PROFILE

PROFILE Profile of , Paul Modrich, and , 2015 Nobel Laureates in Chemistry

James E. Cleavera,1

In 1994, Science magazine heralded “the DNA repair Academy of Sciences (NAS), also recognized DNA re- enzyme” as the molecule of the year, on the basis of pair, now often called the DNA damage response to several significant advances made in mismatch repair encompass its increased breadth. and nucleotide excision repair, both recognized by This year’s awards are to Tomas Lindahl for pio- this year’s Chemistry Nobel awards, that heralds an neering work deriving from his prediction that the in- even better year for the field. This year’s herent instability of DNA in an aqueous, oxygenated for Chemistry recognizes the importance of DNA re- environment required mechanisms for repair (2); to pair as a major player in maintenance of our genomes Paul Modrich for the mechanism of mismatch correc- and its recognition as a significant contribution to the tions caused by replication errors (3); and to Aziz San- of DNA. Although the structure of DNA car for detailed mechanistic study of nucleotide provided a failsafe mechanism by which a damaged excision repair (NER) (4) and (5, 6). The strand could be mended using the sequence informa- award recognizes their contributions to understanding tion on the complementary strand, Frances Crick fa- the chemistry of DNA repair processes (see Fig. 1). mously admitted, “We totally missed the possible role Lindahl started his career as a medical student in of enzymes in repair...” (1). By a remarkable coinci- the (), and after work in dence, the 2015 Lasker awards to Stephen J. Elledge Princeton and the , joined the and Evelyn Witkin, both members of the National faculty at the Karolinska Institute. He then moved to direct the Mutagenesis Laboratory at the Imperial Cancer Research Fund Mill Hill and eventually became pyr=pyr 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 NER photolyase CRY1,2 Mismatch Institute (London). repair Paul Modrich received a PhD degree in 1973 from O6tase ALKB oxidase Stanford University and an SB degree in 1968 from GGR TCR Massachusetts Institute of Technology. He joined BER Duke University’s faculty in 1976 and has been a Howard Hughes Investigator since 1994, pioneering Sancar Lindhal and Sancar Modrich studies on mismatch repair in both bacteria and mam- malian cells. He was elected to the NAS in 1993 and to Fig. 1. Schematic diagram denoting the various pathways of DNA repair for which the National Academy of Medicine in 2003. Lindahl, Modrich, and Sancar were honored. Pyr = pyr, cyclobutane pyrimidine dimers that are substrates for photolyase in most organisms except placental Aziz Sancar was a physician in who came mammals; CRY1, 2, homologous proteins in placental mammals that are involved to the United States to complete his PhD on the in diurnal regulation. Red lozenge, class of large adducts including cyclobutane photoreactivating enzyme of Escherichia coli in 1977 pyrimidine dimers that are substrates for NER; dashed arrows, excision occurs by at the University of Texas at Dallas in the laboratory of cleavage of the phosphodiester bonds in DNA on either side of an adduct. GGR Dr. C Stanley Rupert and subsequently worked with 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, Dean Rupp at Yale. He is Professor of Biochemistry at class of single base modifications (alkylation) that are substrates for BER; dashed the University of North Carolina at Chapel Hill. With arrow, excision occurs by a family of glycosylases that cleaves the sugar his wife, Gwen B. Sancar, who is also a Professor of 6 base bond. Alternative repair of specific single base lesions include the O Biochemistry and at UNC at Chapel Hill, alkyltransferase and the ALKB oxidase. Green wedge, mismatch in a newly synthesized strand repaired by mismatch repair; dashed arrows, excision they continue to support Turkish graduate students and resynthesis can be initiated from single-strand breaks distal from the and promote Turkish–American relations. Sancar was mismatch. elected to the NAS in 2005.

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

242–245 | PNAS | January 12, 2016 | vol. 113 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1521829112 Downloaded by guest on September 26, 2021 Much of the work honored by this year’s Nobel via glycosylases and prizes harks back to early observations in 1950s and apurinic endonucle- 1960s of radiosensitive mutants in E. coli showing that ase (21). Lindahl was radiation damage was more than a hit and miss event but then able to recon- that cellular responses are under genetic and hence bio- stitute BER in vitro chemical control (7, 8). Much of the essential groundwork (22). Lindhal’s labo- was carried out in bacteria and only later in human ratory went on to cells. A molecular approach to DNA repair began with discover a family of Richard B Setlow’s observation that UV light-induced glycosylases with dif- damage (cyclobutane pyrimidine dimers) in DNA could ferent substrate spec- be biochemically characterized. He showed that dimers ificity and identify were actively removed from bacterial DNA over time numerous other en- Tomas Lindahl. Image courtesy of Cancer Research UK. (9). Pettijohn and Hanawalt showed that UV irradiated zymatic components bacteria carried out a form of DNA synthesis that was and novel mechanisms of repair including multiple li- distinct from normal semiconservative replication (10). gases, exonucleases, demethylation via alkyltransferase, In parallel, Robert B. Painter, a coinventor of tritiated and oxidation of cytosine methylation by AlkB (23). thymidine, the radioactive precursor of DNA, discov- Despite the versatility of NER and BER, small ered that UV-irradiated human cells also carried out mismatches that arise during replication errors and in novel DNA synthesis outside of the normal DNA syn- microsatellite repeat sequences appear to escape thesis period (11). These processes were later shown to their detection. These are repaired by the mismatch represent replacement synthesis of the excised dimers. repair system for which Paul Modrich was honored (3). Aziz Sancar, while in Rupp’s laboratory, developed Wagner and Meselson (24), among others, had pre- the maxi-cell method that enabled him to produce viously shown that mismatches were corrected in long purified repair proteins, UVRA, B, and C, from E. coli tracts involving the bacterial gene dam. Modrich was plasmids and reconstitute the bacterial NER process the first to develop a method for detecting mismatch in vitro (12). Unraveling the biochemical details of NER repair in vitro using plasmids with synthetic mis- in human cells began with the development in Lin- matches of various kinds (25). In E. coli, a mismatch dahl’s laboratory, with his colleague Richard Wood, is corrected by excision and resynthesis of the newly of a method for in vitro analysis (13). By an ingenious replicated DNA strand that contains the mismatched use of a pair of plasmids of differing sizes, only one of base. In bacteria, the mismatch in the newly replicated which contained damage, they could discriminate strand is marked by transiently unmethylated adenine specific repair from the background of nonspecific nu- (3, 24), but in mammalian cells may involve transient cleases that had bedeviled previous work in mam- nicks or remnants of RNA. Single-strand breaks in malian cells. Subsequent work in both Lindahl’sand DNA are made many nucleotides distant, 3′ or 5′ from Sancar’s laboratories eventually led to defining the the mismatch, in the new strand, initiate repair numerous components of human NER and recon- through coordinated action of proteins: MutS and structing the complete process in vitro (14, 15). The MutL that act as homo-oligomers and MutH, which is bacterial and mammalian systems are similar in prin- a nuclease. In human cells, a similar mechanism oper- ciple, both being a “cut and patch” process, but use ates, but using heterodimers. The human homolog to different panoplies of proteins and excise different MutS representing 80–90% of the repair activity is sized damaged oligonucleotides. The excised fragment MSH2:MSH6 (MutSα); the human homolog of MutL is 12–13 nt in bacteria and 24–32 nt in humans. NER is MUTLH1:PMS2 (MUTLα), representing 90% of the was found to have wide substrate specificity, possibly activity. Modrich demonstrated that a purified system due to a recognition mechanism using dedicated damage- that could carry out mismatch repair in vitro required specific binding proteins based on distortions to DNA MutSα, MUTLα, exonucease1, RPA, and ATP (3). No rather than precise enzymatic specificity (16). Mellon human homolog of MutH has yet been identified. Ge- (17) and Lindsey-Boltz and Sancar (18) later proposed netic inactivation of the human MutSα or MutLα that arrest of transcription by RNA pol II was an alter- proteins makes cells native and more sensitive damage sensor. NER by resistant to chemo- then had been discriminated into global genome re- therapeutic agents pair (GGR) and the more rapid transcription coupled by blocking apopto- repair (TCR), according to the transcriptional activity sis (26). of the gene regions repaired (19). This revealed a Sancar’sinitial close relationship between NER and transcription reg- work in the United ulation in which the transcription factor TFIIH con- States was with Stanley tained two helicases that unwound DNA around Rupert, who was the damaged site (20). one of the early work- Lindahl realized that repair of single base lesions ers in photoreactiva- was different from NER of bulky lesions, and became tion (PHR), and later the first to isolate a glycosylase that cleaved the uracil- he returned to inves- deoxyribose bond. This led to the discovery of base tigation of this re- excision repair (BER) that removes single base lesions pair system at UNC. Paul Modrich. Image courtesy of Duke Photography.

Cleaver PNAS | January 12, 2016 | vol. 113 | no. 2 | 243 Downloaded by guest on September 26, 2021 In addition to many and their characterization by Sancar and others high- incisive biochemi- lighted an interesting evolutionary divergence (6). The cal experiments in- CRY proteins have a homologous structure and the to the properties of same cofactors as but do not monomer- NER components ize photoproducts. Instead they contribute to diurnal and reconstitution regulation. They interact with other proteins to reg- of NER in vitro (27), ulate circadian rhythms through the proteins Period, he contributed to CLOCK, and BMAL1. Coming full circle, Sancar and resolution of a long- colleagues showed that the NER process was itself standing dilemma in subject to diurnal variations (30). mammalian repair: The characterization of DNA repair at the enzymatic the absence of PHR level has opened the door to many investigations now from placental mam- ongoing into the implications of DNA repair in the Aziz Sancar. Image courtesy of Max Englund (University of North Carolina School of Medicine, Chapel Hill, NC). mals. PHR could maintenance of genomic stability. Its role is integral never be convinc- to discussion of mutagenesis, origins, and treatment of ingly demonstrated cancer, developmental and neurological disease, evolu- in mammalian cells and Sancar demonstrated directly tion, and many other areas. These three investiga- that human cells do not contain a functional photo- tors have contributed mightily to opening these lyase (28). doors with many opportunities for improvement of In most other organisms, photolyase enzymes human health. absorb blue light in reduced flavin-adenine dinucleo- − − tide (FADH ) and transfer an electron from FADH to Acknowledgments pyrimidine photoproducts in DNA, leading to their I am grateful for reviews of this commentary by Drs. monomerization, without excision from the DNA. P.C.Hanawalt,G.C.Walker,andJ.E.Rosen.Inaddition The efficiency of the process is enhanced by an to the references cited below, additional information “antenna” cofactor (folate or deazaflavin) that also was gleaned from the Laureates websites and the Royal harvests the light. Sancar described this electron trans- Swedish Academy of Sciences. Textbook information on fer mechanism in exquisite detail at femtosecond res- DNA repair can be found in “DNA Repair and Mutagen- olution in his inaugural PNAS publication (29). The esis, 2nd edition” (ASM Press) EC Friedberg, GC Walker, discovery of proteins (CRY1, CRY2) W Siede, RD Wood, R Schultz, T Ellenberger.

1 Crick F (1974) The double helix: A personal view. Nature 248(5451):766–769. 2 Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362(6422):709–715. 3 Modrich P (2006) Mechanisms in eukaryotic mismatch repair. J Biol Chem 281(41):30305–30309. 4 Sancar A (1994) Mechanisms of DNA excision repair. Science 266(5193):1954–1956. 5 Sancar A (1994) Structure and function of DNA photolyase. Biochemistry 33(1):2–9. 6 Oztürk N, et al. (2007) Structure and function of animal . Cold Spring Harb Symp Quant Biol 72:119–131. 7 Witkin EM (1946) Inherited Differences in Sensitivity to Radiation in Escherichia Coli. Proc Natl Acad Sci USA 32(3):59–68. 8 Hill RF (1958) A radiation-sensitive mutant of Escherichia coli. Biochim Biophys Acta 30(3):636–637. 9 Setlow RB, Carrier WL (1964) The disappearance of thymine dimers from DNA:an error-correcting mechanism. Proc Natl Acad Sci USA 51:226–231. 10 Pettijohn D, Hanawalt P (1964) Evidence for repair replication of ultraviolet damaged DNA in bacteria. J Mol Biol 9:395–410. 11 Rasmussen RE, Painter RB (1964) Evidence for repair of ultraviolet damaged deoxyribonucleic acid in cultured mammalian cells. Nature 203:1360–1362. 12 Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137(1):692–693. 13 Wood RD, Robins P, Lindahl T (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell 53(1):97–106. 14 Mu D, et al. (1995) Reconstitution of human DNA repair excision nuclease in a highly defined system. J Biol Chem 270(6):2415–2418. 15 Aboussekhra A, et al. (1995) Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80(6): 859–868. 16 Reardon JT, Sancar A (2005) Nucleotide excision repair. Prog Nucleic Acid Res Mol Biol 79:183–235. 17 Mellon I (2005) Transcription-coupled repair: A complex affair. Mutat Res 577(1-2):155–161. 18 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. 19 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. 20 Schaeffer L, et al. (1993) DNA repair helicase: A component of BTF2 (TFIIH) basic transcription factor. Science 260(5104):58–63. 21 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. 22 Dianov G, Lindahl T (1994) Reconstitution of the DNA base excision-repair pathway. Curr Biol 4(12):1069–1076. 23 Lindahl T (2013) My journey to DNA repair. Genomics Proteomics Bioinformatics 11(1):2–7. 24 Wagner R, Jr, Meselson M (1976) Repair tracts in mismatched DNA heteroduplexes. Proc Natl Acad Sci USA 73(11):4135–4139. 25 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. 26 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. 27 Sancar A (1996) DNA excision repair. Annu Rev Biochem 65:43–81.

244 | www.pnas.org/cgi/doi/10.1073/pnas.1521829112 Cleaver Downloaded by guest on September 26, 2021 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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