizing radiation.
flux of 730 r/min, the migration rate returning to its normal value as soon as the radiation is stopped. It is suggested here that these effects are caused by each incident electron of the ionizing radiation momentarily heating the electron atmosphere of a molecule locally to a high temperature, thus creating local fluctuations of the energy levels of the molecule and causing the H radicals or other small molecules to perform random walks between these momentarily fluctuating energy levels. The usual concept of diffusion of atoms is based on the idea that an atom statistically acquires an unusually high energy which enables it to surmount a potential barrier and move to a different site in a static system of energy levels. The migration concept suggested here is that an atom of average energy is enabled to move to a different site by a statistical disturbance of the system of energy levels by incident radiation. Biemann and RIcCloskey’s experiments6 show that a single excitation of the electron system has a very high probability of transporting an H radical from the sugar to the base, a distance of about 5 atomic spacings, or a random walk of about 25 steps. It therefore seems correct to assume that a single excitation of the electron atmosphere of the molecule lasts long enough to permit the H radicals to migrate from the sugar to the nearest pair of bases, and perhaps farther.
+
+
11. Abundance of Radicals Formed in DNA It will clarify our thinking to state quantitatively the abundance of radicals formed in DNA by radiation. In what follows we shall refer to the entire DNA content of a virus (for example) as a single molecule and consider that a structure of molecular weight lo* can be inactivated by formation of a single covalent cross-link between a pair of bases.’ A typical twin helix DNA molecule of molecular weight 6 X 10’ has a D3, of about lo5 rads. This amount of radiation will, on the average, deliver 630 ev/molecule, which with a G of 2.0 will produce about 12 H radicals/molecule, or 6 pairs of H radicals/105 pairs of nucleotides. It is evident from these figures that it is extremely improbable that a second pair of radicals will be formed in a given pair of nucleotides during the irradiation and that therefore the radical attack and cross-linking of a pair of bases must be carried out by a single pair of H radicals produced in an adjacent sugar.
111. Migration of Radicals The H radicals have to migrate from the sugar t o the bases in order to produce inactivation and, from what has been said about the abundance of radicals formed in DNA, it is clear that there is no concentration gradient for conventional diffusion. Biemann and RIcCloskey6have demonstrated the rapid migration of H radicals from the sugar to the base in the mass spectra of nucleosides. It has also been shown by Tikhomirova, Malinskii, and Karpovs that gases migrate through polyethylene 10-16 times faster than normally while the polyethylene is exposed to a p r a y
IV. Inactivation of Virus Two-Strand DNA Figure 1 shows a projection of a short section of a two strand Watson-Crick helix, based on a figure of W i l k i n ~ but , ~ with the bases rotated about 45O, so as to make evident their structure and hydrogen bonding. The bases adenine, thymine, cytosine, and guanine are labeled A, T, C, and G, and the sugar and phosphate, S and P, respectively. Suppose a pair of H radicals is generated in the sugar ring at left center of Figure 1. A minimum random walk of 9 atomic spaces (including one hydrogen bond), about 81 random steps, would carry one of these H radicals through the adenine to the CS of the thymine, where it might add to Ce,breaking the double bond and producing a radical on Cs. The other H radical of the pair might interact with the adenine. We shall attempt to estimate quantitatively the chance that a (7) J. T. Lett, G. Parkins, P. L. Alexander, and M. G. Ormerod, Nature, 203, 593 (1964). (8) N. S. Tikhomirova, Yu. M. Malinskii, and V. L. Karpov, Vysokomolekul. Soedin., 2 , 1335 (1960);see Chem. Abstr., 5 5 , 19312d (1961). (9) M. H. F. Wilkins in “Comprehensive Biochemistry,” Vol. 8, Florkin and Stotz, Ed., Elsevier Publishing Co., New York, N. Y., 1963, 272.
Volume 71, Number 6 M a y 1967
1712
Figure 1. Rligration paths for H radicals in DNA helix.
single pair of H radicals will intereact with both members of this pair of bases so as to produce a crosslink between them. It should be stated explicitly that this calculation is intended only to demonstrate that, with a particular set of assumptions, which are based as far as possible on observed experimental data, the calculated probability of cross-linking a pair of bases is in approximate agreement with the observed value of D37for DSA. The inference should not be drawn that this is intended as a definitive treatment of this difficult problem. The assumptions qeeded for this calculation are as follows. The first assumption specifies the reactive sites for the radicals in the purine and pyrimidine bases, and in the sugar-phosphate strands. The radiation chemistry of aqueous solutions of nucleic acids, as reviewed by ScholesI1O indicates that the 5,6 carbon double bond in a pyrimidine and the 4,5 carbon double bond in a purine are the most probable sites for attack by the H and OH radicals formed in water. The double bond becomes saturated, leaving a new radical on one of the carbons. Other chemical reactions usually follow this initial step. In the present case, we are postulating that the subsequent chemical reaction is the reaction of the two new radicals, one on each base, to form a covalent cross-link between the purine and the pyrimidine. In addition to reacting with the adjacent bases, H radicals will also migrate along the sugarphosphate strands of the twin helix and presumably will react with the singly bonded oxygen atoms in the phosphate groups. For a pair of H radicals originating in the ring of the sugar to the left center of Figure 1, The Journal of Physical Chemistry
KENNETH H. KINGDON
there will thus be 6 reactive sites, with the following atomic bond spacings from the nearest point of the sugar ring: 0 in one phosphate (3), 0 in the other phosphate (4),Cq in adenine (a), and CS(4),CS in thymine @), and Ce (9). Since the number of random walk steps required to traverse a given distance is proportional to the square of the distance and if each atomic spacing is traversed in one step (as discussed in section 111),the number of random steps required to reach each of the above reactive sites will be 9, 16, 4, 16, 64, and 81, respectively. Our second assumption is that the probability of reaching a particular site is inversely proportional to the number of random steps required, so that the probabilities for reaching the various sites are k/9, k/16, k/4, k/16, k/64, and k/81, respectively. The constant lc may be evaluated by setting the sum of the probabilities equal to unity. This scheme of calculation neglects H radicals which pass through the phosphate groups without reacting and those which pass through the adenine and thymine without reacting. For such a pair of H radicals to reach the nearest reactive sites in the A-T base pair at the top of Figure 1 would require random walks of 81 and 400 steps, respectively, so that the neglect of this group is justified for the elementary calculation. H radicals migrating from the sugar ring at left center of Figure 1 may form cross-links between the adjacent adenine and thymine by two mutually exclusive processes." In part a of process 1, the first H radical reacts in the adenine, whereas, in part b, the second H radical passes through the adenine and reacts in the thymine. In part a of process 2, the first H radical passes through the adenine and reacts in the thymine, whereas, in part b, the second H radical reacts in the adenine. The total probability will be the sum of the probabilities for these two mutually exclusive processes. Table I lists the reactive sites, and the probabilities for reaching each site by a random walk, as outlined above. This is followed by the detailed calculations for the two processes. In part a of the first process, the sum of the probabilities has been set equal to unity, and k was found to be 1.94, so that the individual probabilities are as listed. The probability for reaction in A is 0.608, made up of 0.486 on C, and 0.122 on CS,so that 0.608 is the probability for part a of process 1. Part b of process 1 must be divided into two cases: (10) G. Scholes, P r o g r . Biophys. Mol. Biol., 13, 59 (1963). In Scholes' article the reactive bond for radicals in 5 pyrimidine is referred to as the 4,5 double bond. I n the present article this bond is referred t o as the 5,6 double bond, in accordance with most recent references. The pyrimidine ring is symmetrical about a 2-5 line, so that a dual numbering system is possible. (11) The author is indebted to a referee for pointing out this appronch t o the calculation.
INDIRECT INACTIVATION OF DEOXYRIBONUCLEIC ACID
1713
Table I: Chance of H Radical Reaction with A and T Reactive Sites and Probabilities for A + T CSSe
-Adenine-----.
-Oxygen-
Reaction sites Probabilities Process 1, Part a
1 k/9 0.216
2 k/16 0.122
V T h y m i n -
k
fraotion
C6 k/81 0.024
1.94
0.047
3.79
0.80
0.027 0.062 bl b = (0.80 X 0.106) (0.20 X 0.062) 0.097 Probability of process 1: 0.608 X 0.097 = 0.059 0.216 0.122 0.486 0.122 0.030 0.024 0.054 0.222 0.125 0.500 0.125 0 0.025 0.625 0 0.221 0.124 0.498 0.124 0.031 0.622 be = (0.56 X 0.625) (0.44X 0.622) = 0.623 bi Probability of process 2: 0.623 X 0.054 = 0.034 Total cross-links for both processes: 0.059 0.034 = 0.093
2.22
0.20
c 4
k/4 0.486
C6 k/16 0.122
k/M 0.030
0.236
0.059
c 6
0.608 Case b1
0.421
0
0.237
0.106 Case br
0.246
0.138
0.554
+
Process 2, Part a Case bl Case b2
0.035
0
+
+
i=
1.94 2.00
0.56
1.99
0.44
+
+
Reactive Sites and Probabilities for T -Oxygen-
Reaction sites Probabilities
1 k/9
+
-Thymh-
A
-Adenin-
2 C6 co c4 k/16 k/25 k/4 k/49 Probability of process 1 0.098 Probability of process 2 0.056
CS k/36
0.154 Average for sources in both strands: 0.5(0.093
case bl where the first H radical has reacted with the C4 of A so that a new radical exists on CS, and case bz where the first H radical has reacted with Ca of A and the new radical exists on Cq. Case bl will appear in 0.486/0.608 or 0.80 of the process 1 reactions, and case bz, in 0.20. In case bl the probability of reaction on C4 of A is zero, so that setting the sum of the other probabilities equal to unity gives a k of 3.79 and the values listed in the b1 line of the table. The situation is similar for bz. From the probabilities listed for case bl it will be seen that the chance for its occurrence is 0.80 (0.059 0.047), or 0.80(0.106), whereas the corresponding value for case bz is 0.20(0.062). The probability for b is the sum of these, or 0.097, and the probability for process 1 is the product of the probabilities for parts a and b, 0.608 X 0.097, or 0.059. The calculation for process 2 is similar, leading to a probability of 0.034. Hence, the chance of forming a cross-link by both mutually exclusive processes is 0.059 0.034, or 0.093. H radicals will also be produced in the sugar ring at
+
+
+ 0.154) = 0.123
right center of Figure 1 and will migrate through T and A, in that order. The distances to the reactive sites in T and A are now different from before, and the new reaction probabilities are listed in the lower part of Table I. Carrying out the calculation in the same way shows that the probability for process 1 is 0.098, and for process 2,0.056, giving a total of 0.154. Since there is an equal chance that H radicals will originate in the sugar ring in either strand, the total probability for cross-linking the AT pair of bases by a single pair of H radicals is 0.5(0.093 0.154), or 0.123. Within the assumptions of our calculation, this is also the probability that a single pair of H radicals will form a cross-link between a cytosine-guanine pair of bases, so that no allowance need be made for the difference in abundance of the different base pairs. The end result of the calculation therefore is that a radiation dose suffcient to produce on the average a single pair of H radicals in each DNA molecule will inactivate 0.12 of the molecules. The estimation of this probability, together with the
+
Volume 71, Number 8 May 1967
KENNETHH. KINGDON
1714
experimental value of C for DNA, makes possible a direct calculation of D3, for DNA. A dose of 100 ev/ molecule produces experimentally on the average 1 pair of H radicals/molecule.7 This dose is equivalent to 9.60 X 10”/M rads, where M is the molecular weight. With this dose we have estimated above that 0.88 of the molecules survive, so that the exponential survival law gives D37 =
- (9.60 X 101l/M)/ln 0.88 = 7.5 X 1012/M rads
The values of 0 2 7 X M listed by Kaplan and Moses12 for two-strand DNA viruses range from 4.6 X 10la to 6.9 X with an average of 5.8 X 10l2. Our calculation agrees satisfactorily.
V. Inactivation of Diploid Cell DNA I n 1961, Terei13pointed out that, per in vivo molecule of DNA inactivated, the amount of radiation for a diploid cell was over 100 times that required for a twin helix DNA virus of equal molecular weight. This is due in part to the duplication of genes in the diploid cell, but some additional factor seems necessary to account for the great difference. Resides this quantitative difference, there is the qualitative difference that the plot of log (surviving fraction) against dose is sigmoidal for the diploid cell, but linear for the DNA virus. We shall try to explain both of these differences in terms of indirect inactivation by radicals. Langridge, Marvin, Seeds, Wilson, Hooper, Wilkins, and Hamilton14 have pointed out that water molecules may be hydrogen bonded in a single nucleotide of DNA, or between neighboring nucleotides, to establish the secondary structure of a DNA crystal, as studied by X-ray diffraction. We assume that the arrangement of the folded DKA in a diploid cell is similar to that of DNA in the crystal and that water molecules will form hydrogen bonds between neighboring nucleotides. A second assumption is that the diploid cell DNA contains much more water than virus DNA. A third assumption is that in the diploid cell the radiation produces H radicals principally from the sugars, the adsorbed water being so tenuously distributed that the processes which give rise to H and OH radicals in bulk water are much less efficient here. It is planned to discuss the evidence for this in a subsequent publication. Under these assumptions it is clear that the additional water-hydrogen bonds of a diploid cell give the H radicals generated in the sugars many more migration paths than are available in dry DNA. Therefore the chance that a single pair of H radicals shall cross-link an adjacent pair of bases is greatly reduced in the diploid cell, thus accounting in part for its much lower sensitivity per molecule to radiation. MigraThe Journal of Physical Chemistry
tion of H radicals in the bases of virus DNA is two dimensional, whereas in diploid cell DNA the migration is three dimensional, leading to much wider dispersion of a pair of H radicals. It has been suggested often that a likely effect of radiation on DNA is to break hydrogen bonds. Accordingly, it is likely that an initial effect of radiation on diploid cell DNA is to break some of the water H bonds which determine the secondary structure of the DNA. This will lead to local separations of one twin Watson-Crick helix from another, thus converting a small region of three-dimensional migration into two small regions of two-dimensional migration in isolated twin helix DNA, which will be more sensitive to radiation than the three-dimensional region. We assume that inactivation of diploid cells is due entirely to cross-linking of bases by radiation in the isolated twin helix DNA produced by the radiation. The individual strands of a twin helix are not separated, and the isolated twin helices will recombine to the threedimensional structure whenever conditions become favorable. The isolated lengths of twin helix may be thought of as the products of a photochemical dissociation, and the chance of their recombining will be proportional to the square of their concentration. It is not necessary that an isolated length of twin helix should recombine with its original partner. The formation of water H bonds with any other piece of isolated twin helix will give the seeondary structure which protects from radiation and will leave the biological function of the twin helix unimpaired. Let M30 be the initial amount of three-dimensional DNA, M3, and let Mz be the fraction of this converted to two-dimensional DNA after the incidence of D units of radiation, at intensity I = dD/dt. We assume that M z is always small, so that the radiation is always acting on an essentially constant amount of M3. Then, for an additional amount of radiation, dD
dMz
= ciM3odD
dM2 = CiMsodD
- ~2M2~dt
- c2MZ2dD/I
(1)
dMz a =CIM~O -c~M~~/I Integrating
(12) H.S.Kaplan and L. E. Moses, Science, 145,21 (1964). (13) M. Tersi, Nature, 191, 461 (1961). (14) R.Langridge, D.A. Marvin, W. E. Seeds, H. R.Wilson, C. W . Hooper, M. H. F. Wilkins, and I,. D. Hamilton, J. Mol. Biol., 2, 38 (1964); see p 64.
INDIRECT INACTIVATION OF DEOXYRIBONUCLEIC ACID
Since M 2 is assumed zero when D is zero, the integration constant is zero. Solving for Mz and writing K for 1/c1Mac2/I
M2
=
tanh ( K D ) dc2/
(Idfa)
1715
I :o ,
(2)
This gives the production of Mz in a population of diploid cells as a function of total dose D . Even though most of the population becomes inactivated biologically, the production of M z continues at approximately this rate, since 'the inactivated molecules will continue to produce approximately the normal amount of Mz. If a population Ma of diploid cells has been subjected to a dose D , the number inactivated by a further dose dD is dMa = -caM,MzdD Substituting for M z from eq (2) and integrating In Ma = -!!!In cosh ( K D ) c2
+ constant
Since M3 = M30 when D is zero, the constant is In Mae, 80 that
M3
In - = -@ In cosh ( K D ) MU
c2
Since In (M3/M30) is -1 when D is D31, c31/cz is l/ln cosh (KD3.I) and l n -M3 = M30
I
I
2
5
I
IO 20 COSH D/D37
50
Figure 2. Irradiation plots to test eq 3.
- In cosh ( K D )
In cosh (KDs7)
Expressing D in units of (KD37) and inserting the value of In cosh (1) give finally
M3
I
In - = -2.30 In cosh (D/D37) A130
(3)
Thus a plot on log-log paper of surviving fraction as a function of cosh (D/D37) should be linear, with a slope of -2.3. Three examples of inactivation plots, selected at random from the literature, are shown in Figure 2: for hamster cells in tissue culture, from Elkind and Sutton;16 for hela cells in tissue culture, from Puck and Marcus;16 and for normal mouse bone marrow cells, from Till and ~lcCul10ugh.~~ These plots were constructed by measuring the points on published figures. The plots are approximately linear and have the slopes: mouse, -2.0; hamster, -2.1; and hela, -3.5. Two of these slopes are near the calculated -2.3. In the hela cell measurements it appears that there was a strong photoelectric component of backscatter from the glass slides on which the cells were
mounted, so that there might have been a mixture of radiations with different LET. This is possibly the reason for the slope being too large. In order to get more information on the slope, two more readily available plots of data were analyzed: diploid Saccharomyces cerevisiae (X 320 yeast), from Rlortimer;lE and chicken embryo wing bud cells irradiated in ovo, from Philpott, Shaeffer, and T01rnach.l~ Log-log plots of surviving fraction as a function of cosh ( D / D 3 7 ) had slopes of -2.1 and -2.6, respectively. It is concluded from these examples that the normal slope for such plots is about -2.3, in agreement with our eq 3. The two plots last mentioned intersect the axis of ordinates very close to unity, but in Figure 2 there is (15) M. M. Elkind and H. Sutton, Nature. 184, 1293 (1959). (16) T. T. Puck and P. I. Marcus, J. Ezptl. Med., 103, 653 (1956). (17) J. E. Till and E. A. MoCullough, Radiation Res., 14, 213 (1961). (18) R. K. Mortimer, ibid., 9,312 (1958). (19) B. W.Philpott, C. W. Shaeffer, and L. J. Tolmach, ibid., 17, 508 (1962).
Volume 71, Number 6
M a y 1907
KENNETH H. KINGDON
1716
no doubt that the main part of each line intersects the axis of ordinates below 1.0. The intercepts are: hela, 0.78; mouse, 0.58; and hamster, 0.90. The initial portions of the plots are shown on a larger scale at the upper right of Figure 2, and in the enlarged figure nonlinear curves have been fitted to the upper parts of the hela and hamster plots to match the data points as closely as possible. The reason for an initial high sensitivity is thought to be as follows. It has been recognized for many years that “the sensitivity of cells to irradiation is in direct proportion to their reproductive activity.” Since reproductive activity involves the production of new DNA, which presumably is initially not protected with the threedimensional secondary structure due to water H bonds, the high sensitivity of reproducing cells is entirely in line with the ideas discussed above. Moreover, it is likely that the majority of cells will have some isolated DNA helix which has not yet entered the protective secondary structure or which has been withdrawn from that structure for replication purposes. Accordingly, it seems reasonable that the more rapid inactivation shown in the plots of Figure 2 is due to the rapid destruction of isolated DNA helix already in the cell at the start of the irradiation. Elkind and S ~ t t o nstudied ’~ the recovery of irradiated hamster cells as a function of the resting time between two irradiations. Following the ideas advanced above, the rate of recovery, as shown in their Figure 1, would measure the rate a t which Mz recombined to Ma, according to our eq 1. The measured recovery proceeds initially exponentially as a function of time, with a time constant of about 50 min for the amount of M z to fall to l/e. This recombination would therefore have little effect on the fraction surviving, since the largest dose of about 1200 rads was given in less than 2 min. This is in accordance with the usual experience that the fraction surviving does not depend critically on the rate at which the dose is given. One artifact may be noted in connection with survival plots. For diploid cells, plots of log (surviving fraction) as a function of De’/’are quite linear. It is thought
The Journal of Physical Chemistry
that this is due to the fact that log cosh (D/D37)in/ ~ a range of creases proportionally to ( D / D ~ , ) ’over D/D37 from 0.8 to 4.0, which covers the range of dose used in many investigations.
VI. Discussion I n conclusion, since this paper presents some novel ideas about the radiation inactivation of DNA, it may be well to summarize the experimental facts on which the ideas are based and the scope of the understanding to which these ideas lead. The main experimental facts used are as follows. That DNA molecules can be inactivated by some form of cross-linking is very old. Inactivation by cross-linking of bases is discussed by Marmur and Grossman,’ and the “reversible DNA” of Geiduschek2 makes use of the same fact. The great radiation stability of bases in nucleosides and the plentiful generation of H radicals from sugars in nucleosides are based on the mass spectra of Biemann and McCloskey.6 The migration of atoms and molecular fragments in molecules is demonstrated by all mass spectrometry6 and by Tikhomirova, et al.,* in bulk polyethylene. The G value for DNA was given by Lett, et al.’ The principal reaction sitesfor H radicals in the bases are discussed by Scholes’o from the aqueous solution chemistry point of view. X-Ray crystallographic data on the presence of water in DNA are discussed by Langridge, et aZ.14 The major results derived from this experimental basis are as follows: (a) a single mechanism correlating radiation inactivation of virus DNA, inactivation of diploid cell DNA, and restoration effects after irradiation of diploid cells; (b) a direct calculation of D37 for virus DNA, in fair agreement with experiment; (c) a direct calculation of the shape and slope of the inactivation curve for diploid cells, in agreement with experiment.
Acknowledgments. The author is grateful to Dr. Hillel Poritsky for help with the integrations of section V. His thanks are also due to a referee for detailed criticism of the calculations of section IV, which led to the improved treatment given here.
