The Photochemistry, Photobiology, and Repair of Polynucleotides1

The Photochemistry. Photobiology. and Repair of Polynucleotides' R . B. SETLOW I Biology

I

Diuisima.

Oak Ridge National Laboratory. Oak Ridge. Tennessee

I . Introduction . . . . . . . . . . I1 Photoproducts in Polynucleotides . . . . . A General Comments . . . . . . . B. Pyrimidine Dimers . . . . . . . . C . Experiments at Two Wavelengths . . . . D Experiments at One Wavelength . . . . . I11. The Action of Enzymes on Irradiated Polynucleotides . A. Degradation by Nucleases . . . . . . B. Polymerases . . . . . . . . . C Photoreactivating Enzyme . . . . . . D Nucleases Specific for Ultraviolet-Irradiated DNA . IV . The Biological Activity of Ultraviolet-Irradiated DNA . A Short Wavelength Reversal . . . . . . B. Proflavine Reversal . . . . . . . C . Photoreactivation . . . . . . . . D . Dark Repair Enzymes . . . . . . . V Cells and Viruses . . . . . . . . . A Photoreactivation . . . . . . . . B Repair in the Dark . . . . . . . . C . Effects of Ultraviolet on DNA Synthesis . . . D Excision . . . . . . . . . . VI . Steps in the Repair of DNA . . . . . . A. Excision . . . . . . . . . . B Breakdown of DNA . . . . . . . C Resynthesis and Repair Replication . . . . D Rejoining of Strands . . . . . . . VII . Conclusion . . . . . . . . . . References . . . . . . . . . . Note Added in Proof . . . . . . . .

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257 259 259 261 262 265 266 266 267 269 271 272 272 273 273 274 275 275 277 278 280 284 284 286 287 288 289 290 294

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1 Introduction It has been known for a number of years that the responses of biological systems to ultraviolet radiation can be altered by various treatments that follow the irradiation . For example. the survival (the ability to form a colony) of bacteria after irradiation depends upon the medium

' Research sponsored by the U S . Atomic Energy Commission under contract with the Union Carbide Corporation . 257

258

R. B. SETLOW

upon which they are plated, the illumination in the laboratory, and the incubation temperature. The description we give to such observations depends upon the choice of a standard state. For example, if we look upon the most sensitive condition as the normal one, enhanced survival is described as a recovery phenomenon, whereas if the most resistant situation is looked upon as normal, the more sensitive would be described as arising from a sensitization phenomenon. The two points omf view are not really separable. The key to unraveling these difficulties of points of view has been the recognition that the most important structure for cellular proliferation is DNA. Not only is DNA the most important macromolecule in the metabolic hierarchy of cellular growth, but it is a molecule that is relatively easy to study by physical and chemical techniques. The evidence that DNA, at least in simple systems, is the most important target for ultraviolet irradiation has been reviewed by many authors ( 1 4 ) . Thus we may look for answers to our questions concerning recovery or sensitization in investigations concerning the sensitization or repair of DNA, although even in some simple systems [e.g., T-even bacteriophages ( 5 ) ] effects on DNA do not account for all inactivation events. While it is important carefully to examine the evidence for the repair of radiation damage at the level of DNA molecules (6, 7 ) and outline what is known of the presumed steps in repair processes, it is perhaps just as important to indicate what is not evidence for repair of DNA ( 8 , 9 ) . Before analyzing the repair of DNA, it is necessary to know something about the physical changes or lesions in DNA that have biological consequences. We must know what is being repaired. This essay concentrates on one particular chemical change in DNA-the formation of dimers (10, 21) between adjacent pyrimidine residues by the creation of a cyclobutane ring joining t w o sets of 5,6 double bonds. The properties of such dimers have been reviewed elsewhere ( 1 2 ) . There are of course other types of lesions in DNA produced by radiations ( 2 , 3, 13, 14) and by chemical treatment, and there are many lines of evidence that such lesions also may be repaired or ignored ( 9 ) . However, pyrimidine dimers really represent the cornerstone in the argument about repair, not only, as we shall see, as a lesion to repair but also because they represent the best documented case of a change in DNA that is an induced lesion ( 1 5 ) . They are produced in large numbers by the irradiation of DNA with uItraviolet light. They are easy to detect in small amounts. They are stable to acid and enzymatic hydrolysis and they are not reincorporated into DNA once removed from the polymer. Since they are easy to observe, it is a relatively simple matter to trace their distribution through cellular or subcellular fractions. Such an analysis would be very difficuIt for a

PHOTOBIOLOGY AND REPAIR OF DNA

259

chemically altered base in DNA, such as a deaminated cytosine. The resulting uracil would not pair properly, but if it were removed from the DNA by some type of repair mechanism, it could be reincorporated into RNA or DNA, and so its movement-for example, from the acidinsoluble to the acid-soluble fraction of cells-could not easily be followed. Pyrimidine dimers are not the only photoproducts in DNA, and they do not account for all the effects of ultraviolet irradiation on DNA (14). Nevertheless, they have been extensively studied and constitute the physicochemical change that has been best correlated with biological effects. Thus, to build a more or less logical foundation and story concerning the repair of DNA, I shall overstate the case for pyrimidine dimers and consider, in order, the photochemistry of such dimers, their photobiology, and the repair of DNA containing them.

II. Photoproducts in Polynucleotides

A. General Comments There are numerous reviews on the photochemistry of model polydeoxyribonucleotides and polynucleotides and DNA (2, 11-13). It is a complex subject because many of the photoproclucts involve adjacent pairs of bases rather than individual components of the polymer. Thus the photochemical reactions of polynucleotides do not equal the sum of the reactions of the individual bases at infinite dilution. Moreover, the photochemical interactions between pairs of bases seem to depend upon neighboring bases or photoproducts. Thus the photochemistry of adjacent pairs of bases is not the sum of the effects on model dinucleotides. In other words, the whole does not equal the sum of its parts. The photochemistry of polynucleotides depends not only on the composition but also the conformation of the polymer. For example: ( a ) the rate of hydration (16) of cytidine in DNA or of uridine in poly U is suppressed in organized polymers such as native DNA as compared to denatured DNA ( 1 7 ) or in poly U-poly A as compared to pure poly U (18);( b ) the rate of formation of dimers between adjacent pyrimidine residues in DNA depends on the nature of the surrounding bases (1 9), and the rate of dimer formation in poly U depends on its past history (20) (for example, the formation of a dimer increases the probability that a dimer will be formed next to the original one); ( c ) dimer formation is suppressed in bacterial spores ( 2 1 , 22), in dry DNA (22, 23), and in DNA in frozen solutions as compared to DNA in liquid solutions (24-26); ( d ) dimer formation is suppressed by the binding of intercalating dyes to DNA (27, 28).

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R. B. SETLOW

The rate of formation of photoproducts in DNA as a function of the incident radiation flux is wavelength dependent ( 1 2 , 29, 3 0 ) . As a general rule, at any particular wavelength, increasing doses produce increasing amounts of photoproducts. But since the destruction or loss of individual bases or DNA components may result from the production of many different types of photoproducts, it is very difficult to render a correlation between the formation of a particular photochemical product and a biochemical or biological effect, This is especially true since most of the products (even though they have not been structurally identified and may be produced in differing amounts) have similar functional dependencies on incident radiation dose. The finding, for example, that the template activity of DNA in the DNA-dependent RNA polymerase reaction is reduced by ultraviolet irradiation (31, 32) and that such radiation makes pyrimidine dimers is obviously not proof that such dimers affect the template activity even though it may be a reasonable conclusion from the known effects of dimers on enzymatic reactions. Under certain experimental conditions (Sections 11, C, D and 111, C ) cyclobutane dimers may be monomerized by irradiation. Thus they are an exception to the generalization “as the dose increases, the products increase,” and this exceptional behavior permits one to implicate them as affecting biochemical and biological processes. The formation of some types of photochemical products in DNA cannot be of large biological importance because their numbers are too small. For example, if in a system inactivated with a mean lethal dose, one finds much less than one product per molecule, one can safely assume that this type of product is not of major importance in inactivation ( 2 , 1 5) . Thus crosslinks or chain breaks in transforming DNA or in viruses or sensitive bacteria cannot account for the lethal action. However, resistant bacteria (bacteria that can survive high doses of radiation) and mammalian cells with much DNA could be inactivated by such photoproducts. The formation of hydrates, in particular cytidine hydrate, has been looked upon as an attractive mutagenic possibility, but there is no direct evidence that it is formed in native DNA ( 1 7 ) . It could, however, be formed in a single-stranded region corresponding to the replicating region of DNA. This latter product (hydrate) illustrates another difficulty in detecting photoproducts, namely, it is not stable to most of the common hydrolysis procedures. The fact that cyclobutane dimers are observed in acid hydrolysates of polynucleotides does not prove that they existed in the polymer. They might result from some secondary reactions of unknown products during acid hydrolysis. Wang et al. (33) describe experiments that support the latter point of view. Carrier and I ( 3 4 ) and Wang et al. ( 3 5 ) have not

261

PHOTOBIOLOGY AND IWPAIR OF DNA

been able to repeat these experiments. Techniques that do not use acid hydrolysis, such as ultraviolet absorbance (17, 36, 3 7 ) , enzymatic hydrolysis (19, 3 8 ) , and photochemical properties (see Section 11, B ) , support the notion that dimers exist in polynucleotides. A further complication in attempting to correlate photoproducts with biological effects is that the high doses usually used to detect photochemical products are beyond the biological range and secondary reactions may have occurred; for example, the production of uracil from uracil dimers derived by the heat deamination of the primary product, cytosine dimers (39, 4 0 ) .

B. Pyrimidine Dimers The formation of cyclobutane pyrimidine dimers is a photochemically reversible reaction as indicated in Eq. 1. -PyPy-

ki

-

$ -PyPy-

kr

Such reversibility is not observed for crosslinks, DNA-protein links, or hydrate formation. The forward and reverse constants are dependent on environmental conditions and wavelength ( 1 2 ) . Thus, by proper choice of experimental conditions, it is possible to form dimers and subsequently to monomerize them by a different set of conditions and observe whether a similar reversibility obtains in biochemical and biological phenomena. If the photobiological reversibility is similar to the photochemical reversibility, this result may be taken as evidence for the involvement of pyrimidine dimers in the biological effect. The details of the photochemistry of pyrimidine dimers, first discovered in the form of thymine dimers as a radiation product of thymine in has been elucidated by numerous studies frozen aqueous solution ( on model polynucleotides and oligonucleotides (18, 20, 36, 37, 4 - 4 6 ) . The observation of reversible photochemical reactions similar to those shown in Eq. 1 are taken as evidence for the existence of cyclobutanetype pyrimidine dimers in the polymer, although it is only in one case ( thymine-thymine dimer from irradiated DNA) that the structure has been uniquely identified (47, 4 8 ) . The photochemical reactions of model dinucleotides are an interesting subject in their own right ( 4 9 ) , although we shall not discuss them further except to remark that in such model * I n aqueous solution, thymine molecules are too far apart to react readily with each other. As a result, few dimers are formed unless a sensitizer is present ( 4 1 - 4 3 ) or they are held close together, as in frozen solutions or in a polynucleotide. In polynucleotides in frozen solution, the average rate of dimer formation is much less than in aqueous solutions, because the bases presumably are not all in the proper position to react to form a dimer (24, 26).

R. B. SETLOW

262

\

SUGAR

FIG. 1. A schematic diagram of the presumed structure of a thymine dimer in a polynucleotide. [The correct full name of the classical thymine dimer, as given in Chemical Abstracts, is hexahydro-4a,4b-dimethylcyclobuta[1,Zd;4,3-d']dipyrimidine2,4,5,7( 3H,GH)tetrone. (Eds.)]

compounds several stereoisomers of dimers are possible ( 50) , whereas in native DNA only one has been observed-the one indicated in Fig. 1.

C. Experiments at Two Wavelengths The wavelength dependence for the forward and back reactions of Eq. 1 differ because the absorption spectra of the individual pyrimidines and their saturated derivates differ greatly. Figure 2 shows some of these spectra. The formation of a dimer involves the saturation of the 5,6 double bond and hence results in an absorbance decrease at long wavelengths. Dimers exhibit high absorbance only at shorter wavelengths. The quantum yield for monomerizing dimers is approximately one (36, 37, Sl), although it seems to decrease somewhat at longer wavelengths. Thus short wavelengths are very effective in splitting dimers. On the other hand, long wavelengths, because of the small absorption coefficient of dimers, are ineffective in monomerization. The quantum yield for dimer formation in a polynucleotide is only approximately 0.01 (29, 36, 37). However, the high absorption coefficient of the pyrimidines at long wavelengths favors dimer formation. As a result, irradiation by long

263

PHOTOBIOLOGY AND REPAIR OF DNA

12 j4

1

1

R

b 10 X

P

!z

> 8 -

c

a

86

m

m

a

5 4 0

E

2

0

1

210

\

\,THYMINE \,plER

--

230

250

I

270

290

2 o ;

2Ao

2;io

2bo

360

FIG.2. The absorption spectra of thymidine, thymine dimer, cytidine, and dihydrocytidine. A cytosine dimer in a polynucleotide probably has a molecular absorptivity twice that of dihydrocytidine (12, 16).

wavelengths of a polynucleotide with adjacent thymines would preferentially form dimers, and subsequent irradiation with short wavelengths would monomerize most but not all of them as indicated in Eq. 2. 1.O-TT-

long

x 0.2-TT-

+ -

0.8-TT-

short

0.9-TT-

+ _ .

0.1-TT-

It is important to emphasize that all incident wavelengths will make some dimers if one irradiates a polymer containing no dimers. Thus at any wavelength there will exist a steady-state distribution of dimers. At long wavelengths, the distribution favors dimers; at short wavelengths it favors free thymine residues. As a result, it is possible to change the steady-state number of dimers by irradiating with different wavelengths3 This is illustrated in Fig. 3, which shows the formation of dimers by two wavelengths, one long and one short. Figure 3A shows the formation of dimers in a polynucleotide irradiated either with a long wavelength or 'In such a steady-state individual, dimers are constantly made and broken, although the total number of dimers remains constant unless other photoproducts are made that remove the pyrimidines from the reaction. The latter situation obtains in poly U, where hydrates compete with dimers (20, 4 5 ) .

264

R. B. SETLOW

with a short wavelength. Figure 3B shows how the effects of a large dose of a long wavelength may be partially reversed by a short wavelength. Figure 3C emphasizes this latter point by indicating that no reversal is observed unless the initial dose of long wavelengths is large enough to make more dimers than the equilibrium level at the short wavelength

FIG. 3. The formation of thymine-containing dimers in Escherichia coli DNA by two different wavelengths. ( A ) A long and a short wavelength separately. ( B ) A large dose of a long wavelength followed by short wavelength irradiation. ( C ) A small dose of a long wavelength [note the 10-fold scale difference from ( A ) and (B)]followed by a short wavelength compared to the short wavelength alone. From data of WulfF (29).

(52). The mean lethal doses used for the inactivation of most DNAcontaining bacteriophages are indicated by the almost imperceptible solid bar near zero dose in Fig. 3C. Obviously the reversal trick cannot be used on such systems since the doses necessary to do it would leave no survivors. The number of dimers indicated in Fig. 3 may be measured by use of chromatographic techniques after acid (11, 29) or enzymatic hydrolysis (19, 53) of the polymer and by the changes in ultraviolet absorbance in the irradiated polymer (17). These three different methods give approximately the same numbers of dimers, a result indicating that such structures are not formed by the analytical procedure and that they are stable in DNA (some of the other isometric dimer forms observed in model dinucleotides are not stable to acid hydrolysis) (50). Cytosine-containing cyclobutane dimers are qualitatively similar to thymine dimers. However, as can be seen from the spectra in Fig. 2, the absorption coefficient of the cytosine-containing dimer is large. As a result, the rate of the back reaction is much larger than that for thyminethymine dimers and the steady state for cytosine-containing dimers is

265

PHOTOBIOLOGY AND REPAIR OF DNA

shifted in the direction of the free pyrimidines. This quantitative difference between cytosine and thymine allows one, by a special trick outlined below, to show that both types of dimers cause biological damage. The saturated rings formed in a cytosine-containing dimer render the amino group of cytosine labile to hydrolysis, and such dimers are deaminated to uracil-containing dimers as indicated by both photochemical and chromatographic analyses of polymers containing only cytosine (.as, 54).

D. Experiments at One Wavelength The photochemical reversal of pyrimidine dimer formation may be effected at a single wavelength by changing the polymer configuration. The dye proflavine, when bound to DNA, changes the dimensions of the polymer and inhibits the formation of dimers but not their destruction (27, 28). Thus the steady-state distribution at high doses in the presence of proflavine is shifted far to the side of the monomers compared to the distribution in the absence of proflavine. Thus the results in Fig. 3B may also be achieved by an initial irradiation at any wavelength in the absence of proflavine followed by continued irradiation in its presence. Dimers are monomerized according to the scheme shown in Eq. 3. -PyPy-

A

-

' -PyPy-

-

A

proflavine

(3)

-PyPy-

Since dimers containing cytosine are monomerized much more effectively than those of the thymine-thymine type, it is possible, at a wavelength such as 280 mp, selectively to split cytosine-containing dimers and to show that such dimers are biologically important ( 5 5 ) . - The formation of the different types of pyrimidine dimers (CC, CT in DNA has not been extensively studied. Table I gives the and

m)

TABLE I CYCLOBUTANE-TYPE PYRIMIDINE DIMERS I N ULTRAVIOLE'PIRRADIATED DNA's (40)

DNA Hemophilus influenzae Escherichia coli Micrococcus lysodeikticus

A

+T

(%I 62

50 30

Wavelength (mp)

265 280 265 280 265 280

Flux (ergs/mm2) 2 4 2 4 2 4

x x x x x

x

103 10" 103 104 103 104

Dimers per nucleotide ( X lo2)

Percentage in

CC

CT

TT

0.27 2.06 0.20 1.34 0.14 0.69

5 3 7 6 26 23

24 19 34 26 55 50

71 78 59 68 19 27

266

R. B. SETLOW

numbers of dimers formed at two radiation doses: a relatively small one ( approximately 10 times that used for the inactivation of bacteriophages), and a large one in the range often used for the determination of photoproducts and photochemical properties. The different dimers are formed with digerent efficiencies and at rates that are proportional to the average nearest-neighbor frequency of the DNA, even though the probability of forming a particular dimer seems to depend on the neighboring bases (19).

111. The Action of Enzymes on Irradiated Polynucleotides A. Degradation by Nucleases Irradiated native DNA, subsequently denatured by heat, is resistant to exonucleases specific for denatured DNA (19, 5 6 ) . Part of the resistance arises because individual strands have become crosslinked and hence the molecule cannot be dissociated by heat, and part of the resistance arises because of the changes in the individual strands of the polymer, Thus the kinetic analysis of the degradation of this material is complicated because of the two sources of its nuclease resistance. Obviously any conclusions as to molecular mechanisms in such a system are di5cult. It is somewhat simpler to analyze the degradation of DNA irradiated in the denatured state. Exonucleases act more slowly on such an irradiated polymer and the limit digest of such an irradiated polynucleotide after treatment with DNase I and venom phosphodiesterase consists of mononucleotides plus enzyme-resistant sequences (19). The majority ofthese sequences are trinucleotides whose structure is of the form pNpTpT. Such trinucleotides are completely resistant to either venom or spleen diesterase. If, however, the cyclobutane ring is broken by short-wavelength irradiation, the trinucleotide is digestible. The distribution of the four bases among such trinucleotides does not seem to be random; relatively more are formed in which N is A or T. Since the formation of a dimer takes place with a low quantum yield, it is reasonable to suppose that small changes in local configuration will affect the rate of formation drastically, and therefore this nonrandomness is best attributed to an influence of the surrounding bases of the structure rather than to an indication of a nonrandom distribution of bases in sequences of this type. Similar nuclease-resistant sequences are observed in irradiated native DNA, but other unidentified sequences are also observed, although they do seem to be trinucleotides as judged by chromatographic mobility ( 19). The nuclease-resistant sequences containing dimers are not observed in such large numbers if the irradiated native DNA is subsequently exposed to the action of the photoreactivating

267

PHOTOBIOLOGY AND REPAIR OF DNA

enzyme plus light of wavelength 365 mp (19). Such treatment monomerizes dimers and this accounts for the increased hydrolysis of irradiated DNA by exonucleases after photoreactivating treatment. Irradiated ribopolynucleotides also contain sequences resistant to nuclease degradation. For example, transfer RNA after UV irradiation yields longer sequences in subsequent ribonuclease digestion than are found in digests of unirradiated transfer RNA (57), and the limit digest ( 5 3 ) contains sequences of the form UpUpUp and of irradiated poly U UpUpUp (hydrate). Pearson and Johns ( 18) observed that such resistant sequences included clusters of dimers with a frequency much greater than predicted from random dimer formation. The probability of forming a second dimer next to the first was greater than expected, a result indicating either some type of energy transfer mechanism or that the first dimer alters the structure of the polynucleotide enough so that the quantum yield for the formation of a subsequent dimer in the neighborhood of the first is increased. The ribonuclease digestion of irradiated poly U has been described as a process of excision akin to that observed in vivo for resistant bacteria ( 5 3 ) . Obviously it is not an excision process since the appearance of nuclease-resistant sequences is observed only in the limit digest and results from the degradation of all the polymer except the dimer sequences, which remain as undigested pieces, whereas the excision process represents the specific removal of dimers without the attendant removal of large numbers of mononucleotides. The excision process is quite the opposite of the formation of nuclease-resistant sequences.

B. Polymerases Ultraviolet irradiation of DNA inhibits its priming activity in both the RNA (31, 3 2 ) and DNA polymerase ( 5 8 ) systems of Escherichia coli, but it has not been demonstrated that such inhibition arises from the production of pyrimidine dimers. The calf thymus DNA polymerase utilizes denatured DNA as a template ( 5 9 ) , and irradiation of denatured DNA inhibits the polymerization reaction. The inhibition produced by long wavelengths may be partially reversed by subsequent short wavelength irradiation (60). This is direct evidence that pyrimidine dimers inhibit the polymerization process in this enzymatic system. The greatest inhibition was observed in DNA of high A T content, a result to be expected because thymine dimers are formed with higher probability than dimers containing cytosine. The product formed by the calf thymus polymerase with an irradiated primer contains relatively more guanine than adenine and, in this sense it represents a mutant product although there is no evidence that such a reaction occurs in uiuo. Further evidence

+

268

R. B. SETLOW

that pyrimidine dimers inhibit the rate of synthesis as well as giving rise to aberrant products comes from analyses of the nearest-neighbor frequencies (61) in the products formed from irradiated primers. At high doses, the products are deficient in A-A sequences indicating that the principal block is a thymine-thymine dimer (62). The data that indicate that pyrimidine dimers in a DNA template inhibit polymerization catalyzed by the DNA polymerase and result in the synthesis of a noncomplementary product are not subject to a unique interpretation because the enzyme preparations are impure. DNA polymerase preparations may have small amounts of terminal deoxynucleotidy1 transferase activity (an enzyme that adds nucleotides to the ends of existing chains) (59). As irradiation inhibits the polymerase activity and does not seem to affect the so-called addition activity, the impurity takes on added significance when the effects on irradiated primers are analyzed. Thus two models shown in Fig. 4 are qualitatively consistent

B

-----__

Tci

E------*

FIG.4. Models to explain the slow polymerization in the presence of irradiated primer in the calf thymus DNA-polymerase system ( 6 0 ) . In ( A ) the polymerization past a h e r is slow, and probably noncomplementary. In ( B ) polymerization stops at a dimer, but random and addition continues. The experimental observations are consistent with a mixture of the two models.

with the kinetic and analytical data. In model A, a dimer acts to slow down polymerization. On this model, synthesis past the dimer takes about the same length of time as synthesis past 100 normal nucleotides. In this slowly synthesized region, one can imagine that A-A sequences are not incorporated opposite a thymine dimer and hence the product has a “wrong” base composition and nearest-neighbor frequency. In model B, a dimer acts as an absolute block to further polymerization and the incorporation that is observed with highly irradiated primers is accounted for by end addition to the template strand. This newly incorporated material obviously will have sequences more or less independent of the primer. Physical data on the molecular weights of the template and the product are needed to distinguish between the two possibilities, but the kinetic and analytical data fit model A better. The extension of this reasoning to polymerase systems that use double-stranded DNA as templates is not clear.

PHOTOBIOLOGY AND REPAIR OF DNA

269

Ultraviolet irradiation also inhibits the template activity of polyribonucleotides in the RNA-dependent RNA synthesis. Pyrimidine dimers inhibit template activity, and hydration products in these polymers ( a complication of little importance in the DNA-dependent polymerase systems) seem to act as miscoding bases, so that uracil hydrate codes in part for G ( 6 3 ) and cytosine hydrate tends to code for A ( 6 4 ) . The existence of two types of photoproducts, dimers and hydrates, with only one of them, dimers, photochemically reversible introduces complications in the photochemical analysis. For example, at high doses, and at all wavelengths, dimers tend to be converted ultimately to hydrates ( 4 5 , 4 9 ) , and therefore the precise measurement of the relative numbers of all types of photoproducts is difficult without careful and elaborate analysis. Such analyses were not carried out in the original work on the irradiation of RNA polymers and have led to criticisms ( 2 ) concerning the quantitative description of the effects of ultraviolet radiation on polyribonucleotides in terms of dimers and hydrates. The use of such irradiated polymers in amino acid incorporating systems is beyond the scope of this essay. Such studies are complicated because radiation not only can change the template properties of such a polymer, but can also change its binding to ribosomes and the rate of degradation of the polymer by contaminating nucleases (65).

C. Photoreactivating Enzyme In many biological systems, the effects of ultraviolet irradiation are photoreactivable; that is, the effects of ultraviolet are reversed in part by subsequent irradiation with light of wavelength greater than 330 mp (reviews in references 2, 3, 6 6 ) . Photoreactivation as thus defined is obviously different from the two-wavelength process, long wavelength followed by short wavelength, discussed above. It is important because it occurs in biological systems in the low dose range and because a great deal is known about the molecular mechanisms of enzymatic photoreactivation. Thus this process acts as a bridge between the purely photochemical and the purely photobiological events. The biological phenomenon of photoreactivation is complicated by the existence of what has been termed “indirect photoreactivation” ( 6 7 ) , a process whose nature is not completely understood, but which seems to be analogous to the restoring effects of long wavelength irradiation administered before the ultraviolet (cf. Section V, A). There are three aspects of photoreactivation that are important: ( a ) Photoreactivation occurs in &TO. Illumination of ultraviolet-irradiated transforming DNA in the presence of enzyme extracts from various systems results in an increase in the transforming activity. This observa-

270

R. B, SETLOW

tion really represents the definition of enzymatic photoreactivating activity in vitro. ( b ) Photoreactivating enzyme preparations in the presence of long wavelength ultraviolet act by monomerizing pyrimidine dimers. There is no evidence that such enzyme preparations have any other activity ( see below), ( c ) Illumination of ultraviolet-irradiated bacteria with photoreactivating wavelengths results in the destruction of pyrimidine dimers in vivo in cells in which photoreactivating enzyme activity can be demonstrated. It is presumed that the dimer destruction represents the monomerization of dimers. Photoreactivating activity has been isolated from many different materials: e.g., bacteria, yeast, mold (68, 69) sea urchins ( 7 0 ) ,amphibia, fish, and chick embryo ( 7 1 ) .Most of these systems have not been purified or analyzed to the same extent as the enzyme from yeast, The action spectra for in vitro photoreactivation by the yeast ( 72) and E . coli enzymes ( 2 ) are alike and are similar but by no means identical to that for direct photoreactivation of E . coli in vivo (73). The ability of photoreactivating enzyme to act on polynucleotides other than transforming DNA may be assessed by observing the competition for the enzymatic activity between transforming DNA and irradiated polynucleotides ( 74) containing known photoproducts. Polymers that compete with the enzyme are irradiated polynucleotides that contain adjacent pyrimidine residues ( 75). The competing ability of these irradiated polymers may be removed by illumination in the presence of the enzyme. At the photochemical level, Wacker ( 7 6 ) was able to show that a little over 10%of the thymine dimers is destroyed by photoreactivating enzyme from yeast, a demonstration that unfortunately proves little. W u B and Rupert ( 7 7 ) showed that all the thymine-containing dimers in the acid-insoluble fraction of DNA are destroyed by photoreactivating treatment. In these early experiments, it was dBcult to demonstrate that dimers were monomerized because of the difficulty of observing the small increase in the radioactivity of thymine associated with the disappearance of dimers. (If 1%of the thymine radioactivity were in dimers and 99%in thymine, it would be very difficult to observe the absolute increase in thymine activity associated with the Ioss of 1%of the activity in dimers.) The identification of the destruction of dimers with their monomerization has been shown by two independent types of investigation. The first utilized irradiated poly dI-poly dC ( 4 6 ) . In this polymer the cytosine dimers formed by ultraviolet irradiation may be deaminated to uracil dimers by elevated temperature. The subsequent treatment of this polymer with photoreactivating enzyme destroys the uracil dimers and leads to the production of an equivalent amount of uracil, a component very easy to observe against the background of

271

PHOTOBIOLOGY AND REPAIR OF DNA

labeled cytosine in such a polymer. The following scheme indicates how monomerization was observed.

-cccc-

uv

-cEcC,W

Heat

-C K C -

C,W

-PR

cuuc-

c, u

In a very careful experiment using purified yeast photoreactivating enzyme, Cook (78) showed that the radioactivity lost from thyminecontaining dimers is observed in the thymine itself. Thus there is little question that photoreactivating enzyme acts in uitro and presumably in uiuo by the monomerization of pyrimidine dimers. The rates of monomerization of dimers depend on the type of pyrimidine ( 4 0 ) , and such monomerization has only been demonstrated for deoxyribopolynucleotides. The rates of monomerization seem to be greater for native than for denatured DNA (79), and the minimum length of poly T that can act as a substrate for the photoreactivating enzyme seems to be 9 (80). Thus it has not been possible to use very simple models, such as dinucleotides, to investigate the action of this interesting enzyme-an enzyme that to date has been purified over 105-fold (81) .

D. Nucleases Specific for Ultraviolet-Irradiated DNA There is a large body of experimental evidence from in viuo studies indicating that damage produced by ultraviolet irradiation of DNA can be repaired. It is reasonable to suppose that there exist specific enzymes that can act on such DNA, presumably either because they recognize specific lesions such as dimers, or, more probably, because they use as a substrate the denatured region in the neighborhood of a lesion such as a dimer. Extracts have been prepared from Micrococcus Eysodeikticus that have some of the expected specificities. For example, such preparations degrade ultraviolet-irradiated DNA more rapidly than unirradiated DNA (82-85). They make single-strand breaks in the irradiated replicative form of 4x174 (86) and also breaks in other irradiated DNA's (83). The numbers of breaks approximately equal the numbers of dimers, and, as judged by the increased rate of digestion with venom phosphodiesterase, the breaks seem to be on the 5' side of dimers ( 3 4 ) . PNPTpT

Cruder extracts not only insert single-strand breaks, but are able specifically to excise oligonucleotides containing pyrimidine dimers from

272

R. B. SETLOW

irradiated DNA (87). This excision property requires at least two separable components ( 3 4 ) , one of which is the UV-specific endonuclease? The other component could be another nuclease or a combination of polymerase and nuclease activities. ( See Section VI. )

IV. The Biological Activity of Ultraviolet-Irradiated DNA We have seen in the previous section that cyclobutane-type pyrimidine dimers affect many interactions between enzymes and polynucleotides. It is reasonable to suppose that the alteration of the DNA structure caused by dimers will affect the biological activity of DNA. It is difficult to verify this expectation because, as pointed out earlier, increasing doses of irradiation make more of all types of photoproducts. Nevertheless a particular photochemical attribute of pyrimidine dimers-that of monomerization under various experimental conditions-gives a clue as to the type of experiment that can be performed to show that dimers affect biological activity, If the reversal of biological activity has the same wavelength dependence and kinetics as the monomerization of pyrimidine dimers, we may take such reversal as evidence for the importance of pyrimidine dimers in the inactivating event. There are three ways in which this reversal has been accomplished: ( a ) short wavelength reversal, ( b ) irradiation in the presence of pro%avine,and ( c ) enzymatic photoreactivation. The first two can be done only at high doses, as pointed out with reference to the discussion concerning Fig. 3.

A. Short Wavelength Reversal Short wavelength reversal has been observed for transforming DNA (52, 88). DNA inactivated with a large dose at long wavelengths may subsequently be reactivated by irradiation at shorter wavelengths ( Fig. 5). The reactivation is more effective at 239 mp than at 265 mp, and the reactivation depends, as expected, on the order in which the irradiations are given. The kinetics are quantitatively similar to those of dimer splitting. It is clear that short wavelength reversal cannot restore all the biological activity of DNA, for at least two reasons. First, short wavelength does not monomerize all dimers and second, other lesions undoubtedly contribute to the inactivation at high doses. The latter lesions are not reversible by a short wavelength. Quantitative analysis indicates that, at high doses, between 50 and 70%of the biological inactivation may be accounted for by pyrimidine dimer formation. Similar results have been obtained for Bacillus subtilis DNA ( 2 , 52) although one early ‘Two components are also necessary for the degradation of irradiated DNA by Micrococcus Zysodeikticus extracts ( 85). The excision and degradation reactions are not identical but may have some steps in common.

273

PHOTOBIOLOGY AND REPAIR OF DNA

, , 280mp

-9

z a a

1-

t-

a

-.97j=

0

-.96

c d

0.01

'0.

b

p--.

0

W

----.

/239rnp

*,'

-

LL 0

cn

\ ,

-

"\0

4

r

1

I

1

1

1

I

I

I

I

b

l

I

f

FIG.5. The effects of sequential irradiation with two wavelengths on the transforming activity of Hemphilus influenme DNA (cathomycin marker); 0, 280 mp; 0, 239 KIP. Since the conversion from h e r to monomer is accompanied by an increase in absorbance (Fig. 2), the symbol represents the extent of dimer monomerization (88). attempt (89) to demonstrate short wavelength reversal failed, probably because the dose level was much too high.

B. Proflavine Reversal Proflavine inhibits the formation of dimers but does not affect their monomerization (27, 28). Thus transforming DNA irradiated at long wavelengths and then irradiated further with long wavelengths in the presence of proflavine may be reactivated as the result of the monomerization of dimers containing cytosine ( 5 5 ) . The kinetics of this phenomenon correspond to those observed for the photochemical monomerization of cytosine-containing dimers and thus are evidence for the importance of dimers in the inactivation of transforming DNA and in particular for the importance of cytosine-containing dimers in such inactivation. Quantitatively, the data indicate that cytosine-containing dimers are as important as thymine-thymine dimers in inactivation.

C. Photoreactivation Transforming DNA inactivated with relatively low doses of ultraviolet irradiation may be reactivated by enzymatic photoreactivation. Such treatment reduces the initial effective ultraviolet dose by 90%. The

274

R. B. SETLOW

photoreactivable sector is 0.9 (90). This is an indication that in the lowdose region, 90% of the inactivation may be ascribed to the formation of pyrimidine dimers. Enzymatic photoreactivation overlaps that produced by short wavelength reversal ( 9 1 ) . Since both processes are known to monomerize pyrimidine dimers, these data indicate that the photoreactivation of the biological activity involves only dimer monomerization. The large photoreactivable sector indicates the importance of pyrimidine dimers in the inactivation of transforming DNA. It must be remembered that DNA's such as that of H . influenme have a high A T content. Other DNA's may have smaller photoreactivable sectors because, since the thymine-thymine dimers are formed most efficiently, the irradiation of a high G + C DNA would require a higher dose to form equivalent numbers of pyrimidine dimers and more nonphotoreactivable lesions would be expected to be formed. Confirming and compelling evidence for this point of view comes from the photoreactivation of the competing activity of model polynucleotides. Ultraviolet-irradiated polynucleotides containing adjacent pyrimidines compete for the photoreactivating enzyme and this competition is eliminated by prior treatment of the irradiated polymers with enzyme extracts plus light. The rate of elimination of competition is similar to that for dimer monomerization ( 7 5 ) . For example, cytosinecytosine dimers in poly dI-poly dC are monomerized more slowly than uracil-uracil dimers in the same polymer, and it is observed that the competing ability of the polymer containing cytosine dimers is photoreactivated at a slower rate. Irradiation conditions, such as frozen solutions or high doses at low wavelengths, that favor the formation of other photoproducts compared to cyclobutane dimers will result in a low photoreactivable sector. In such cases pyrimidine dimers may be relatively unimportant in inactivation, and it is possible to identify other physicochemical changessuch as DNA-protein links (25) and the spore-type photoproduct (92, %)-as a lesion. The similar changes with temperature of the inactivation sensitivity of a single-stranded viral DNA and the estimated dimer formation indicate that, at room temperature, 2 5 0 % of the inactivation arises from dimers ( 9 4 ) .

+

D. Dark Repair Enzymes Enzyme extracts similar to those that act specifically on ultravioletirradiated DNA also are effective in promoting the reactivation of ultraviolet-irradiated DNA's containing biological activity. Elder and Beers (95) report that extracts of Micrococcus lysodeikticus can reactivate ultraviolet-irradiated transforming DNA. Even more convincingly,

PHOTOBIOLOGY AND REPAIR OF DNA

275

Rorsch and his colleagues (86, 96) have shown that such extracts reactivate the ultraviolet-inactivated replicative form of +X174. Such extracts clearly can do at least the first step in repair, namely, make one break in the ultraviolet-irradiated DNA, but it is not clear whether the extracts or the cells used to titer these biologically active systems perform the next steps. It is an anomaly that such activities are observed in extracts of sensitive strains of M . lysodeikticus (86, 97).

V. Cells and Viruses The implication of pyrimidine dimers in the inhibition of biochemical systems and in the effects on the biological activity of DNA in vitro is relatively simple because of the demonstration that these activities obey the same photochemical relations as the formation and monomerization of pyrimidine dimers in DNA. Moreover, enzymatic photoreactivation reactivates ultraviolet-irradiated transforming DNA by a mechanism that seems to be identical with the monomerization of pyrimidine dimers. However, the extension of the importance of pyrimidine dimers to more complicated biological systems, such as cells and viruses, is much more difficult,The purely photochemical reversal tricks have not been utilized because they require doses so large that the biological system is completely killed. Thus we have to rely on somewhat different types of reasoning. Two completely different lines of evidence indicate that pyrimidine dimers may be associated with lethality and with mutation, although it should be clear that their exact role in these processes and how they produce their end results are not known.6 The evidence has been reviewed extensively elsewhere by J. K. Setlow (2, 3) and has also been reviewed recently for mutation induction in E . coli by Witkin (99). The evidence we shall discuss briefly relates to photoreactivation (light repair), dark repair, and the genetic control of radiation sensitivity.

A. Photoreactivation The existence of the phenomenon of photoreactivation (discussed at the enzymatic level in Sections 111, C and IV, C ) is evidence for the importance of pyrimidine dimers in inactivation and mutation production, even though the details of the situation may be complicated as in phage (100) and Neurospora (IOI), for which UV-induced mutants of many different types are photoreactivable. However, three warnings must go with arguments on the use of photoreactivation. ( a ) Many biological

' I t is worth emphasizing again that the formation of pyrimidine dimers cannot explain dl the effects of ultraviolet radiation on polynucleotides. In some systems, such as bacterial spores ( 2 1 , 2 2 ) or cells irradiated while frozen (2,5, 98) they seern to be unimportant.

276

R. B. SETLOW

systems may exhibit indirect photoreactivation, a phenomenon not related to dimer monomerization but more to some generalized nonenzymatic effect on cellular components ( 6 7 ) . One may distinguish, to a large extent, indirect from direct photoreactivation by the wavelengths at which each is effective, at least in E . coli, and by the fact that enzymatic photoreactivation has a much larger temperature coefficient than indirect photoreactivation. A further distinction may be made by administering the photoreactivating illumination before ultraviolet irradiation. If such a treatment results in a restoration of the biological system, it is presumably indirect. ( b ) Most of the observations of biological photoreactivation are scored many hours after the initial ultraviolet irradiation, because what one observes is colony formation or mutant production. Therefore it is especially difficult to correlate events at these late times with macromolecular events during the illumination itself ( 102). ( c ) Some cells or systems may exhibit no photoreactivation. It is obviously not possible in such a case to give a clear interpretation of the results, because the systems may have no photoreactivating enzyme or, even if they do, the enzyme may not be able to reach pyrimidine dimers. Thus, for example, if the dimers were in denatured DNA or in small pieces of DNA, they would be acted on at a slower rate than dimers in native DNA. If there is no photoreactivation, no interpretation is possible on such information alone. In only a few cases has the observation of photoreactivation of biological properties been correlated with the destruction (presumably monomerization) of dimers in uivo. Monomerization is observed in E . coli (79,103,104) [but not in a mutant lacking photoreactivating enzyme (105)] and in Bacillus mguterium (92). However, there are no reported data correlating the kinetics of photoreactivation of colony-forming ability with the kinetics of dimer monomerization. The inhibition of DNA synthesis by ultraviolet irradiation is also photoreactivable (103, 106). The effect is observed immediately after radiation treatments, and although the data are not extensive the dose reduction factor for inhibition of synthesis is similar to that for dimer monomerization (103). In vivo destruction of dimers has been observed in Paramecium (107) and in amphibian cells in tissue culture (108). Both systems also show biological photoreactivation, but no good correlation has been made between the biological and the biochemical measurements. Extracts of many other metazoan (but not mammalian) cells have photoreactivating activity ( 7 1 ) as measured on irradiated transforming DNA, but in uivo photoreactivation has not been reported. Photoreactivation of Tetrahymena and of repair replication in Tetrahymena has been reported ( 1 0 9 ) .

PHOTOBIOLOGY AND REPAIR OF DNA

277

B. Repair in the Dark The radiation sensitivity of microorganisms is under genetic control (86, 110-113). The differences in sensitivity are not associated with different DNA conformations because both resistant and sensitive strains are similar as far as the induction of dimers is concerned (79, 103, 104). This is not the place to review this subject, except to note that such control of sensitivity to ultraviolet light is similar in many respects to the control of sensitivity to ionizing radiation and to chemical agents, although there are obvious differences in the responses of the mutants to deleterious agents. Nevertheless, there are data indicating that pyrimidine dimers can be repaired or ignored in radiation-resistant cells, and thus these data also indicate that dimers are lesions in bacteria. Radiation sensitive and resistant mutants are known in E . coli, B. subtilis ( 1 1 4 l l S ) , T4 phage (117, 118), and Micrococcus radiodurans (119),among others. In the first three cases, much is known about the genetics of the situation. The big impetus to the subject of dark repair and genetic control came about with the discovery by Ruth Hill (120) of an exquisitely sensitive mutant of E . coli, which she called E. coli BS+ This discovery started the search for other sensitive mutants, and large numbers of them in E. coli have been identified by many investigators. It is simplest (but obviously an oversimplification) to group these mutants into two categories: resistant bacteria that are also host-cell reactivating, and sensitive bacteria that are not host-cell reactivating (66). ( Host-cell reactivation refers to the ability of a cell to effect the reactivation of ultravioletirradiated bacteriophages during the infectious process. ) E . coli ELl is Hcr-, and irradiated bacteriophages such as T3 phage show a higher sensitivity when assayed on this host than on a host such as E. coli B or B/r (121). When the irradiated double-stranded replicative form of 4x174 is titered with spheroplasts of Hcr+ strains, it shows a higher survival than on Hcr- spheroplasts (122, 123). Neither the virus nor its single-stranded DNA shows such an effect. The variations in ultraviolet sensitivity of the bacteria themselves vary widely within each group because of the many ways in which cells may die as a result of ultraviolet irradiation (6, 7, 86). These other complicating events, such as filament formation and induced viruses, are perturbations to the separation of cells into the two categories, so that, as far as radiation sensitivity is concerned, there may actually be some overlap among the two groups. Nevertheless the distinction is a useful one. It is useful because one may think of the resistant bacteria as being resistant because they can repair or bypass some of the radiation damage.

278

R. B. SETLOW

Some irradiated resistant strains of E. coli show higher survival when held in buffer before plating on agar (review in ref. 66). This phenomenon, called liquid-holding restoration, and host-cell reactivation and photoreactivation are not additive. They overlap (66, 124, 125). All the reactivations seem to act on similar types of lesions-lesions that we have identified with pyrimidine dimers. This identification is made stronger by the investigation of DNA’s containing bromouracil. In such DNA’s very few dimers are formed (126),since bromouracil does not participate in such photoproducts (11)and the various recovery phenomena are not found ( 1 2 7 ) . Thus pyrimidine dimers are implicated in these recovery phenomena. ( A particular protection phenomenon associated with the ultraviolet irradiation of DNA’s containing bromouracil is concerned with irradiation carried out in the presence of sulfhydryl agents. Under such irradiation conditions, bromouracil DNA’s may actually be more resistant than unsubstituted DNA’s ( 128). Presumably the formation of lethal bromouracil photoproducts is suppressed under these conditions. ) A conceptual difficulty with these analyses of various types of restoration is that they are scored or observed much later than the initial radiation (102). Repair in the dark is normally not as effective as photoreactivation, as evidenced by the fact that even ultraviolet resistant E . coli strains are photoreactivable.

C. Effects of Ultraviolet on DNA Synthesis Pyrimidine h e r s act as inhibitors to DNA synthesis in vitro and, because it is known that ultraviolet irradiation stops DNA synthesis in vim, it is reasonable to look upon such dimers in the bacterial DNA’s as the blocks to synthesis. This is especially the case since such inhibition is photoreactivable and the amount of photoreactivation is similar to the monomerization of dimers (103). Such a response may be observed immediately after the irradiations. It involves no long time lag and indicates that pyrimidine dimers inhibit DNA synthesis in duo. The inhibition of DNA synthesis by irradiation may be used to divide the various strains of E. c d i into three categories (6 , 129, 130) (Fig. 6 ) . The Hcrstrains are much more sensitive as far as this inhibition is concerned. The lags or inhibitions of synthesis are photoreactivable. A detailed analysis of such incorporation curves, as is shown in Fig. 6, is complicated by changes in the intracellular pools, breakdown of DNA (132, 133), and the production of new growing points (134, 135). Nevertheless it is apparent that the Hcr+ cells of E . coli recover quickly from radiation and that the very resistant bacteria, M . radiodurans, also recover after very large numbers of dimers in their DNA. These recoveries take place in the dark and are evidence for mechanisms of dark repair or bypass of the

279

PHOTOBIOLOGY AND REPAIR OF DNA

FIG.6. The typical effects of ultraviolet radiation (265 ma) on subsequent DNA synthesis in irradiated cultures. The numbers next to the curves and the values of DNrepresent doses in ergs/mm2. Parts ( a ) and ( b ) represent Hcr- cells ( 6,129, 131

-

lesions that induce the lag in DNA synthesis. Any specific interpretation is complicated by the fact that even in uitro one is not sure whether to consider a dimer as an absolute block to synthesis or only a relative block as indicated in Fig. 4. It seems clear that dimers inhibit DNA synthesis and that the Hcr+ cells recover quickly. Such cells must either ignore or remove the blocks ( 8 ) , as indicated in Fig. 7. If dimers were only ignored in the Hcr+ cells, one would expect that the slow polymerization past them would not -?r.

-.JT.-

-

m-

A: PHOTOREACTIVATION

FIG.7. Schematic ways in which cells may cope with dimers.

280

R. B. SETLOW

result in the almost complete inhibition of synthesis followed by the very rapid resumption indicated in Fig. 6. However, such a mechanism may well exist and be responsible for the apparent recovery and high mutability of some of the Hcr- strains similar to those indicated in Fig. 6b ( 8 , 99, 136). If this is the case, it seems to be a mechanism that operates well only at low doses. Rupp and Howard-Flanders (137, 138) have shown that the DNA initially synthesized at a slow rate in H c r ceIls acts as if it had strand breaks opposite dimers on the template strand. The breaks disappear with time, possibly as a result of recombinational events or by polymerization and rejoining reactions. There is excellent evidence that pyrimidine dimers in DNA, in U ~ U O , can be repaired in the dark. The evidence is of two forms. ( a ) The ability to monomerize dimers by photoreactivating illumination in sioo is lost with time in the resistant strains, but not in the sensitive ones ( 8 , 79).6 Thus either the dimers are parts of different structures than they were before, such as denatured DNA or smalI pieces, or they are no longer accessible to the enzyme. ( b ) As time goes on after irradiation, the dimers appear in the acid-soluble fraction of the cells and disappear from the insoluble fractions (79, 104). Thus the dimers become parts of small molecules in Hcr+ cells, and, more significantly, the time it takes for the dimers to disappear from the DNA corresponds closely to the time to resume DNA synthesis (139). The disappearance has been observed in resistant strains of E. coli ( 6 , 79, 104) and B . subtilis (83, 140), in B. megaterium ( 0 2 ) , M . radioduruns (141), H . influenxae (142), cells infected with T4 phage (143, 144), some mammalian cells ( 1 4 5 ) , Paramecium (146) and Tetrahymena (147). It has been termed "excision" and now deserves more of our attention, since it seems to form the basis for the molecular mechanism of repair of damage to DNA.

D. Excision If pyrimidine dimers are lesions, then it is an attractive possibility that they are removed bodily from the DNA of resistant cells, whereas the sensitive ones cannot remove them. It is worthwhile to look at the detailed experimental procedures by which excision is measured to see what the various complications and pitfalls in the interpretation are. A typical experimental procedure is shown in Table 11. One obtains three fractions, the acid-insoluble and soluble fractions of the cells and the medium. Each of these fractions may contain thymine-thymine dimers The concomitant loss in the ability to photoreactivate biological activity may be interpreted as indicating that the dimers are no longer in a position to cause biological damage; i.e., they are no longer in DNA.

PHOTOBIOLOGY AND REPAIR OF DNA

281

and uracil-thymine dimers, the latter arising by deamination from C ~ O sine-thymine dimers. It is possible to measure the amounts of the various products in each fraction, although the many components of the medium compared to the trace amounts of dimers make it more difficult to rneasure precisely the number of dimers in this fraction than in the others. In many cases, it is found that less than 10% of the radioactivity is in the TABLE I1 P R O C E DTO ~ EMEASUREDISTRIBUTION OF DIMERS 1. Label cells with thymin&H, transfer to nonradioactive medium 2. Irradiate with W

3. Incubate cells for various times 4. Separate cells from medium 5. Fractionate cells into acid-soluble and acid-insoluble parts 6. Analyze the medium and cellular fractions for thymine and thymine-containing dimers (by hydrolysis, chromatography, and radioactive assay)

medium, and so, as a first approximation, the medium may be ignored, especially if the number of dimers in cells equals the number originally present. In resistant E . culi, dimers are lost from the insoluble fraction and gained by the soluble fraction of cells, while the thymine content of each changes little. Thus in this case, a convenient measure of excision is the change in the ratio of radioactivity in dimers to thymine in the insoluble fraction, Figure 8 shows such data for the resistant and sensitive strains of E. coli and for the very resistant species, M. radiodurans. The dimer-containing molecules in the acid-soluble fraction have chromatographic mobilities similar to those of the nuclease-resistant sequences described in Section 111, A. Thus dimers appear in the soluble fraction not as free dimers, but as parts of small oligonucleotides. A simple picture of the excision process is shown in Fig. 9, step ( 3 ) . Excision, in this picture, is looked upon as the removal of small oligonucleotides containing dimers from the DNA. However, this is not the only possible picture, because during the excision process net DNA synthesis has stopped and actually some degradation of the DNA is observed. Thus it is possible to describe the results shown in Fig. 8 by saying that there is complete degradation of the DNA to mononucleotides and dimercontaining oligonucleotides followed by reincorporation of the mononucleotides into acid-insoluble material. The dimers that are not reincorporated remain acid-soluble. Figure 9, steps ( 1 ) and (Z),describes this state of affairs. Of course, the scheme of degradation and resynthesis could be much more subtle. If degradation, for example, could proceed along one strand of DNA at a time with resynthesis following closely behind, the end result would be the same.

-

282

R. B. SETLOW

FIG. 8. Left: Excision of dimers from the acid-insoluble fraction of ultravioletresistant cells at various times of incubation in growth medium after irradiation. For Escherichia coli 15 T;0, incubation with thymine; 0, incubation without thymine. Right: Lack of extensive excision, even at low doses, in Hcr- strains of E . coli. The L given on the right-hand ordinate ( 3 4 , 79, 141 ). initial doses at 265 ~ J are

If, during the process of repair, there was much random breakdown of DNA, perhaps initiated by the single-strand breaks that seem to precede the final steps in repair, the resulting thymidylic acid, thymidine or thymine could ultimately appear in the medium or acid-soluble fraction, and the loss of thymine from the acid-insoluble fraction could be almost as great as that of dimers. The ratio of dimers to thymine would not fall rapidly in the acid-insoluble fraction even though there was a big Ioss in the absolute numbers of dimers. If the oligonucleotides that contain THYMINE IN ACID-INSOLUBLE

(1)

(2)

(3)

.,, ,.:x,. t;f+5 + ;!$

yx,<;Ltfi;’

“ z == -t

-

DECREASES

DIMERS/THYMINE IN ACID-INSOLUBLE -CONSTANT

INCORPORATION INCREASES

DECREASES

EXCISION -CONSTANT

DECREASES

FIG.9. Ways in which dimers may be removed from DNA (8). The end result of step 3 is the same as that of steps 1 and 2.

PHOTOBIOLOGY AND REPAIR OF DNA

283

dimers did not leak out of the cells as rapidly as did mononucleotides, the ratio of dimers to thymine would increase in the acid-soluble fraction of cells. Thus, if there is a large amount of breakdown as indicated by the appearance of much of the radioactive label in the medium, it becomes difficult to interpret the data as indicating specific excision. Such difficulties exist at high UV doses and for some of the so-called recombinationless mutants at low doses. Data for these mutants do not indicate excision, because during the time of such an experiment there has been an -50% loss of thymine from the acid-insoluble fraction and equal loss of dimers (148). The thymine appears mostly in the medium, the dimers mostly in the acid-soluble fraction of the cells. These results are most simply interpreted as indicating general rather than specific breakdown. The fact that the Rec- strains are, in many cases, Ncr+ (130) indicates that such strains can excise dimers, although the present analytical data are not convincing. One is on dangerous ground in giving a unique interpretation, unless there are large changes in the ratio of dimers to thymine and there has not been much loss of radioactivity from the acid-insoluble fraction. Obviously such experiments must be done at dose levels that correspond to reasonable survival of the cells because one would like to show that the excision process is a property of surviving cells and not of an irradiated but dying population. The strongest evidence that excision is involved in repair is that it involves pyrimidine dimers (this is really a circular argument), that it takes place in a time shorter than the lag period produced by ultraviolet irradiation, and that it is not found in radiation-sensitive cells, even at very Iow doses (Fig. 8). Two genes, v and x, that control radiation resistance are known in T4 phage ( 1 1 7 ) .The action of the v gene overlaps photoreactivation (118), and one might guess that its action is associated with excision. Recent experiments have shown that it is (143, 144), although an earlier worker (149) was not able to detect excision. Thus E. coli strains infected with T4 v+ phage show excision of thymine dimers from the phage DNA, but no excision is observed in cells infected with irradiated T4 v-. It is of interest that the excision observed in this system is independent of the host cell, as would be expected from the observation that the T-even phages are not subject to host-cell reactivation (121, 150). The simplest biological description of this system is that the v+ phage brings in information to rescue its own DNA and to excise dimers from any other DNA in the cell. Thus mixed infection of v+and v- phages results in the excision of dimers from the v- DNA and a rescue of such irradiated phages. One of the most resistant bacteria is M . rudioduruns. It is able to excise 10 times as many dimers as E. coli and still show 100% survival (141). The excised dimers do not remain in the cells but are found in the sur-

284

R. B. SETLOW

rounding medium. Detection is easy only if one knows what to look for. Hence, if M. radwdurans had been the first bacterial strain in which the fate of dimers was investigated, one might have concluded erroneously that dark repair involved the destruction of dimers (as in photoreactivation) rather than their excision. The efficient repair of dimers means that M . radiodurans dies from some other type of damage and indeed the action spectrum for killing is significantly different from the absorption spectrum of thymidine or DNA ( 151 ) . The excision of dimers has not been detected in Chinese hamster (152, 153) or in mouse L cells (154) but excision of about half the dimers was observed in cells derived from human tissues (145).However there are no comparative data to indicate that such excision enhances survival in mammalian cells. Such cells survive hundreds of thousands of dimers and one would be justified in concluding that pyrimidine dimers are not lesions in mammalian DNA,

VI. Steps in the Repair of DNA Our discussion of repair by excision of dimers has obviously been simplsed. The repair of DNA at the molecular level must involve steps other than excision if the DNA is to be returned to its original base sequence and configuration. Steps that seem to be associated with repair of DNA and that have been postulated to be an integral part of the repair mechanism are excision, breakdown of DNA, repair replication, and rejoining of strands. The direct evidence for the interrelations among these various steps is tenuous, and the temporal and spatial ordering of them is obscure. We consider below some of the details for each of these steps and the evidence that they are associated with repair.

A. Excision The molecular events associated with the act or acts of excision may be looked on in two ways (155) as shown in Fig. 10. In scheme B the dimer-containing oligonucleotide is removed as a unit and a gap is left in the DNA chain. In scheme A excision is preceded by a single-strand break on one side of a dimer, followed by DNA synthesis that tends to push off the dimer-containing oligonucleotide. In scheme B, the DNA chain seems to be in a more vulnerable configuration than scheme A. In scheme A, the excision process requires several different enzymatic actions. In E. coli, excision is an exponential process (139) and is as rapid in chromosome regions that have been newly synthesized as in older regions ( 3 4 ) . Thus excision in vivo takes place rapidly and seems to be a random

285

PHOTOBIOLOGY AND REPAIR OF DNA

&LESION

/

INCISION

-x

+

I

-

+

I

----

x

DEGRADATION

-----

REJOINING

A B FIG.10. Two schematic pathways illustrating the possible sequences in the repair of DNA (an outgrowth of the discussions given in ref. 155).

process. It is inhibited by acridine dyes (139) and caffeine (144)-two agents that inhibit dark repair ( 6 6 ) .The rate of excision is decreased in E . coli incubated in the absence of glucose (79, 144) or the presence of KCN (144),but glucose deprivation does not affect excision in M. radioduruns ( 1 4 1 ) . However, excision is unaffected by chloramphenicol (129, 144) and is little affected by the absence of substances such as thymine, uracil, and amino acids in bacteria that require these for normal growth (Table 111). Mutations at three separate genetic loci in E . coli render cells Hcr and result in a loss of the ability to excise dimers ( 1 1 2 ) . Whether these loci control individual enzymes in the steps of excision or operate in some other fashion is not known. Excision in vitro by extracts of M. lysodeikticus requires at least two heat-labile components (34,144). One might be tempted to suppose from the rapid excision in the absence of thymine in thymine-requiring bacteria (Fig. 8) that DNA synthesis is not necessary for excision. This is not a fair deduction because associated with excision is the breakdown of DNA, and even a small amount of breakdown could contribute to an internal pool of nucleotides sufficient to account for the small amount of synthesis necessary for the excision process as described in Fig. 10A. The fact that the absence of glucose inhibits excision whereas excision

286

R. B. SETLOW

TABLE 111

EXCISION OF DIMERS FROM Escherichia coli 15 TAU

(6)O

Percentage of thymine activity Incubation time (minutes) ~

Incubation medium ~~

0

30

~~~

in

GT

in TT

~

Complete Complete - uracil - uracil - amino acids - uracil - amino acids - glucose

0.044

1

i

0.024 0.028

0.088 0.035 0.049

0.024

0.056

0.038

0.070

a Cells were labeled with thymine-*H and irradiated with 200 ergs/mm* of 265 mp radiation. Complete medium contains glucose, salts, uracil, casamino acids, and tryptophan. No thymine was added to the medium after irradiation since it does not affect excision (see Fig. 8).

may be observed in in vitro systems that do not contain glucose indicates that excision in E . coli needs some metabolically labile cofactor or represents some coordinated type of synthetic activity.

B. Breakdown of DNA Bacterial strains that are able to excise dimers show extensive breakdown of DNA following UV irradiation (130, 132). Much of the original thymine-8H labeled DNA appears in the medium as thymidine or thymine and is easy to observe experimentally. Breakdown in B. subtilis is inhibited by removal of glucose ( 1 5 6 ) . It is an especially valuable parameter to follow, because in general it is a property of Hcr+ cells, but not Hcr- cells, following ultraviolet irradiation. A molecular picture of breakdown is that a break inserted in a DNA chain is enlarged following excision or the first step in excision. If a break is not inserted, as in an Hcrcell, no breakdown will be observed. The observation of DNA breakdown is well correlated with cells that can repair UV damage, but there are no direct data indicating that such a breakdown represents a necessary step in the repair process. Such observations are made on cultures of irradiated bacteria and, to simplify the measurements, the radiation doses are usually large. As a result, the majority of the cells do not survive, and one could take the point of view that DNA breakdown represents the degradation in dead cells and has nothing to do with repair, Following ultraviolet irradiation there is also breakdown observed in cells of E . coli B,-l ( 1 3 3 ) . The amount of breakdown is independent of dose over a large range and is not observed until an appreciable time

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after DNA synthesis has been inhibited. It probably arises from random degradation of the DNA, since in this strain it appears as if one or only a few strand breaks produced by X-rays lead to extensive degradation of the bacterial DNA ( 1 5 7 ) . In the case of other strains, in particular strains other than Rec- or E x r strains (86), DNA breakdown after X-irradiation is much smaller ( 158).

C. Resynthesis a n d Repair Replication One would suspect a priori that the gaps resulting from the removal of dimers would be filled in by polymerization under the direction of a DNA polymerase or that such an enzyme would be associated, as indicated in scheme A of Fig, 10, with the polymerization underneath a dimer-containing oligonucleotide. The observed repair replication of Pettijohn and Hanawalt (9, 159, 160) fits this concept. Experiments to observe repair replication are performed in the following fashion. Cells whose DNA synthesis has been inhibited, say, by ultraviolet radiation, are placed in medium containing radioactive bromodeoxyuridine instead of thymine. Before normal, rapid, DNA synthesis resumes, the bacterial DNA is isolated and fractionated by equilibrium centrifugation in cesium chloride. Much of the incorporated bromouracil is found at a density corresponding to normal DNA or at densities intermediate between the normal DNA and a hybrid of one normal strand and one heavy strand. After sonication of such DNA's to reduce their molecular weight, bromouracil is still found near normal densities and therefore the pieces in which the bromouracil may be localized are small compared to several million. Denaturation of the DNA followed by cesium chloride centrifugation also results in bromouracil observed in normal densities. Thus the bromouracil is incorporated into individual DNA strands in a manner unlike that in normal DNA replication. This aberrant incorporation is observed after ultraviolet irradiation of Hcr', but not B,-* (ISI),and its magnitude is less in cells that have been exposed to photoreactivating light (160). It seems intimately associated with the repair of damage (9). The above data fit a picture consistent with the incorporation of bromouracil into regions originally occupied by dimers or into enlarged regions that originally contained pyrimidine dimers. The aberrant synthesis described as repair replication has an alternative explanation that depends on the observation that after ultraviolet radiation there is DNA breakdown and new growing points are introduced into the chromosome (134,135).Such points can represent regions of synthesis into which bromouracil as well as thymine arising from the breakdown of the remainder of the DNA is incorporated, since breakdown of DNA gives rise to an internal pool of thymidylic acid. The

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newly synthesized material will contain mostly thymine and little bromouracil, since thymine is preferentially incorporated, and thus the labeled bromouracil would be at a light or an intermediate density. This is a dScult argument to refute as an alternative explanation of repair replication, especially because it is difEcult to be quantitative about the size of the intracellular pools that originate from DNA breakdown, and the origin of the degraded DNA among chromosomes or bacteria is not known. Clearly what is needed is to stop “normal” replication absolutely and show that the newly incorporated material contains many sequences of bromouracil. Repair replication has been detected in repairing strains of E. coli that have been treated with chemical mutagens (162, 163). This is the best evidence that the repair systems are general and work on many types of changes in DNA-not only pyrimidine dimers. Such replication has also been observed in mammalian cells (164) and in Tetruhymenu (109).

It seems clear that some type of replication analogous to our picture of repair replication must take place if the excised regions are to be resynthesized. Whether the observed repair replication represents only this synthesis or whether it includes some normal synthesis from new growing points that incorporates nucleotides from DNA breakdown has not been demonstrated.

D. Rejoining of Strands During the time that DNA synthesis is inhibited in resistant strains of E. coli, approximately 500 pyrimidine dimers are excised per chromosome strand after an incident dose of 200 ergs/mm2. Thus in 60 minutes approximately loo0 breaks per chromosome are introduced before normal synthesis resumes. The continued existence of such breaks would obviously be an inconvenience to a surviving cell, if only because there are enough of them so that in a random process two of them could be close enough together in opposite strands to give rise to a double-strand break.7 Thus if the breaks were not repaired, a double-strand break-preswnably a lethal event-would ensue. Moreover, the continued existence of gaps in DNA would result in the accessibility of DNA to nuclease attack and, as has been pointed out, to severe dif€iculties in normal replication (165). All these are reasons for assuming that the gaps or

‘ If there are 500 random breaks in each strand of length 1 mm, the probability of finding a break in a given segment 30 A long (the approximate pitch of a double helix) equals 15 X lo4, and opposite a particular break in strand A, there is this probability that there will be a break in strand B. Hence the number of pairs of breaks within 30 A of one another is 500 X 15 X lo‘, or 0.75.

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holes do not remain but are repaired. McGrath and Williams (166) have described a technique for detecting large single-stranded pieces of bacterial DNA, pieces whose length is approximately one-fifth that of the chromosome length. This technique has been used to measure strand breaks that appear during the excision process in resistant cells. The data indicate that approximately 1520 strand breaks exist at any time even though approximately 30 dimers are being excised per minute (6, 7). Thus, the repair of single-strand breaks comes quickly after the excision, although such data give no information about the spatial events or the sequence of ordered or disordered steps in the excision process. The appearance and subsequent disappearance of these single-strand breaks is observed in Hcr+strains and is not observed, in general, in Hcr- strains, although a low level of such breakage repair has been observed in E. coli Bs-3 ( 1 6 7 ) . Polynucleotide joining enzymes using DPN as a cofactor have been isolated from both excising and nonexcising strains of E . coli (168-171), and a similar activity, but with ATP as a cofactor, is found in T4-infected E . coli (172).

VII. Conclusion The presumed steps in the repair of damage to DNA-excision, breakdown, repair replication, replication, and rejoining of strands-have all been observed. They may be put together to make a convincing argument that this is the method by which pyrimidine dimers are removed and the DNA is repaired. The generality of the phenomena lies in observations indicating that there seems to be excision of products arising from treatment of DNA in vivo with nitrogen mustard and sulfur mustard (review in ref. 173), that breakdown of DNA follows many chemical and radiation treatments (104, 158, 174), and that repair replication is observed in bacteria treated with nitrogen mustard ( 162) and nitrosoguanadine (163). Thus the scheme may be extended to systems other than those containing pyrimidine dimers, although it is only in the latter case that the steps have been followed in detail. Isolated enzyme systems (175) that can do many of the steps in these hypothetical repair systems are known. Enzymes specifx for UVirradiated DNA have been described (Sections 111, D and IV, C ) . Exonuclease I11 in E. coli could be the degrading enzyme; the DNA polymerase as normally isolated could be the repair polymerase, and the polynucleotide ligases could be the joining enzymes. However, neither the nature of the individual enzymes that act nor the sequence of steps involved in repair in vivo are known. Nevertheless, the results from the

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interactions among photochemists, photobiologists, molecular biologists, and geneticists provide convincing evidence that damage to DNA can be repaired by an excision-replacementtype of mechanism.

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NOTEADDEDIN PROOF The fact that the yield of photochemical products depends on the conformation of DNA is clearly indicated by the work of Hosszu and Rahn ( N l ) , which shows that the yield of cyclobutane dimers in native DNA decreases abruptly in going through the melting temperature. The yield of dimers in denatured DNA decreases uniformly as the temperature increases. Photoreactivating activity has been observed in extracts of blue-green algal cells ( N 2 ) , and PPLO can be added to the list of organisms in which repair replication is found ( N 3 ) after ultraviolet irradiation. Photoreactivating illumination reduces the magnitude of the repair replication. Repair replication in E. coli takes place at elevated temperatures in a mutant whose chromosomal synthesis is temperature sensitive ( N 4 ) . Thus Couch and Hanawalt suggest that normal and repair synthesis use different enzyme systems. Single-strand breaks are observed in the DNA of irradiated T4v+ phage after the phage infect resistant or sensitive strains of E . coli ( N S ) . Such breaks are not found in T4v- phage. (See Section VI, D for analogous data for bacterial DNA. ) Kozinski and Lorkiewicz interpret their phage data to indicate that the enzymes of the host cell can work on T4v’ but not T4v- DNA. A simpler description (page 283) that is consistent with both the biological and the excision data is that repair

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is independent of the host and that the v', but not v-, phage brings in information necessary to effect repair of its own DNA.

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N1. N2. N3. N4.