Profile of Tomas Lindahl, Paul Modrich, and Aziz Sancar, 2015 Nobel Laureates in Chemistry James E. Cleavera,1

In 1994, Science magazine heralded “the DNA repair enzyme” as the molecule of the year, on the basis of several significant advances made in mismatch repair and nucleotide excision repair, both recognized by this year’s Chemistry Nobel awards, that heralds an even better year for the field. This year’s Nobel Prize for Chemistry recognizes the importance of DNA repair as a major player in maintenance of our genomes and its recognition as a significant contribution to the biochemistry of DNA. Although the structure of DNA provided a failsafe mechanism by which a damaged strand could be mended using the sequence information on the complementary strand, Frances Crick famously admitted, “We totally missed the possible role of enzymes in repair. . .” (1). By a remarkable coincidence, the 2015 Lasker awards to Stephen J. Elledge and Evelyn Witkin, both members of the National







ALKB oxidase

Mismatch repair



Lindhal and Sancar


Fig. 1. Schematic diagram denoting the various pathways of DNA repair for which Lindahl, Modrich, and Sancar were honored. Pyr = pyr, cyclobutane pyrimidine dimers that are substrates for photolyase in most organisms except placental mammals; CRY1, 2, homologous proteins in placental mammals that are involved in diurnal regulation. Red lozenge, class of large adducts including cyclobutane pyrimidine dimers that are substrates for NER; dashed arrows, excision occurs by cleavage of the phosphodiester bonds in DNA on either side of an adduct. GGR and TCR are two branches of NER that act on nontranscribed and transcribed strands of DNA, respectively; TCR involves cofactors for RNA pol II. Purple disk, class of single base modifications (alkylation) that are substrates for BER; dashed arrow, excision occurs by a family of glycosylases that cleaves the sugar base bond. Alternative repair of specific single base lesions include the O6 alkyltransferase and the ALKB oxidase. Green wedge, mismatch in a newly synthesized strand repaired by mismatch repair; dashed arrows, excision and resynthesis can be initiated from single-strand breaks distal from the mismatch.

Academy of Sciences (NAS), also recognized DNA repair, now often called the DNA damage response to encompass its increased breadth. This year’s awards are to Tomas Lindahl for pioneering work deriving from his prediction that the inherent instability of DNA in an aqueous, oxygenated environment required mechanisms for repair (2); to Paul Modrich for the mechanism of mismatch corrections caused by replication errors (3); and to Aziz Sancar for detailed mechanistic study of nucleotide excision repair (NER) (4) and photolyase (5, 6). The award recognizes their contributions to understanding the chemistry of DNA repair processes (see Fig. 1). Lindahl started his career as a medical student in the Karolinska Institute (Stockholm), and after work in Princeton and the Rockefeller University, joined the faculty at the Karolinska Institute. He then moved to direct the Mutagenesis Laboratory at the Imperial Cancer Research Fund Mill Hill and eventually became director of the Imperial Cancer Research Fund’s Clare Hall laboratories that became part of Cancer Research UK. He was elected Fellow of the Royal Society in 1988 and is currently emeritus group leader at the Francis Crick Institute (London). Paul Modrich received a PhD degree in 1973 from Stanford University and an SB degree in 1968 from Massachusetts Institute of Technology. He joined Duke University’s faculty in 1976 and has been a Howard Hughes Investigator since 1994, pioneering studies on mismatch repair in both bacteria and mammalian cells. He was elected to the NAS in 1993 and to the National Academy of Medicine in 2003. Aziz Sancar was a physician in Turkey who came to the United States to complete his PhD on the photoreactivating enzyme of Escherichia coli in 1977 at the University of Texas at Dallas in the laboratory of Dr. C Stanley Rupert and subsequently worked with Dean Rupp at Yale. He is Professor of Biochemistry at the University of North Carolina at Chapel Hill. With his wife, Gwen B. Sancar, who is also a Professor of Biochemistry and Biophysics at UNC at Chapel Hill, they continue to support Turkish graduate students and promote Turkish–American relations. Sancar was elected to the NAS in 2005.


Department of Dermatology, University of California, San Francisco, CA 94143 Author contributions: J.E.C. wrote the paper. The author declares no conflict of interest. 1 Email: [email protected].

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Much of the work honored by this year’s Nobel prizes harks back to early observations in 1950s and 1960s of radiosensitive mutants in E. coli showing that radiation damage was more than a hit and miss event but that cellular responses are under genetic and hence biochemical control (7, 8). Much of the essential groundwork was carried out in bacteria and only later in human cells. A molecular approach to DNA repair began with Richard B Setlow’s observation that UV light-induced damage (cyclobutane pyrimidine dimers) in DNA could be biochemically characterized. He showed that dimers were actively removed from bacterial DNA over time (9). Pettijohn and Hanawalt showed that UV irradiated bacteria carried out a form of DNA synthesis that was distinct from normal semiconservative replication (10). In parallel, Robert B. Painter, a coinventor of tritiated thymidine, the radioactive precursor of DNA, discovered that UV-irradiated human cells also carried out novel DNA synthesis outside of the normal DNA synthesis period (11). These processes were later shown to represent replacement synthesis of the excised dimers. Aziz Sancar, while in Rupp’s laboratory, developed the maxi-cell method that enabled him to produce purified repair proteins, UVRA, B, and C, from E. coli plasmids and reconstitute the bacterial NER process in vitro (12). Unraveling the biochemical details of NER in human cells began with the development in Lindahl’s laboratory, with his colleague Richard Wood, of a method for in vitro analysis (13). By an ingenious use of a pair of plasmids of differing sizes, only one of which contained damage, they could discriminate specific repair from the background of nonspecific nucleases that had bedeviled previous work in mammalian cells. Subsequent work in both Lindahl’s and Sancar’s laboratories eventually led to defining the numerous components of human NER and reconstructing the complete process in vitro (14, 15). The bacterial and mammalian systems are similar in principle, both being a “cut and patch” process, but use different panoplies of proteins and excise different sized damaged oligonucleotides. The excised fragment is 12–13 nt in bacteria and 24–32 nt in humans. NER was found to have wide substrate specificity, possibly due to a recognition mechanism using dedicated damagespecific binding proteins based on distortions to DNA rather than precise enzymatic specificity (16). Mellon (17) and Lindsey-Boltz and Sancar (18) later proposed that arrest of transcription by RNA pol II was an alternative and more sensitive damage sensor. NER by then had been discriminated into global genome repair (GGR) and the more rapid transcription coupled repair (TCR), according to the transcriptional activity of the gene regions repaired (19). This revealed a close relationship between NER and transcription regulation in which the transcription factor TFIIH contained two helicases that unwound DNA around the damaged site (20). Lindahl realized that repair of single base lesions was different from NER of bulky lesions, and became the first to isolate a glycosylase that cleaved the uracildeoxyribose bond. This led to the discovery of base excision repair (BER) that removes single base lesions


via glycosylases and apurinic endonuclease (21). Lindahl was then able to reconstitute BER in vitro (22). Lindhal’s laboratory went on to discover a family of glycosylases with different substrate specificity and identify numerous other en- Tomas Lindahl. Image courtesy of Cancer Research UK. zymatic components and novel mechanisms of repair including multiple ligases, exonucleases, demethylation via alkyltransferase, and oxidation of cytosine methylation by AlkB (23). Despite the versatility of NER and BER, small mismatches that arise during replication errors and in microsatellite repeat sequences appear to escape their detection. These are repaired by the mismatch repair system for which Paul Modrich was honored (3). Wagner and Meselson (24), among others, had previously shown that mismatches were corrected in long tracts involving the bacterial gene dam. Modrich was the first to develop a method for detecting mismatch repair in vitro using plasmids with synthetic mismatches of various kinds (25). In E. coli, a mismatch is corrected by excision and resynthesis of the newly replicated DNA strand that contains the mismatched base. In bacteria, the mismatch in the newly replicated strand is marked by transiently unmethylated adenine (3, 24), but in mammalian cells may involve transient nicks or remnants of RNA. Single-strand breaks in DNA are made many nucleotides distant, 3′ or 5′ from the mismatch, in the new strand, initiate repair through coordinated action of proteins: MutS and MutL that act as homo-oligomers and MutH, which is a nuclease. In human cells, a similar mechanism operates, but using heterodimers. The human homolog to MutS representing 80–90% of the repair activity is MSH2:MSH6 (MutSα); the human homolog of MutL is MUTLH1:PMS2 (MUTLα), representing 90% of the activity. Modrich demonstrated that a purified system that could carry out mismatch repair in vitro required MutSα, MUTLα, exonucease1, RPA, and ATP (3). No human homolog of MutH has yet been identified. Genetic inactivation of the human MutSα or MutLα proteins makes cells resistant to chemotherapeutic agents by blocking apoptosis (26). Sancar’s initial work in the United States was with Stanley Rupert, who was one of the early workers in photoreactivation (PHR), and later he returned to investigation of this repair system at UNC. Paul Modrich. Image courtesy of Duke Photography.

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In addition to many incisive biochemical experiments into the properties of NER components and reconstitution of NER in vitro (27), he contributed to resolution of a longstanding dilemma in mammalian repair: the absence of PHR from placental mamAziz Sancar. Image courtesy of Max Englund (University mals. PHR could of North Carolina School of Medicine, Chapel Hill, NC). never be convincingly demonstrated in mammalian cells and Sancar demonstrated directly that human cells do not contain a functional photolyase (28). In most other organisms, photolyase enzymes absorb blue light in reduced flavin-adenine dinucleotide (FADH−) and transfer an electron from FADH− to pyrimidine photoproducts in DNA, leading to their monomerization, without excision from the DNA. The efficiency of the process is enhanced by an “antenna” cofactor (folate or deazaflavin) that also harvests the light. Sancar described this electron transfer mechanism in exquisite detail at femtosecond resolution in his inaugural PNAS publication (29). The discovery of cryptochrome proteins (CRY1, CRY2)

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and their characterization by Sancar and others highlighted an interesting evolutionary divergence (6). The CRY proteins have a homologous structure and the same cofactors as photolyases but do not monomerize photoproducts. Instead they contribute to diurnal regulation. They interact with other proteins to regulate circadian rhythms through the proteins Period, CLOCK, and BMAL1. Coming full circle, Sancar and colleagues showed that the NER process was itself subject to diurnal variations (30). The characterization of DNA repair at the enzymatic level has opened the door to many investigations now ongoing into the implications of DNA repair in the maintenance of genomic stability. Its role is integral to discussion of mutagenesis, origins, and treatment of cancer, developmental and neurological disease, evolution, and many other areas. These three investigators have contributed mightily to opening these doors with many opportunities for improvement of human health.

Acknowledgments I am grateful for reviews of this commentary by Drs. P. C. Hanawalt, G. C. Walker, and J. E. Rosen. In addition to the references cited below, additional information was gleaned from the Laureates websites and the Royal Swedish Academy of Sciences. Textbook information on DNA repair can be found in “DNA Repair and Mutagenesis, 2nd edition” (ASM Press) EC Friedberg, GC Walker, W Siede, RD Wood, R Schultz, T Ellenberger.

Crick F (1974) The double helix: A personal view. Nature 248(5451):766–769. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362(6422):709–715. Modrich P (2006) Mechanisms in eukaryotic mismatch repair. J Biol Chem 281(41):30305–30309. Sancar A (1994) Mechanisms of DNA excision repair. Science 266(5193):1954–1956. Sancar A (1994) Structure and function of DNA photolyase. Biochemistry 33(1):2–9. Oztürk N, et al. (2007) Structure and function of animal cryptochromes. Cold Spring Harb Symp Quant Biol 72:119–131. Witkin EM (1946) Inherited Differences in Sensitivity to Radiation in Escherichia Coli. Proc Natl Acad Sci USA 32(3):59–68. Hill RF (1958) A radiation-sensitive mutant of Escherichia coli. Biochim Biophys Acta 30(3):636–637. Setlow RB, Carrier WL (1964) The disappearance of thymine dimers from DNA:an error-correcting mechanism. Proc Natl Acad Sci USA 51:226–231. Pettijohn D, Hanawalt P (1964) Evidence for repair replication of ultraviolet damaged DNA in bacteria. J Mol Biol 9:395–410. Rasmussen RE, Painter RB (1964) Evidence for repair of ultraviolet damaged deoxyribonucleic acid in cultured mammalian cells. Nature 203:1360–1362. Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137(1):692–693. Wood RD, Robins P, Lindahl T (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell 53(1):97–106. Mu D, et al. (1995) Reconstitution of human DNA repair excision nuclease in a highly defined system. J Biol Chem 270(6):2415–2418. Aboussekhra A, et al. (1995) Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80(6): 859–868. Reardon JT, Sancar A (2005) Nucleotide excision repair. Prog Nucleic Acid Res Mol Biol 79:183–235. Mellon I (2005) Transcription-coupled repair: A complex affair. Mutat Res 577(1-2):155–161. Lindsey-Boltz LA, Sancar A (2007) RNA polymerase: The most specific damage recognition protein in cellular responses to DNA damage? Proc Natl Acad Sci USA 104(33):13213–13214. Mellon I, Bohr VA, Smith CA, Hanawalt PC (1986) Preferential DNA repair of an active gene in human cells. Proc Natl Acad Sci USA 83(23):8878–8882. Schaeffer L, et al. (1993) DNA repair helicase: A component of BTF2 (TFIIH) basic transcription factor. Science 260(5104):58–63. Lindahl T (1974) An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci USA 71(9):3649–3653. Dianov G, Lindahl T (1994) Reconstitution of the DNA base excision-repair pathway. Curr Biol 4(12):1069–1076. Lindahl T (2013) My journey to DNA repair. Genomics Proteomics Bioinformatics 11(1):2–7. Wagner R, Jr, Meselson M (1976) Repair tracts in mismatched DNA heteroduplexes. Proc Natl Acad Sci USA 73(11):4135–4139. Lu AL, Clark S, Modrich P (1983) Methyl-directed repair of DNA base-pair mismatches in vitro. Proc Natl Acad Sci USA 80(15): 4639–4643. Hickman MJ, Samson LD (1999) Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Proc Natl Acad Sci USA 96(19):10764–10769. Sancar A (1996) DNA excision repair. Annu Rev Biochem 65:43–81.

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28 Li YF, Kim ST, Sancar A (1993) Evidence for lack of DNA photoreactivating enzyme in humans. Proc Natl Acad Sci USA 90(10): 4389–4393. 29 Kao Y-T, Saxena C, Wang L, Sancar A, Zhong D (2005) Direct observation of thymine dimer repair in DNA by photolyase. Proc Natl Acad Sci USA 102(45):16128–16132. 30 Kang TH, Reardon JT, Sancar A (2011) Regulation of nucleotide excision repair activity by transcriptional and post-transcriptional control of the XPA protein. Nucleic Acids Res 39(8):3176–3187.


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Profile of Tomas Lindahl, Paul Modrich, and Aziz Sancar, 2015 Nobel Laureates in Chemistry.

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