.. - aleminde IT UNCLASSIFIED ORNL . . UTL IM la . . 85 TOF2 LEGAL NOTICE This report was prepared as an account of Government sponsored work. Neither the United States, nor the Commission, nor any person acting on behalf of the Commission: A. Makes any warranty or representa- tion, expressed or implied, with respect to the accuracy, completeness, or usefulness of the information contained in this report, or that the use of any information, appa- ratus, method, or process disclosed in this report may not infringe privately owned rights; or B. Assumes any liabilities with respect to the use of, or for damages resulting from the use of any information, apparatus, method, or process disclosed in this report. As used in the above, "person acting on behalf of the Commission” includes any em- ployee or contractor of the Commission, or employee of such contractor, to the extent that such employee or contractor of the Commission, or employee of such contractor prepares, disseminates, or provides access to, any information pursuant to his employ- ment or contract with the Commission, or his employment with such contractor. . - . **...-.. or.. O A . powinna L ....... DTIE MICROCARD ISSUANCE DATE Si 8 / 26 - YU 1964 elv-f485 . CONF-611-12 L1 51964 Photochemistry of Nucleic Acids, and Its Biological Implications* - .E MASTER s John Jagger Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 1 servering long wind -- LEGAL NOTICE ---- TWO report nowepared un account of Coven wrat opoumored work. Nolubor he United daw, sur the Commiswa, por las persoa kun on bolall of Coownloa: A. 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Rapporteur's report, Session No. 2, Fourth International Photobiology Congress, Odord, Bagland, July 27-30, 1964 k Facsimile Price $_ Microfilm Price $1 o 25 Available from the Office of Technical Services Department of Commerce Washington 25, D. C. : ' . *Research sponsored by the U.S. Atomc Energy Commission wder contract with the Union Carbide Corporation. Running head: Photochemistry and Biological. Implications Send proof to: Dr. John Jagger : Biology Division Oak Ridge National Laboratory P. O. Box Y Oak Ridge, Temesbee 37831 ........ . ....... - .-- •-- Papers Discussed & M. Chebbin The effect of light on clover yellow mosaic virus. I. Photoreactivation. II. Photoinhibition. Botary Department, Montana State University, Missoula, Montana, U.S.A. D. Pittman Photoreactivation of RNA lesions. I. Utraviolet inactivation and differential photoreactivation of zero-point and delayed mutations of an RNA-determinant : extrachromosomal factor in yeast. Microbiology Department, Southern Illinois University, Carbondale, Dlinois, © U.S.A. . Moroson, O. Gallego, and P. Alexander Viscosity and byperchromi city studies 01 DNA extracted from UV irradiated E. coli 15 r. Biophysics Division, Sloan-Kettering Institute, New York, U.S.A., and Chester Beatty Research Institute, London, Great Britain. K. C. Smith The photochemical interaction of deoxyribonucleic acid and protein in vivo and its biological importance. Department of Radiclogy, Stanford University School of Medicine, Palo Alto, California, U.S.A. J. S. Cook Enhanced ultraviolet sensitivity of cleavage intervals in sand dollar zygotes .. following incorporation of 5'-bromodeoxyuridine. Mount Desert Island Biological Laboratory, Bar Harbor, Maine, and New York Universiti School of Medicine, New York, U.S.A. . .. . . .. . -- .---. ) P. C. Hanawalt and D. E. Pettijohn Replication of ultraviolet damaged DNA in bacteria. Biophysics Laboratory, Stanford University, Stanford, California, U.S.A. 8) J. Jagger and R. 8. Staffo.d Direct and indirect photoreactivation in Escherichia coli. Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, U.S.A. ", P. van de Putte, C. A. van Sluis, and A. Rðrach Genetic analysis of photoreactivation, dark reactivation and resistance to ultraviolet light in Escherichia coli. Medisch Biologisch Laboratorium RVO-TNO, Rijswijk, 2.., The Netherlands. I. Introduction Wavelengths below 3000 Å form a unique region of the photobiologicaui spectrum, the final effects on living cells being almost always deleterious. In this respect, their action resembles that of ionizing radiation. Chief among these effects, and the only one we consider here, 18 cell killing, Unlike our less fortunate Pellows in the ionizing radiation Meld, we have known for over thirty years the nature of the primary chromophore Por killing. The pioneering work of Gates (1930), showed that this was nucleic acid. One might have thought that such knowledge would rapidly lead to elucidation of the mechanism of ultraviolet killing, but little has been learned of the mechanism until quite recent times. In 1960, Beukers and Berends showed that ultraviolet could aimerize & frozen solution of thymne, and, further, that the same wavelength that produced the dimers could then split them upon melting of the solution. This finding inaugurated a resurgence of interest in the action of ultraviolet on biological materials and marks the beginning of the present phase of research, in which information on the molecular modes of action of ultraviolet is being accumulated at a remarkable rate. Way did it take thirty years to reach the point where we began to understand the mechanism of ultraviolet action? I think there are at least two important reasons. One is that ultraviolet, like ionizing, radiation 18 extremely efficient in killing cells. Doses of the order of 10° erg mms are needed to alter measurably the physical properties of large nucleic acids in vitro. Even tiny chemical changes cannot usually be detected with doses of less than about 104 erg 2. In contrast, the bacterium Dacherichia coli B, shows a 37% survival dose (Dzy) of about 100 erg moºs, and some bacteria have a Day as low as 1 erg mm2 (HU and Simson, 1961). Apparently, it was only through a combination of luck and the fact that clever people kept probing the matter, that the induction of thymine dimers was discovered. This induction 18 sensitive, drastic in its biological consequences, and, though ordinarily yielding a stable product, can nevertheless be reversed . in at least two ways. The point, to which we shall have occasion to refer later in this discussion, is that the so-called "biological doses" of ultraviolet (below about 1000 erg mm) are far below the doses required for most observable effects in vitro, and the products that we sees are therefore difficult to find. The other factor which contributed to the slow development of knowledge of mechanisms is that a considerable amount of biological information had to be accumulated. During the thirty-year period I mentioned, many protection and recovery phenomena, including photoreactivation (Rupert, 1964) and dark recovery, were discovered and characterized. "Dark recovery" includes phenomena like host-cell reactivation of bacteriophage (see Farm, 1963) and liquid holding recovery of bacteria (Roberts and Aldous, 1949). The point I wish to make 18 that, while we are all, in the last analysis, seeking to understand biological phenomena in molecular terms, this understanding cannot be obtained before we recognize the biological phenomena themselves. As a plant depends on the sun for both energy and control, so molecular biology must always turn to biology for (1) the source of all problems it seeks to Bolve, (2) much of the information that contributes to molecular solutions, evad (3) determination of the biological importance of effects that are discovered in the test tube. The series of papers that we are about to discuss 18 primarily concerned, not as the first part of the session title Indicates, with photochemistry of nucleic acids, but, as the second part indicates, with the biological implications of this photochemistry. We know that wtraviolet acts on nucleic acids in cells, and we know a good deal now about the photochemistry of this interaction in vitro. The question we ask here 18, which test-tube reactions are of . importance to the survival of the cell? We shall use certain abbreviations. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are familiar ones. Radiation of wavelength 2300-3000 Å, which 1.8 bighly lethal to smered cells, we call "ffar uitraviolet" (far w), and radiation of 3000-3800 A, which 18 relatively nonlethal, but highly effective for photoreactivation and induction of growth-division delay, we call "near ultraviolet" (near UV). If the term "U" 18 not modified, "far UV" 18 implied, and usually the wavelength 18 2537 Å. "Photoreactivation" 18 abbreviated as "PR". Discussion of the papers is in three parts. The first two papers concern effects of UV on RNA. Although it is believed that the primary effects of w in small cells are on DNA, this 18 a first-order approximation, and we are · reaching a level now where it behooves us to consider other possible effects. The next three papers deal with ultraviolet effects on protein-DNA linkage. Action spectra and other information have implicated nucleic acids as both chromophores and sites of primary lethal damage, but usually with varying degrees of protein involvement. The role of protein is not at all understood. The last three papers deal with recovery from UV damage to DNA, a subject that has implications reaching far beyond the relatively narrow Held of utraviolet photobiology.. Such repair ability may be crucial in the maintenance of species integrity over long periods of time. Without this stability, evolution would be arricuit if not trapossible. 2. Efects on RNA It seems likely that the nucleic acids are the only molecules which suffer photoreactivable damage (Jagger, 1958). There 18 no question that such damage. 18 produced in DNA. Is it produced in RNA? 'The evidence 18 largely affirmative. All of the plant viruses so far tested, except the rigid rods, are photoreactivable. Furthermore, the only rigid rods that have been examined ia this respect, tobacco mosaic virus and tobacco rattle virus; do not show PR 38 the intact virus 18 irradiated with UV, but do chow PR 1f their extracted RNA, 18 irradiated. Thus, the plant viruses without exception can be made to exhibit PR. Clover yellow 100saic virus 18 of particular laterest, since it is a flexible rod and therefore might be expected to show a behavior intermediate between that of the spheres and the rigid rods. However, this virus, like two other flexible rods, potato virus X and cabbage black ringspot virus, has now been shown by Chesoin?) to exhibit PR after irradiation of the intact virus. Thus, the flexible rods behave like the spheres . In site of all this evidence for the photoreactivability of RNA, no one has yet demonstrated a PR enzyme that works on RNA. Consequently, one cannot help wondering if plant virus photoreactivation may not be a basically different phenomenon, caused by some other response of the plant to photoreactivating light, which is a normal component of sunlight. An example of the sort of effect that is disturbing is shown by the data of Chessin for clover yellow nobal.C virus, shows 10 Mgure 1. Though at some UV doses there 18 unquestionably higher survival of irradiated virus when the plant 18 10 . the light, klus the fact that the slope of the curve in the light 18 much less steepthan in the dark, stuu thore 18 a very large lethal effect of the light alone (zero UV dose). Chesain thought this might be caused by a photodynamic action of the chlorophyll that 18 present in the virus bus pension. · However, he found that a colorless Inoculum, containing no chlorophy 21, caused the same sensitivity to photoreactivating light. Thus the cause of the lethality remains unknown. Pittman) has studied photoreactivation of kiling and mutation in haploid yeast. The mutation 18 the famous one leading to respiratory deficiency, resulting in the 60-called "petite” colonies. A variety of Indirect evidence, mostly genetic, has indicated that the mutation is nongenic. Pittman studied . both zero-point mutants, which give rise to entirely petite colonies, and delayed mutants, which yield one-half or ona-quarter mutant sectors. He also examined cultures in two states, stationary phase, consisting of 72-hour broth cultures, and a phase just at the inception of budding, obtained by growing stationary cells in fresh medium for 1 hour, at which time the DNA content has roughly doubled and the RNA content roughly quadrupled. Figure 2 illustrates his findings. Stationary-phase cells show PR of Kiling and of zero-point mutation, but no PR of delayed mutation. Cells at the Inception of budding are much more resistant to killing and to the induction of zero-point mutation, but are more sensitive to delayed mutation. The PR picture here is the same as for the stationary cells, namely, that PR of killing and of zero-point mutation occurs, while PR of delayed mutation does not. The interpretation of these findings 18 not clear. Pittman Assumes that both types of mutation are caused by UV danage to RNA, thus reflecting a different class of damage from that leading to killing, which is presumably 10 DNA. However, it 18 tempting to imagine that both zero-point mutations and killing are caused by damage to DNA. There are several reasons why this might be so: (1) roughly the same photoreactivable sector 18 found for both killing and zero-point mutation in both growth phases studied, (2) the enzyme · 10 yeast extracts that photoreactivates DNA has been shown by Rupert (1964) not to combine with UV-damaged RNA, and (3) the mutation of E. coll B/s to : resistance to page 12 was shown by Kelner (1949) to show PR of zero-point mutation but no PR of delayed mutation, and there 18 no reason to expect that the mutation in this system occurs in RNA. Finally, the evidence seems clear that the "petite" mutation of yeast 18 nongenic, but this does not say that it does not occur in DNA. Indeed, a variety of recent papers report the normal presence of DNA in cytoplasm, so that the mutation could even have a cytoplasmic locus and stu reside in DNA. Thus the results of Pittman, though very intriguing, remain unexplained at this time. 3. Protein Involvement Many action spectra for UV damage to cells are not identical to the absorption spectrum of nucleic acid, but suggest possible involvement of protein. It 18 also known that the chromosomes of larger cells contain protein. These facts have suggested to many workers that part of the lethal effect of w may involve protein, either as a chromophore, or as a substance that might bind to bites of u damage in DNA. Some experimental evidence for such DNA-protein binding 18 now appearing. Moroson, Gallegn, and Alexander “ have irradiated 10g-phase E. coli 15 T™ and then removed and studied the DNA, using detorgent Lysis. As the E cose increase8, the amount of DNA that can be extracted from the cells decreases. Furthermore, at relatively low doses (1600-6600 erg mm), the extracted and purified DNA has a higher viscosity than DNA from wirradiated cells. They suggest that this effect 18 caused by DNA-protein linkage, but offer no further evidence bearing on this matter. It would seem that their , suggestion could be easily tested by seeing whether proteolytic enzymes eliminate the increased viscosity. At doses roughly ten-folů higher, the viscosity falls to much lower levels than that of the control, and they attribute this to main-chain scission of the DNA. Such doses also decrease the hypochromic effect, but, contrary to exper cation, they do not alter the melting temperature. Finally, all of the effects they observe by irradiating cells occur at doses as much es 50 times lower than those required to produce the same effects by in vitro irradiation of DNA. This should rem.nd us of the fact that test-tube reactions cannot always be extrapolated to the biological situation. These results, however, all suffer from the possibility that the actual damage to DNA within the cell may be relatively moderate, becoming serious oniy as a result of the unnatural events that occur during extraction and purification of the DNA. It would be interesting to see if photoreactivation of the cells causes any reduction in the observed effects. If not, then they probably are involved in a minor fraction of the UV biological effect. These phenomena are of great interest, since they occur at doses close to the biologi.cal range. However, it 18 probable that much broader studies Will be required before we can say with assurance that they are biologically important. A simlar, but more detailed, study has been made by Smith (). He finds, in agreement with Moroson et al., that when E. coli is irradiated with increasing doses of UV, a proportional decrease 18 observed in the amount of DNA that can be extracted from the cells wità detergent. In a Com gradient, about 80% of the unextractable DNA floats on top of the gradient with protein, and about 50% of this DNA can be freed with trypsin. These results indicate that the unextractable DNA 18 bound to protein. Figure 3 shows how the percent recovery of DNA decreases with UV dose, the behavior being similar for strains B/r, B, and B, T. Thirty percent of the DNA in these strains 3,8 quite sensitive to the cross-linking, a dose of 1800 erg mm, which leaves 1% survival of B/s, causing a 10% drop 10 recoverable DNA. The top line in Figure 3 shows the loss of thymine due to thymine-dimer formation; the cross-linking reaction appears to be much more sensitive than thymine-dimer formation. Various experiments with thymine-requiring strains indicate that cro88-linking is related to the growth cycle. That portion of the genome that is being actively copied 18 the most sensitive to cross-linking. In vitro experiments also show cross-linking. DNA irradiated in the presence of bovine serum albumin shows extensive cross-linking, and some is obtained if either the DNA or the bovine serum albumin 18 irradiated separately before mixing, suggesting a remarkable stability of the molecular configuration that leads to cross-linking. These experiments show clearly that UV produces lesions in vivo that result in cross-linking of DNA with protein upon extraction by detergent. They also show that the same phenomenon occurs if irradiation of DNA and/or protein 18 carried out in vitro. Questions arise, however, regarding the biological significance of the reaction. For one thing, the cross-linking has only been shown to occur with extraction by detergent; it does not occur 1f the cells are first ground with alumina and then treated with detergent (Smdth, 1964), although Smdth (personal communication) believes this to be due to the drastic shearing caused by this treatment. Secondly, the phenomenon 18 not photoreactivable. U biological damage in these strains 18 roughly . 70% photoreactivable, suggesting that the cross-linking can involve not more than 30% of the biological damage. As with the experiments of Moroson et al., these results of Smith could possibly be artifacts caused by the extraction procedures. There 18 no doubt that w increases the susceptibility of the DNA to cross-linking. The question that arises 18 whether or not the cross-linking, actually occurs in the intact cell. Another possibility 18 that the cross-linking reflecte repair by dark enzymes that are still attached to the DNA at the time of extraction. Smith finds (personal commuication) that the same effect 18 found with strains B/r, which has dark recovery ability, and with Bale which shows no evidence of dark recovery (Harm, 1963). This would seem to eliminate the possibility of attachment of dark repair enzymes. However, it also shows that cross-linking bas nothing to do with the wusual sensitivity of Biol The next paper we shall discuss concerns effects of UV on echinoderm It concerns cells in which protein 18 intimately associated with the chromosomes. It has been shown in bacteria (Greer, 1960) and in phage (Sauerbier, 1961) that incorporation of 5-bromouracil or 5'-bromodeoxyuridine (BUAR) in place of thymine or thymdaine (TUR) in the DNA leads to a greatly increased sensitivity to radiation throughout the far ultraviolet and to a newly acquired sensitivity at the shorter end of the near wtraviolet, around 3100 A. 'There 18 also a concomitant sharp drop in photoreactivability. Cook) has extended this work to echinoderm wygotes (the sand dollar, Echinarachnius parma). Figure 4 shows, at the top, the normal time course of the first and second cleavagos in a pomulation fertilized at time zero. The middle part of the figure shows that addition of TaR and BUAR 81,multaneously at 30 moutes, followed by UV at 70 minutes, ceuses no delay in the first cleavage, but a large delay in the second cleavage. Falle no PR 18 seen in the first cleavage, an almost complete PR of the delay in second cleavage 18 observed. This behavior 18 similar to that found 18 no TdR or BUAR 18 added, suggesting that . using TR at all 18 that the cells do not survive 1f exposed indefinitely to BUAR alone.) If the BUAR 18 presented to the cells 20 minutes before Tar, it becomes incorporated, and the bottom of the figure shows that the second cleavage is even further delayed and PR 18 elind dated. The additional delay in second cleavage 18 much greater than appears from this figure; in terms of dose modification, it is usually about a factor of three. Thus, this system behaves in a manner parallel to that of phage and bacteria. Further experiments by Cook show that, although the first division can be delayed by U and this delay photoreactivated, provided the treatments are given early enough, uptake of BUAR does not affect either of these phenomena. Since the uptake of BUAR can occur during DNA replication in preparation for the first division, it is clear that BUSR, which affects only the second division, shows its effects only 11 It is present in parental DNA at the time of replication. A final point of great interest 18 that PR can occur at a time when the chromosomes are condensed, showing that the PR enzyme has access to the UV lesions even under these conditions. 4. Light and Dark Recovery We now move on to discussion of papers that concern protection and recovery processes. One of the most important concepts that is now emerging from this type of work 18 that a very great fraction indeed of U damage 18 probably rendered harmless by the call itself. Thus, it seems likely that the 100-fold difference in sensitivity of E. coli strains B and B-1(AL) reflects merely a difference in ability of the cells to recover from the initial damage, which is probably identical in both strains. This means, of . course, that when we observe the survival of typical cells, we see a population that, even under our "control" conditions, has already undergone a great deal of repair. The additional repair that we induce by various treatments may in some instances be little more than minor enhancements of recovery processes that are already very active. This complicates our study of recovery phenomena. However, it aj.co means that these phenomena are much more important than was previously thought, and they may have a very general application to genetic damage, whether induced by UV or by other agents. Hanawalt and Pettijohn) have made extensive studies of the physical nature of the DNA synthesized by E. coll during growth after w Irradiation. Cells of a thymine-requiring mutant (TAU-bar) were fed thymine or 5-bromouracil (a thymine analog), either stable or radioactive, in various combinations before and after U. The DNA was then extracted and examined in a Csal gradient. All their experiments involved a w dose of 500 erg mm-<. They fount that DNA synthesis was rather erratic after w. However, there was a steady loss with time of some of the thymide that was originally present in the DNA. This brings to mind the thymine dimer "excision" after uv, reported by Setlow and Carrier (1964) and by Boyce and Howard-Flanders (1964). Further experiments showed that, in cells incubated in nutrient medium for several hours after UV, DNA synthesis 18 quite abnormal. Normal incorporation of 5-bromouracil would lead to DNA of hybrid density, in which one strand 18 light and one heavy. After UV, however, the new DNA bas a density intermediate between normal and bybrid, suggesting that if one strand 18 light the other 18 heavy only in certain regions. This again fits with the notion of "excision" followed by new synthesis of certain parts of one or the other strand of a two-stranded DNA. Further more, they find evidence that this "cut-and-patch" phenomenon occurs throughout the genome. They then designed experiments to test the four models shown in Figure 5. In this figure, new DNA 18 indicated by the jagged line. The first model (A) strates normal synthesis, involving a single "growing point". Model B Lllustrates intermolecular cross-linking that has occurred after considerable synthesis within ons of the molecules. Now, with either of these models, the DNA extracted from a cell, which 18 usually broken down into several hundred hybrid density. This is not found, even after further breakage by moderate sonication. These two models are therefore rejected. Models C and D :Involve localized replication, either occurring in parallel on both strands, as in C, or of he "cut-and-patch" type, as in D. NOW, sonication plus thermal denaturation (separation of strands) should yield some heavy fragments if model C is correct. Such heavy fragments are not found, but fragments of hybrid density or of density between hybrid and normal are found. It is concluded that model C 18 wrong and model D 18 right. Finally, thermal denaturation alone, without sonication, causes little change in molecular weight, indicating that the phosphodiester backbone 18 not broken, and again contradicting model C and supporting model D. These findings provide support for the earlier observations of thymine dimer "excision" arter u. It was supposed in this earlier work that, if thymine dimers were excised, and if this led to viable cells, then the cut-out sections mist in some way have been patched up with the proper bases. The experiments of Harawalt and Pettijohn provide the firot clear evidence that patching does indeed take place in cells. They do not prove, however, that . the patched regions are truly back to normal. Finally, Hanawalt and Pettijohn have shown that photureactivation prevents all this wusual synthesis, presumably caused primarily by the presence of thymine dimers, which are known to be split by photoreactivation. It 18 of interest that the "dark repair" ("cut-and-patch"), even after several hours of post-UV growth, does not bring the cells back to the point of producing normal DNA, as does photoreactivation. This implies that dark repair and photoreactivation do not overlap completely, which is contrary to indications in the literature (Castellani, Jagger, and Setlow, 1964). A p2861ble explanation is that much of the abnormal synthes18 observed by Hanawalt and Pettijohn may be unrelated to the dark repair processes that lead to higher survival. Their w dose leaves only 4 x 10°4 surviving fraction, loss of thymine efter UV 18 drastic, and repair processes are expected to be complete after 90 minutes in nutrient medium, whereas their observed synthesis becomes progressively aberrant for hours after this time. It would seem that studies at lower w doses and under conditions where biological recovery could be studied in parallel, would provide more information on the biological significance of these processes . It has been found by Jagger and Stafford %) that there are two distinct types of photoreactivation 10 E. coli B. Before we can discuss their findings, however, we must first review what 18 known of the phenomenon called "photoprotection". It 18 generally known that photoreactivation involves a treatment with near ultraviolet or viblble radiation after UV, and that this treatment results in higher survival of the cells. In some systems, however, a higher survival may be obtained by such a treatment before w, and this effect has : been called "photoprotection". Jagger and coworkers have shown that the rate of photoprotection does not saturate at high dose rates of the protecting radiation, nor does the reaction show much dependence upon temperature. This behavior is quite different from that of the usual photoreactivation and suggests that the initial reaction in photoprotection 18 purely photochemical and does not involve enzymes, as does the usual photoreactivation. Futhermore, the action spectrum for photoprotection, which is much narrower than that for photoreactivation (see Figure 6) is identical to that for the induction of growth delay in the near ultraviolet. These and other data suggest that photoprotection operates by inactivating components of the electron transport system, thereby inducing both a growth delay and a division delay. This permits more time for dark repair systems within the cell to act upon the UV damage in pucleic acid, and this tbcam leads to higher survival. Thus, photoprotection acts in a very indirect way to repair UV damage, while photoenzymatic photoreactivation acts in a very direct way. Now, if this hypothesis of the mechanism of photoprotection is correct, then there seems to be no reason why one should rot be able to induce the required growth-division delay by near wtraviolet treatment after as well As before u. In most calls, such an "indirect photoreactivation" would be difficult to detect, because it would be masked by the usual "direct photoreactivation", caused by photoenzymes. However, a mutant of E. coli B, called "phr", was isolated a few years ago by Dr. W. Harm. This mutant does not contain the photoreactivating enzyme. However, Jagger and Stafford bave deronstrated photoreactivation in this mutant. This observation alone shows that there are two different mechanisms of photoreactivation in E. coli B. . Jagger and Stafford have shown that photoreactivation in this mutant 18 similar in every respect examined to photoprotection and dissimilar to the usual enzymatic photoreactivation. For example, it shows no saturation at high dose rate and only a sligiit temperature dependence. It occurs at 3342 A, but not at 4047 Å (see Figure 6). Finally, 3341 Å radiation induces a growth delay, but 4047 Å does not. Therefore, they conclude that they have observed in this organism an "indirect photoreactivation" that is similar in mechanism to photoprotection, and that therefore does not utilize a "photoreactivating enzyme". The question immediately arises whether or not this indirect photoreactivation may not be peculiar to this one mutant. They therefore examined photoreactivation in the parent strain, E. coli B. As expected, there was a high photoreactivation at both 3341 Å and 1047 Å. At 4047 A, there was a large dose-rate saturation and also a large temperature dependence, as expected for direct enzymatic photoreactivation. However, at 3341 Å, there was very little dose-rate saturation and only a small temperature coefficient, suggesting that photoreactivation at this wavelength is largely indirect. Recent support for these conclusions comes from examination of the fate of thymine dimers in the DNA of irradiated cells, In strain B phr", the photoreactivation at 3341 Å does not split thymne dimers (of course, 18 the calls are plated and allowed to lievelop, their dimers should be excised by dark enzymes). In the parent strain B, photoreactivation at 4047 Å splits most of the dimers, but 3341 Å splits many fewer dimers. While the exact extent of indirect photoreactivation at 3341 A has not yet been dearly determined, there seems little doubt that photoreactivation in the mutant 18 entirely indirect and that photoreactivation in the parent 18 almost entirely direct at 4047 A, but shows an Indirect component at 3341 A. One consequence of this work is that existing action spectra for photoreactivation must be re-examined in the light of possible contributions of indirect photoreactivation. For example, the spectrum of Jagger and Latarjet (1956) was done with starved cells in the logarithmic growh phase. Such cells are photoprotectable, and hence probably show indirect photoreactivation. Complications due to indirect photoreactivation might be avoided in two ways. Either one can work with a cell that does not show photoprotection (such as E. coli Bs-2) or have the cell in a state (such as the stationary plase for B/r) where it does not show photoprotection. Alternatively, one may obtain action spectra using both high and low dose rates, or both high and low temperatures. The difference between these spectra should then correspond to the absorption of the photoreactivation enzyme. This work should also serve to alert people to the fact that photoreactivation 18 not synonymous with thymine-dimer splitting except in very special cases. as the other hand, it is likely that thymine-dimer splitting is the major event in most photoreactivation. Several laboratories in the past few years have directed their efforts toward location on the bacterial chromosome of the genes controlling sensitivity to U. Van de Putte, vous Sluis, aad Rörsch) have been active in this effort. 22 . A summary of their findings is shown in Figure 7, where the circle represents the E. coli chromosome, and biochemical markers are indicated by symbols connected to the circle by short radial lines (e.g., threo and try). Markers affecting w sensitivity in strain B are shown inside the circle (syn, B.-22 phr, M) and those affecting the W sensitivity of strain K12 are shown outside the circle (hcr, dirz, darg). In E. coli B, the marker controlling production of the photoreactivating enzyme is located near the galactose marker. The marker controlling the shift to the sensitivity of strain Be 18 located between the markers for methionine and threonine, while the marker (syn) controlling a similar shift to a sensitivity like that of strain Rs-1 18 located some distance away, between the markers for xylose and streptomycin resistance. Finally, a merker controlling fulament production (ful), which is believed to be related to radiation sensitivity, 18 located near the marker for the photoreactivation enzyme. Clearly, markers for various factors that influence UV sensitivity are scattered widely on the genetic map, and even markers that seem very closely related in terms of phenotypic expression, e.g., syn and B-2, are found to lie at some distance from each other. In 12 strains, the marker for host cell reactivation (her), which appears to be phenotypically related to the syn and B. 2 markers, does indeed lie in the same general region of the chromosome. On the other hand, another mutant to UV sensitivity (dar.) 18 some distance away from this region. The marker concerned with fllamext formation in K12 (dir.) 18 not too far away from the Mul marker for strain B. The findings just described are from only one laboratory. If I were to include those of other laboratories, I could draw a quite complicated picture. This would reveal the same things thai are demonstrated by the simpler diagram that we have bere, namely, (1) that markers affecting UV sensitivity are scattered over the entire genetic map, (2) that phenotypically similar markers (e.g., B6-2 and dar,) may be widely separated, and (3) that apparently identical markers in different strains generally occur in the same regions, but not in identical positions. I think the interpretation we may draw from this 18 that (1) sensitivity to UV 18 affected by many genetic factors and (2) at least some of these factors interact. We cannot leave the work of van de Putte, van Sluis, and Rörsch without mention of their recent demonstration of a dark recovery in vitro. Their test system was the replication of bacteriophage 6x174 in bacterial spheroplasts. The DNA of this phage was isolated in the double-stranded form, irradiated with w, held for various times in an extract of cells of Micrococcus lysodeikticus, then allowed to infect the host spheroplasts. The host cells were hor”, that 18, unable to conduct host cell reactivation, and therefore probably lacked a dark recovery enzyme. However, the phage DNA incubated with the lysodeikticus extract showed higher survival, in proportion to the time of incubation with the extract. Therefore, the extract contains a repair factor. This factor is precipitable with anmonium sulfate, 18 inactivated by trypsin or by heat, and 18 nondialyzable. Thus it appears to be a protein and, since it catalyzes a biological reaction, may be further considered to be an enzyme. Thus, we now have systems that can conduct photoreactivation in vitro and also systems that can conduct dark repair in vitro, and all theses systems appear to utilize enzymes. Although the active components are probably present in only very small concentrations 10 cells, it nevertheless appears to be only a matter of time before they will be isolated and characterized. 5. Summary Many of the papers we have just reviewed concern actions of us on molecules other than DNA. Because of its genetic role, as well as its ability the to absorb radiation, DNA 18 certainly the most important biological target for w. However, RNA absorbs U quite as well as DNA, and must therefore also be considered. Two of the papers have dealt with effects of Won RNA. One : supports the interesting generality that every RNA plant virus that has been studied in this regard shows photoreactivation under the appropriate conditions, photoreactivate while the other suggests that it may be possible to see mutations produced in the RNA of yeast cells. Of course, there is a good deal of other evidence suggesting that damage to cellular RNA can be photoreactivated. The involvement of prote:n in UV'effects on cells is a very difficult thing to study. The papers we have considered here show clearly that UV irradiation of bacteria does make their DNA susceptible to cross-linking with protein, but they have not provided the final proof that this actually occurs inside cells. The non-photoreactivability of the phenomenon implies that such cross-linking cannot make up more than a minor fraction of the UV damage. Nevertheless, it could be a significant fraction, and we may hope that these studies will be actively pursued. The matter of extrapolation from one biological system to another arises frequently. For example, do our findings with bacteria apply to mammalian cells? PR studies suggest that yeast and bacteria behave in somewhat parallel fashion, and, even more striking, that echinoderm eggs and bacteria bebave similarly with regard to sensitization by BUAR and photoreactivation. These limited obser rations tend to support the idea that one can extrapolate from one biological system to another in a surprieing number of instances. We have considered some papers that deal with both light and dark i'ecovery in bacteria. It has been shown that an effect that apparently encourages dark recovery 18 caused by PR light, and that therefore there are two entirely different mechanisms of PR in some bacteria. This points up the error that people frequently fall into, namely of equating PR and thymine dimer splitting. PR does split thymine dimers in most systems, but it is rarely, 11 ever, the only PR effect. Dark riepair enzymes have now clearly been found in cell extracts. At least some dark recovery may act by cutting out thymine dimers and patching up the holes with complementary DNA, and we are now beginning to see more and more evidence that this amazing process actually does occur. We have seen also that the genetic basis of UV sensitivity 18 complex and this 18 a problem that is not likely to be solved without a great deal more work. 6. closing Remarks We have considered in this session a rather heterogeneous group of papers. It is difficult, therefore, to draw general factual conclusions. I think, however, that we might make some observations on the course of research in this field and the direction it may take in the future. The Straviolet photochemistry of the nucleic acids 18 now virtually a subject in itself. Much has been learned in the last five years, and vigorous work in this direction continues. Utraviolet photoblology of a descriptive type has also reached a certain maturity, as the result of some thirty years of research. The problem now 18 1:0 bring the two together, and it is a problem indeed. Many sophisticated studies have been made using all the modern methods of biochemistry and physical chemistry that are applicable. The workers who conduct these studies are to be congratulated for their perserverence and ingenuity. Nevertheless, a large proportion of these studies run into serious stumbling blocks when it comes to proof that such-and-such a molecular reaction studied in vitro plays such-and-such a role in vivo. Such proofs are not at all easy to come by. I feel that their demonstration in most instances requires an attitude of respect for and thorough knowledge of both the physical and the biological facts. Experts entirely on one side or the other are not likely to bridge the gap. A final point I should like to make 18 the necessity of considering all biological effects, regardless of their magnitude or ubiquity. In 1937, Hollaender and Claus observed a recovery of cells from UV by holding them in distilled water. It was a small effect. judging from subsequent literature, hardly anyone noticed it. In 1949, Roberts and Aldous rediscovered the effect, showed it could be quite large, but found that it occurred only in certain bacterial strains. It then became known by a few photobiologists who did not mind being accused of esoteric interests, but it seems quite certain that photochemists were unaware of the phenomenon. Now, essentially this same phenomenon 18 being used to explain hundred-fold differences in UV sensitivity, and, as I have noted in the Introduction, it promises to be one of the most important and far-reaching discoveries of ultraviolet photobiology. This, to me, is a very interesting little history. References R• Beukers and W. Berends 1960 Blochim. Blophys. Acta 41, 550. R. P. Boyce and P. Howard-Flanders 1964 Proc. Natl. Acad. Sci. U.S. 51, 293. A. Castellant, J. Jagger, and R. B. Setlow 1964 Science 113, 117. F. I. Gates 1930 J. Gen. Fhysiol. 14, 31. S. Greer 1960 J. Gen. Microbiol. 22, 618. W. Harm 1963 2. für Vererbungslehre 94, 67. R. F. Hull and E. Simson 1962 J. Gen. Microbiol. 24, 1. A. Hollaender and W. Maus 1937 Bulletin Natl. Research Council, No. 100, Natl. Acad. Sci., U.S., Washington, D. C. J. Jagger 1958 Bacteriol. Revb. 22, 99. J. Jagger and R. Latar jet 1956 Anni inst. Pasteur 21, 858. J. Jagger and R. S. Stafford 1962 Photochem. Photobiol. 1, 245. A. Kelner 1949 J. Bacteriol. 58, 521. R. B. Roberts and E. Aldous 1949 J. Bacteriol. 31, 363. C. S. Rupert 1964 Photopbysiology, vol. 2, ed. by A. C. Giese (Academic), Chapter 19. W. Sauerbier 1960 Virology 15, 465. R. B. Setlow and W. L. Carrier 1964 Proc. Natl. Acad. Sci., U.S. 21, 226. K. C. Smith 1962 Biochem. Biophys. Research Communications 8, 157. K. C. Smith 1964 Photopbysiology, vai. 2, ed. by A. C. Giese (Academic), Coapter 20. 27 Legends Fig. 1 Survival of local lesions in Boraphrena globosa induced within 13 days by clover yellow mosaic virus. Circles refer to plants kept dark for 24 hours after UV irradiation, triangles to plants left in sunlight. Experiments started at noon. (From data of Chessin.) Fig. 2 Survival and mutation to respiration deficiency in yeast after UV irradiation of cells in the stationary phase (parts 1 and 2) and at inception of budding (parts 3 and 4). In parts 1 and 3, solid circles represent survival to uv, open circles survival to UV plus maximum photoreactivation. In parts 2 and 4, circles refer to zero-point mutations, squares to delayed mutations, solid symbols to treatment with uv, open symbols to UV plus maximum photoreactivation. Source of photoreactivating light was a 500-watt projection bulb with a one-inch filter of 0.2 N Cusq. (Pittman.) Fig. 3 Extractability with sodium lauryl suliate of free DNA from E. coli strains B/r, B, and B,1º, as a function of UV dose to the bacterium. Also shown is the loss of recovery of thymine from DNA of E. coli B/r, taken as a measure of rate of formation of thymine dimers. (From Smith, 1962.) Fig. 4 cieavage delay and its photoreactivation after UV irradiation of sand dollar zygotes. Upper curve - no irradiation. Middle curve - irradiated, but 5 mg/ml thymidine (TdR) added at same time as I wo/mi 5'-brunadeaxyur idine (BUdR), thus preventing BUdR incorporation. Bottom curve - irradiated, TdR added 20 min after BUdR, thus permitting BUAR incorporation into DNA. Cells in light were exposed from immediately after UV until completion of experiment. Temperature held at 13°. Steps were taken to minimize inactivating effects of photoreactivating light on BUdR-DNA. (Cook.) --. 2000 mm . . Ni 9 NATIF . JUNCLASSIFIED ORNL . .. . Р 85 * 1S - -- - - - - - S . T ! - ..:. :. 1964 8 / 26 DATE ISSUANCE MICROCARD DTIE • - - . . 28 Fig. 5 Possible models for the arrangement of density label (5-bromouracil) in DNA extracted from UV-irradiated E. coli TAU-bar. The jagged line represents regions conta ining the density label. (A) molecules conta ining a single point of replication or transition, (B) linked hybrid and normal molecules, (c) a molecule containing nultiple points of semi-conservative replication, and (D) molecules with sections of repair reconstruction in one or both strands. (llanawalt and Pettijohn). . KA Fig. 6 Action spectra for photoprotection from killing in E. coli 8 (solid line) and for photoreactivation of killing in E. coli B/r (broken line). Curves are normalized to 200 at wavelength of peak efficiency. (from Jagger and Stafford, 1962, and Jagger and Latar jet, 1956.) Fig. 7 Genetic map for chromosome of E. coll. Biochemical markers represented by symbols connected to circie by slıort radial lines. Markers affecting UV sensitivity (no radial lines) shown inside the circle for E. coli B and outside the circle for E. coli K12. (Van de Putte, Van Sluis, and Rörsch.) C'MESSIN -- - . . Lii - LI 1 . 1 . . I . Suevival -- . . • . - . . . . - 358.61 VAG:n ..A. SEMI-LOGARITHMIC KCUFFEL A ESSCA CO. 2 CYCLES X 70 DIVISIONS Tilililil .. . 1 . SNS T.. . . . .. - - Li. I. i III co Fio. O uw bese sec.) > Ellulit : T +PR . 10. O . - STATIONARY PHASE IF INCEPTION OF BUDDING 0,0, "ZERO POINT" MUTATIONS 0,01 DELAYED MUTATIONS 0,0: DARK 0,03 + PR LIGHT (60 MIN) 0,0°: "ZERO POINT" MUTATIONS 0,0. DELAYED MUTATIONS 0,0 : DARK 0,0:7 PR LIGHT (60 MIN) 487 PERCENT MUTANTS STATIONARY . PHASE INCEPTION OF BUDDING ON - 100 o 200 300 400 600 0 100 200 300 400 500 UV DOSE (SEC) Fic.a To THYMINE (8/1) Lond °. DNA- F16.3 Porcont. Recovery • Bir 99% Killing(8/11 2.0 . Ergs/mm2(x 10°*) . . . ... .. :.mooch. :: .. .. :.:; momomowym ---------- ---....... FIRST CLEAVAGE SECOND CLEAVAGE 100 CONTROL PERCENT 50 CLEAVED 100 LIGHT DARK PERCENT TAR + BUDR CLEAVED 100 PERCENT DARK Y LIGHT BUDR TAR UV CLEAVED 50 100 200 F16.4 MINUTES AFTER FERTILIZATION --. J. JAOOLA and R. S. STARTOND 1001 -..- At the hig! P. aerugina P. aerugin. C PR (8/1) - .. 10,793 - PP(8) .. - - - RELATIVE EFFICIENCY PER INCIDENT QUANTUM . It is phenomen A possible ultraviolet and the vi to be mos may well - - . - . . . . . - .. (0) .. . 3000 3200 3400 3600 3400 4000 4200 WAVE LENGTH (A) 4400 ... mamm ogamaphommes more para mais ' N It was PP shows Two of the the same is possible one being high targe onc, is no caused by difference as great i on the u.Y The rol the chrool action spy to differe among th: to daylig mediated Phorcy involve cl that there act upon If this wel oy its in An a action so riscs. Nel ... iii! ... Fisio محامينات threo pro C met 's-2 hcr isol xyl v phrt gal fil pyr syn try Sm F16.7 argo? dal5 his 4 END 1.