The FASEB Journal • Milestone
Tritiated Thymidine: Xeroderma Pigmentosum and DNA Repair James E. Cleaver,1 Department of Dermatology, University of California–San Francisco, San Francisco, California, USA In 1965, I was a postdoc at the Massachusetts General Hospital, recently arrived from the United Kingdom and faced with a major crisis. My supervisor, on whose grant my livelihood depended, was abandoning the United States for New Zealand. I knew no one and had no prospects back in England. My wife and I had arrived by tramp steamer the previous year with less than $10 in our pockets. A major power outage proved our saving grace. In November 1965, the electric power grid failed across most of the northeastern United States. I was at a meeting of the Biophysical Society in Philadelphia and could not leave. Marooned with me was Robert Painter, who had a vacancy in his new laboratory in San Francisco. This lucky encounter resulted in a long-lasting collaboration and career. My graduate work in Cambridge, UK, had employed [3H] thymidine and autoradiography. I had failed to appreciate how new it then was, and how seminal Painter’s role had been in this tracer’s development and subsequent major applications. In fact, when Rasmussen and Painter published a 1964 Nature paper showing that [3H] thymidine would label all cells after doses of ultraviolet (UV) light (1), my then Ph.D. supervisor Charles L. Smith said, “I do not understand that, but Painter knows what he’s doing, so it must be true!” Little did I realize, not only was it true, but it would be the cornerstone of many subsequent discoveries, least of all my own. [3H] thymidine was first synthesized at Brookhaven National Laboratory by a team led by Walter (Pete) Hughes (2). Two motivations appear to have prompted its development: one was the interest in irradiating cells from the inside, influenced by the Atomic Energy Agency (AEC), a major source of funds; the other was the realization that the short range of the [3H]  particle coupled with autoradiography would afford greater resolution of the location of DNA synthesis in nuclei and on chromosomes than [32P], which had been in use for some years for such work (Fig. 1). [3H] has a half-life of 12.26 yr; the  particles have a maximum energy of 18 keV and a mean energy of 5.7 keV, with a mean range in tissue of 0.45 m and in the autoradiographic emulsion of 0.13 m). [3H] thymidine was initially synthesized by catalytic exchange from the carboxyl group of labeled acetic acid to thymidine, followed by purification (3, 4), 2742
Figure 1. Autoradiograph of human epithelial cells labeled for 4 h with high-specific-activity [3H] thymidine showing heavily labeled cells in S phase and unlabeled cells in G1 and G2 phases.
although the precise experimental details were not published. Commercially, several more specific forms of [3H] thymidine became available, with the label either on the 6 position of the pyrimidine ring or in the 5-methyl group. The latter provided the highest specific activity when all three of these hydrogens were replaced with [3H]. This version was invaluable when we investigated the low levels of incorporation in later experiments with cells exposed to UV light. In due course, [14C] thymidine appeared on the scene. One company’s unpublicized technique for manufacture of [14C] thymidine was revealed when a batch appeared to increase photosensitivity (5). This was the result of contamination in manufacture by which synthesis was started from bromodeoxyuridine, some of which persisted into the final product. [3H] THYMIDINE AT WORK Once [3H] thymidine became available commercially, initially by Schwarz Laboratories and then for many 1 Correspondence: Department of Dermatology, University of California-San Francisco, San Francisco, CA 94143, USA. E-mail: [email protected] doi: 10.1096/fj.14-0702ufm
0892-6638/14/0028-2742 © FASEB
years by that company’s successor, Schwarz Mann, its use expanded, and a remarkable series of discoveries ensued. The cell cycle and its division into mitosis, Gap1, DNA synthesis, and Gap2 [i.e., M, G1, S (for synthesis) and G2] phases were originally defined by Howard and Pelc (6), using [32P] labeling of Vicia faba root tips. Cell cycle analysis and measurement of the duration of the phases rapidly followed when [3H] thymidine became available (7, 8). The theoretical biologist Henry Quastler had the penetrating idea that the kinetics by which labeled cells entered mitosis was a “record” of their previous interphase staging at the time the pulse label was given (9). The labeling of chromosomes in S phase; their semiconservative segregation at cell division; the opposite polarity of the two subunits of the chromosome, were soon discovered by Taylor using [3H] thymidine (2, 10, 11). Taylor struggled with the segregation patterns, which implied that each chromosome is a single DNA molecule, and he invented elaborate hypotheses. His results were later validated by the discovery of uninemy (12). Later, [3H] thymidine was instrumental in linking heterochromatin to inactive genes and DNA synthesis at the end of S phase (13–15). The concept of cell kinetics in whole tissues rapidly developed with the idea of a replicating “stem cell” population and a subsequent maturating population, exemplified by analysis of [3H] thymidine labeling in the villi of the small intestine (3). Given the interests of the developers of [3H] thymidine, it is understandable that there was immediate interest in using it to study the effects of ionizing radiation (16 –18). Radiation-induced suppression of [3H] thymidine incorporation demonstrated the vulnerability of the cell cycle and DNA synthesis to relatively low doses. These radiation-induced cell cycle delays prefigured much recent work in which the intricacies of signal transduction and DNA repair have filled in the details. A similar use of [3H] labeled cytidine showed that RNA was synthesized in the nucleus, but much was found in the nucleolus prior to export to the cytoplasm (19 –21). I recollect a lively discussion in Cambridge when Henry Harris first showed that much of the [3H]-labeled RNA was also, incomprehensibly at the time, degraded in the nucleus. This observation found explanation many years later with the discovery of split genes and RNA processing. To some amusement, there were a series of papers describing “pitfalls” of both high-specific-activity and of low-specific-activity [3H] thymidine (22–24). Both derived from the simple relationship, mM ⫽ (mCi/ml)/ (Ci/mmol). Low specific activity meant high concentration that resulted in thymidine toxicity that could be reversed with addition of deoxycytidine (4). High specific activity was consequently a low concentration but a high tritium dose rate (25, 26). Estimating actual doses from tritium decays proved conceptually tricky because the actual intracellular volume and path within which XERODERMA PIGMENTOSUM AND DNA REPAIR
the energy dissipated was difficult to define (27). We approached this problem experimentally by measuring DNA breaks in frozen cells labeled with [3H] thymidine and comparing the breakage with that from ionizing radiation (28). This gave an equivalence of 0.48 rad/decay, close to the calculated equivalence when [3H] thymidine was assumed to produce a uniform distribution of ionizations within the mammalian nucleus (27).
SERENDIPITY AND SUNLIGHT By the time I joined Robert Painter in San Francisco, I had parlayed my early work with [3H] thymidine into a small book (4), thus getting this bug out of my system early, earning a small amount of cash, and becoming a little better known in the field. It was with Painter that we made what is probably the final discovery uniquely facilitated by the use of [3H] thymidine. Bob had previously reported on the induction of extensive labeling of cells after exposure to UV light (1), which Djordevic and Tolmach named unscheduled DNA synthesis (UDS; ref. 29 and Fig. 2). It seemed reasonable to assume that UDS represented DNA repair, especially because Regan, Trosko, Kasschau and Carrier had shown that UV photoproducts were excised from human cells over time (30, 31). But we had no mutants to demonstrate functional significance; these came much later in the hands of David Busch (32) and Larry Thompson (33). The latter developed out of pioneering work led by Siminovich on mammalian temperature-sensitive mutants for cell cycle progression (34). At this point, serendipity played into our hands. The San Francisco Chronicle at the time had an outstanding science correspondent, David Perlman, and in 1967 he described a human cancer that was
Figure 2. Autoradiograph of human epithelial cells irradiated with UV light (⬃5 J/m2, 254 nm) and then labeled for 4 h with high-specific-activity [3H] thymidine showing heavy Sphase labeling in 6 cells and light labeling in the remaining cells due to nucleotide excision repair. 2743
inherited but also caused by sun exposure: xeroderma pigmentosum (XP). When I suggested this might be a human mutant in DNA repair, Bob Painter commented, “It’s a crazy idea, but at your stage you’ve nothing to lose!” But he did not interfere; mentoring comes in many forms, and this was among the best. We soon showed that UDS measured with [3H] thymidine was reduced in cells from patients with XP; we had discovered one of the first examples of a human mutation that predisposed to cancer and identified its mechanism (35, 36). Michael Bishop, to become a Nobelist and later Chancellor of the University of California–San Francisco, commented that “While I was still in medical school James Cleaver recognized xeroderma pigmentosum as a deficiency in the repair of DNA damage caused by ultraviolet light . . . I have been a believer in the somatic mutation hypothesis of cancer ever since.” (37). Although first impressions of UDS were that the distribution of silver grains in autoradiographs represented a subnuclear distribution of repaired sites (Fig. 2), this was an illusion caused by the randomness of the [3H]  decays and their range in tissue and the recording emulsion. For UDS and many other uses, [3H] thymidine has now been replaced by fluorescent markers, which show repair distributed more uniformly at the resolution of light microscopy (38). Fluorescent markers provide higher resolution without the messy and expensive problems of radiation safety and disposal. But in its time, [3H] thymidine was revolutionary and led to many important biological discoveries. I was fortunate to be part of this era and to have worked for many years with Bob Painter, who published his own reminiscences (39). Work that was fueled in part by the U.S. Atomic Energy Agency’s emphasis on radiation biology and the innovation of the Brookhaven team in synthesizing [3H] thymidine. I owe a great deal to my early mentors in Cambridge, UK, Charles. L. Smith and Philip. P. Dendy. In San Francisco, I am indebted to Robert Painter, Shelly Wolff, and Harvey Patt, who guided and indulged me for many years. I am also grateful to Gregory H Thomas, who carried out the photography for Figs. 1 and 2 and provided much early technical support. Financial support came from the Atomic Energy Agency, which became the Energy Research and Development Agency and finally the Department of Energy. Their unrestricted approach to basic research during early times gave us the opportunity to discover and explore new worlds (. . . “to boldly go where no one has gone before!”).
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4. 5.
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8. 9.
10. 11.
12. 13.
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REFERENCES 23. 1.
2744
Rasmussen, R. E., and Painter, R. B. (1964) Evidence for repair of ultraviolet damaged deoxyribonucleic acid in cultured mammalian cells. Nature 203, 1360 –1362
Vol. 28
July 2014
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Taylor, J. H., Woods, P. S., and Hughes, W. L. (1957) The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. Proc. Natl. Acad. Sci. U. S. A. 43, 122–128 Hughes, W. L., Bond, V. P., Brecher, G., Cronkite, E. P., Painter, R. B., Quastler, H., and Sherman, F. G. (1958) Cellular proliferation in the mouse as revealed by autoradiography with tritiated thymidine. Proc. Natl. Acad. Sci. U. S. A. 44, 476 –483 Cleaver, J. E. (1967) Thymidine Metabolism and Cell Kinetics, North-Holland, Amsterdam Elkind, M. M., and Ley, R. D. (1976) Spurious photolability of DNA labeled with (14C)-thymidine. Biochem. Biophys. Res. Commun. 68, 691–698 Howard, A., and Pelc, S. R. (1953) Synthesis of deoxyribonucleic acid in normal and irradiated cells and its relation to chromosome breakage. Heditary 6 (Suppl.), 261–273 Lajtha, L. G., and Oliver, R. (1959) The application of autoradiography in the study of nucleic acid metabolism. Lab. Invest. 8, 214 –221 Lajtha, L. G. (1963) On the concept of the cell cycle. J. Cell. Physiol. 62(Suppl. 1), 143–145 Quastler, H., and Sherman, F. G. (1959) Cell population kinetics in the intestinal epithelium of the mouse. Exp. Cell Res. 17, 420 –438 Taylor, J. H. (1989) DNA synthesis in chromosomes: implications of early experiments. BioEssays 10, 121–124 Taylor, J. H. (1991) Tritium-labeled thymidine and early insights into DNA replication and chromosome structure. Trends Biochem. Sci. 19, 479–487 Kavenoff, R., and Zimm, B. H. (1973) Chromosome-sized DNA molecules from Drosophila. Chromosoma 41, 1–27 Rowley, J., Muldal, S., Gilbert, C. W., Lajtha, L. G., Lindsten, J., Fraccaro, M., and Kaijser, K. (1963) Synthesis of deoxyribonucleic acid on x-chromosomes of an XXXXYY male. Nature 197, 251–252 Gilbert, C. W., Muldal, S., Lajtha, L. G., and Rowley, J. (1962) Time-sequence of human chromosome duplication. Nature 195, 869 –873 Gilbert, C. W., Muldal, S., and Lajtha, L. G. (1965) Rate of chromosome duplication at the end of the deoxyribonucleic acid synthetic period in human blood cells. Nature 208, 159 –161 Lajtha, L. G., Oliver, R., Kumatori, T., and Ellis, F. (1958) On the mechanism of radiation effect on DNA synthesis. Radiat. Res. 8, 1–16 Painter, R. B., and Robertson, J. S. (1959) Effect of irradiation and theory of role of mitotic delay on the time course of labeling of HeLa S3 cells with tritiated thymidine. Radiat. Res. 11, 206 –217 Painter, R. B., Mcalpine, V. W., and Germanis, M. (1961) The relationship of the metabolic state of deoxyribonucleic acid during X-irradiation to HeLa S3 giant cell formation. Radiat. Res. 14, 653–661 Perry, R. P. (1960) On the nucleolar and nuclear dependence of cytoplasmic RNA synthesis in HeLa cells. Exp. Cell Res. 20, 216 –220 Perry, R. P., and Errera, M. (1961) The role of the nucleolus in ribonucleic acid-and protein synthesis. I. Incorporation of cytidine into normal and nucleolar inactivated Hela cells. Biochim. Biophys. Acta. 49, 47–57 Feinendegen, L. E., Bond, V. P., Shreeve, W. W., and Painter, R. B. (1960) RNA and DNA metabolism in human tissue culture cells studied with tritiated cytidine. Exp. Cell Res. 19, 443–459 Painter, R. B., and Rasmussen, R., E. (1964) A pitfall of low specific activity radioactive thymidine. Nature 201, 409 –410 Lajtha, L. G. (1958) Pitfalls of specific radioactivity in measuring synthesis of deoxyribonucleic acid. Nature 181, 1609 Oliver, R., and Lajtha, L. G. (1960) Hazard of tritium as a deoxyribonucleic acid label in man. Nature 186, 91–92
The FASEB Journal 䡠 www.fasebj.org
CLEAVER
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Drew, R. M., and Painter, R. B. (1959) Action of tritiated thymidine on the clonal growth of mammalian cells. Radiat. Res. 11, 535–544 Drew, R. M., and Painter, R. B. (1962) Further studies on the clonal growth of Hela S3 cells treated with tritiated thymidine. Radiat. Res. 16, 303–311 Strauss, B. S. (1958) The genetic effect of incorporated radioisotopes: the transmutation problem. Radiat. Res. 8, 234 –247 Cleaver, J. E., Thomas, G. H., and Burki, H. J. (1972) Biological damage from intranuclear tritium: DNA strand breaks and their repair. Science 177, 996 –998 Djordjevic, B., and Tolmach, L. J. (1967) Responses of synchronous populations of HeLa cells to ultraviolet irradiation at selected stages of the generation cycle. Radiat. Res. 32, 327–346 Trosko, J. E., and Kasschau, M. R. (1967) Study of pyrimidine dimers in mammalian cells surviving low doses of ultraviolet radiation. Photochem. Photobiol. 6, 215–219 Regan, J. D., Trosko, J. E., and Carrier, W. L. (1968) Evidence for excision of ultraviolet-induced pyrimidine dimers from the DNA of human cells in vitro. Biophys. J. 8, 319 –325 Busch, D., Greiner, C., Lewis, K., Ford, R., Adair, G., and Thompson, L. (1989) Summary of complementation groups of
33.
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UV-sensitive CHO cell mutants isolated by large-scale screening. Mutagenesis 4, 349 –354 Thompson, L. H., Rubin, J. S., Cleaver, J. E., Whitmore, G. F., and Brookman, K. (1980) A screening method for isolating DNA repair-deficient mutants of CHO cells. Somatic Cell Genet. 6, 391–405 Thompson, L. H., Mankovitz, R., Baker, R. M., Till, J. E., Siminovich, L., and Whitmore, G. H. (1970) Isolation of temperature-sensitive mutants of L-cells. Proc. Natl. Acad. Sci. U. S. A. 66, 377–384 Cleaver, J. E. (1968) Defective repair replication in xeroderma pigmentosum. Nature 218, 652–656 Cleaver, J. E. (1969) Xeroderma pigmentosum: a human disease in which an initial stage of DNA repair is defective. Proc. Natl. Acad. Sci. U. S. A. 63, 428 –435 Bishop, J. M. (2003) How to Win the Nobel Prize: An Unexpected Life in Science, Harvard University Press, Cambridge, MA, USA Nakazawa, Y., Yamashita, S., and Lehmann, A. R. (2010) A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives. DNA Repair (Amst.) 9, 506 –516 Painter, R. B. (1990) On tritium, DNA, and serendipity. Radiat. Res. 121, 117–119
The opinions expressed in editorials, essays, letters to the editor, and other articles comprising the Up Front section are those of the authors and do not necessarily reflect the opinions of FASEB or its constituent societies. The FASEB Journal welcomes all points of view and many voices. We look forward to hearing these in the form of op-ed pieces and/or letters from its readers addressed to [email protected]. XERODERMA PIGMENTOSUM AND DNA REPAIR
2745
Tritiated Thymidine: Xeroderma Pigmentosum and DNA Repair James E. Cleaver,1 Department of Dermatology, University of California–San Francisco, San Francisco, California, USA In 1965, I was a postdoc at the Massachusetts General Hospital, recently arrived from the United Kingdom and faced with a major crisis. My supervisor, on whose grant my livelihood depended, was abandoning the United States for New Zealand. I knew no one and had no prospects back in England. My wife and I had arrived by tramp steamer the previous year with less than $10 in our pockets. A major power outage proved our saving grace. In November 1965, the electric power grid failed across most of the northeastern United States. I was at a meeting of the Biophysical Society in Philadelphia and could not leave. Marooned with me was Robert Painter, who had a vacancy in his new laboratory in San Francisco. This lucky encounter resulted in a long-lasting collaboration and career. My graduate work in Cambridge, UK, had employed [3H] thymidine and autoradiography. I had failed to appreciate how new it then was, and how seminal Painter’s role had been in this tracer’s development and subsequent major applications. In fact, when Rasmussen and Painter published a 1964 Nature paper showing that [3H] thymidine would label all cells after doses of ultraviolet (UV) light (1), my then Ph.D. supervisor Charles L. Smith said, “I do not understand that, but Painter knows what he’s doing, so it must be true!” Little did I realize, not only was it true, but it would be the cornerstone of many subsequent discoveries, least of all my own. [3H] thymidine was first synthesized at Brookhaven National Laboratory by a team led by Walter (Pete) Hughes (2). Two motivations appear to have prompted its development: one was the interest in irradiating cells from the inside, influenced by the Atomic Energy Agency (AEC), a major source of funds; the other was the realization that the short range of the [3H]  particle coupled with autoradiography would afford greater resolution of the location of DNA synthesis in nuclei and on chromosomes than [32P], which had been in use for some years for such work (Fig. 1). [3H] has a half-life of 12.26 yr; the  particles have a maximum energy of 18 keV and a mean energy of 5.7 keV, with a mean range in tissue of 0.45 m and in the autoradiographic emulsion of 0.13 m). [3H] thymidine was initially synthesized by catalytic exchange from the carboxyl group of labeled acetic acid to thymidine, followed by purification (3, 4), 2742
Figure 1. Autoradiograph of human epithelial cells labeled for 4 h with high-specific-activity [3H] thymidine showing heavily labeled cells in S phase and unlabeled cells in G1 and G2 phases.
although the precise experimental details were not published. Commercially, several more specific forms of [3H] thymidine became available, with the label either on the 6 position of the pyrimidine ring or in the 5-methyl group. The latter provided the highest specific activity when all three of these hydrogens were replaced with [3H]. This version was invaluable when we investigated the low levels of incorporation in later experiments with cells exposed to UV light. In due course, [14C] thymidine appeared on the scene. One company’s unpublicized technique for manufacture of [14C] thymidine was revealed when a batch appeared to increase photosensitivity (5). This was the result of contamination in manufacture by which synthesis was started from bromodeoxyuridine, some of which persisted into the final product. [3H] THYMIDINE AT WORK Once [3H] thymidine became available commercially, initially by Schwarz Laboratories and then for many 1 Correspondence: Department of Dermatology, University of California-San Francisco, San Francisco, CA 94143, USA. E-mail: [email protected] doi: 10.1096/fj.14-0702ufm
0892-6638/14/0028-2742 © FASEB
years by that company’s successor, Schwarz Mann, its use expanded, and a remarkable series of discoveries ensued. The cell cycle and its division into mitosis, Gap1, DNA synthesis, and Gap2 [i.e., M, G1, S (for synthesis) and G2] phases were originally defined by Howard and Pelc (6), using [32P] labeling of Vicia faba root tips. Cell cycle analysis and measurement of the duration of the phases rapidly followed when [3H] thymidine became available (7, 8). The theoretical biologist Henry Quastler had the penetrating idea that the kinetics by which labeled cells entered mitosis was a “record” of their previous interphase staging at the time the pulse label was given (9). The labeling of chromosomes in S phase; their semiconservative segregation at cell division; the opposite polarity of the two subunits of the chromosome, were soon discovered by Taylor using [3H] thymidine (2, 10, 11). Taylor struggled with the segregation patterns, which implied that each chromosome is a single DNA molecule, and he invented elaborate hypotheses. His results were later validated by the discovery of uninemy (12). Later, [3H] thymidine was instrumental in linking heterochromatin to inactive genes and DNA synthesis at the end of S phase (13–15). The concept of cell kinetics in whole tissues rapidly developed with the idea of a replicating “stem cell” population and a subsequent maturating population, exemplified by analysis of [3H] thymidine labeling in the villi of the small intestine (3). Given the interests of the developers of [3H] thymidine, it is understandable that there was immediate interest in using it to study the effects of ionizing radiation (16 –18). Radiation-induced suppression of [3H] thymidine incorporation demonstrated the vulnerability of the cell cycle and DNA synthesis to relatively low doses. These radiation-induced cell cycle delays prefigured much recent work in which the intricacies of signal transduction and DNA repair have filled in the details. A similar use of [3H] labeled cytidine showed that RNA was synthesized in the nucleus, but much was found in the nucleolus prior to export to the cytoplasm (19 –21). I recollect a lively discussion in Cambridge when Henry Harris first showed that much of the [3H]-labeled RNA was also, incomprehensibly at the time, degraded in the nucleus. This observation found explanation many years later with the discovery of split genes and RNA processing. To some amusement, there were a series of papers describing “pitfalls” of both high-specific-activity and of low-specific-activity [3H] thymidine (22–24). Both derived from the simple relationship, mM ⫽ (mCi/ml)/ (Ci/mmol). Low specific activity meant high concentration that resulted in thymidine toxicity that could be reversed with addition of deoxycytidine (4). High specific activity was consequently a low concentration but a high tritium dose rate (25, 26). Estimating actual doses from tritium decays proved conceptually tricky because the actual intracellular volume and path within which XERODERMA PIGMENTOSUM AND DNA REPAIR
the energy dissipated was difficult to define (27). We approached this problem experimentally by measuring DNA breaks in frozen cells labeled with [3H] thymidine and comparing the breakage with that from ionizing radiation (28). This gave an equivalence of 0.48 rad/decay, close to the calculated equivalence when [3H] thymidine was assumed to produce a uniform distribution of ionizations within the mammalian nucleus (27).
SERENDIPITY AND SUNLIGHT By the time I joined Robert Painter in San Francisco, I had parlayed my early work with [3H] thymidine into a small book (4), thus getting this bug out of my system early, earning a small amount of cash, and becoming a little better known in the field. It was with Painter that we made what is probably the final discovery uniquely facilitated by the use of [3H] thymidine. Bob had previously reported on the induction of extensive labeling of cells after exposure to UV light (1), which Djordevic and Tolmach named unscheduled DNA synthesis (UDS; ref. 29 and Fig. 2). It seemed reasonable to assume that UDS represented DNA repair, especially because Regan, Trosko, Kasschau and Carrier had shown that UV photoproducts were excised from human cells over time (30, 31). But we had no mutants to demonstrate functional significance; these came much later in the hands of David Busch (32) and Larry Thompson (33). The latter developed out of pioneering work led by Siminovich on mammalian temperature-sensitive mutants for cell cycle progression (34). At this point, serendipity played into our hands. The San Francisco Chronicle at the time had an outstanding science correspondent, David Perlman, and in 1967 he described a human cancer that was
Figure 2. Autoradiograph of human epithelial cells irradiated with UV light (⬃5 J/m2, 254 nm) and then labeled for 4 h with high-specific-activity [3H] thymidine showing heavy Sphase labeling in 6 cells and light labeling in the remaining cells due to nucleotide excision repair. 2743
inherited but also caused by sun exposure: xeroderma pigmentosum (XP). When I suggested this might be a human mutant in DNA repair, Bob Painter commented, “It’s a crazy idea, but at your stage you’ve nothing to lose!” But he did not interfere; mentoring comes in many forms, and this was among the best. We soon showed that UDS measured with [3H] thymidine was reduced in cells from patients with XP; we had discovered one of the first examples of a human mutation that predisposed to cancer and identified its mechanism (35, 36). Michael Bishop, to become a Nobelist and later Chancellor of the University of California–San Francisco, commented that “While I was still in medical school James Cleaver recognized xeroderma pigmentosum as a deficiency in the repair of DNA damage caused by ultraviolet light . . . I have been a believer in the somatic mutation hypothesis of cancer ever since.” (37). Although first impressions of UDS were that the distribution of silver grains in autoradiographs represented a subnuclear distribution of repaired sites (Fig. 2), this was an illusion caused by the randomness of the [3H]  decays and their range in tissue and the recording emulsion. For UDS and many other uses, [3H] thymidine has now been replaced by fluorescent markers, which show repair distributed more uniformly at the resolution of light microscopy (38). Fluorescent markers provide higher resolution without the messy and expensive problems of radiation safety and disposal. But in its time, [3H] thymidine was revolutionary and led to many important biological discoveries. I was fortunate to be part of this era and to have worked for many years with Bob Painter, who published his own reminiscences (39). Work that was fueled in part by the U.S. Atomic Energy Agency’s emphasis on radiation biology and the innovation of the Brookhaven team in synthesizing [3H] thymidine. I owe a great deal to my early mentors in Cambridge, UK, Charles. L. Smith and Philip. P. Dendy. In San Francisco, I am indebted to Robert Painter, Shelly Wolff, and Harvey Patt, who guided and indulged me for many years. I am also grateful to Gregory H Thomas, who carried out the photography for Figs. 1 and 2 and provided much early technical support. Financial support came from the Atomic Energy Agency, which became the Energy Research and Development Agency and finally the Department of Energy. Their unrestricted approach to basic research during early times gave us the opportunity to discover and explore new worlds (. . . “to boldly go where no one has gone before!”).
2.
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4. 5.
6.
7.
8. 9.
10. 11.
12. 13.
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21.
22.
REFERENCES 23. 1.
2744
Rasmussen, R. E., and Painter, R. B. (1964) Evidence for repair of ultraviolet damaged deoxyribonucleic acid in cultured mammalian cells. Nature 203, 1360 –1362
Vol. 28
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