A Personal Tribute to 2015 Nobel Laureate Paul Modrich

DNA Repair 37 (2016) A14–A21

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A Personal Tribute to 2015 Nobel Laureate Paul Modrich Guo-Min Li ∗ Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, University of Southern California, Los Angeles, CA 90033

On October 7, 2015, at approximately 3 AM PST, I awoke to the sound of my cell phone ringing. The call was from Beijing, China. My friend in Beijing was calling to tell me that my former postdoctoral mentor, Paul Modrich, had been awarded the 2015 Nobel Prize in Chemistry, along with Tomas Lindahl and Aziz Sancar, for mechanistic studies of DNA repair; in Paul’s case, for studies of DNA mismatch repair (MMR). I thanked my friend for the exciting news, after which my friend passed the phone to the Editor-in-Chief of a popular social media-based science journal in China. For the next hour, as I answered the Editor’s questions, I reflected on the evolution of the field of MMR, Paul’s well-deserved Nobel Prize, and my experience as a young scientist working in Paul’s laboratory. The editor’s questions brought me back to the early 1990s, when I was finishing my Ph.D. training with Louis Romano in the Chemistry Department at Wayne State University. At the time, I was studying the mechanism of mutagenesis, and I had become interested in cellular mechanisms that prevent mutations. After extensive literature searching, I began to focus on DNA MMR, and its role in maintaining replication fidelity, when I learned of the brilliant studies conducted by Paul Modrich and his colleagues. As a result, I decided nearly immediately to apply for a postdoctoral position in Paul’s laboratory. The following is what was known about MMR at the time and why I wanted to pursue my postdoctoral training with Paul. In the 1950s, several geneticists reported that the frequency of genetic exchange was unusually high in localized regions of some fungal and bacteriophage genomes, a phenomenon referred to as high negative interference of homologous recombination [1]. In the early 1960s, Robin Holliday [2] and Evelyn Witkin [3] suggested that these unstable genomic regions corresponded to sites of “hybrid” or “heteroduplex” DNA containing DNA mispairs. Later, Matthew Meselson and Maurice Fox independently provided direct evidence for what is now known as MMR, performing transfection experiments with mispair-containing heteroduplex DNA [4–6].

∗ Corresponding author. Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, 1450 Biggy Street, 3506 Norris Research Tower, Los Angeles, CA, 90033, Office: (323) 442-7436. E-mail address: [email protected] http://dx.doi.org/10.1016/j.dnarep.2015.12.005 1568-7864/© 2015 Published by Elsevier B.V.

A critical question at the time was how the MMR system distinguishes the parental strand (containing the correct information) from the nascent DNA strand (containing the incorrect base) in a newly replicated DNA duplex. Given that an E. coli strain that lacks dam methylase, an enzyme that methylates the adenine of the sequence d(GATC), has a mutator phenotype [7], and the fact that d(GATC) is transiently under-methylated in nascent DNA [8], it was postulated the hemi-methylated d(GATC) site could be the strand discrimination signal for MMR [6]. In collaboration with Paul Modrich, Matthew Meselson and colleagues showed that this was indeed the case [9]. The genetic studies by Miroslav Radman and Barry Glickman revealed the involvement of mutH, mutL, mutS and uvrD genes in the methyl-directed repair pathway [10]. However, it is the biochemical studies conducted by Paul Modrich and colleagues that elucidated the mechanism of the methyl-directed MMR in E. coli. When I first read about the work on MMR carried out in Paul’s lab, I was very impressed by his lab’s many remarkable achievements. First, the functional in vitro MMR assay developed by Paul and A-Lien Lu [11], is possibly the most elegant and powerful, but simple assay design one could imagine. This assay uses an ingeniously designed DNA substrate, a recombinant circular plasmid DNA containing a hemi-methylated d(GATC) site as the strand-discrimination signal, and a single mismatch placed in the overlapping recognition sequence of two restriction endonucleases. The presence of the mismatch renders the DNA resistant to hydrolysis by either enzyme, with correction conferring sensitivity to one endonuclease or the other depending on the strand that is repaired. This assay allowed Paul to assess the strand-specificity of repair, based on a simple agarose gel assay for sensitivity to restriction endonuclease cleavage. Paul and A-Lien demonstrated that the assay is an effective in vitro measure of in vivo capacity for methyl-directed MMR, as they showed that the repair occurs in the unmethylated DNA strand, while the methylated DNA strand acts as the template for repair of the mismatch [11]. In my opinion, it is this powerful and elegant assay that paved the way for the discoveries made by Paul’s laboratory over the next 30 years. Second, using the powerful in vitro MMR assay, Paul’s lab went on to identify and characterize the components of the E. coli methyl-directed MMR pathway; in particular, MutS, MutL and MutH were characterized by Shin-San (Michael) Su (a graduate student) [12], Michelle Grilley (a graduate student) [13] and Katherine

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Welsh (a postdoctoral fellow) [14], respectively. Building on this accomplishment, Robert (Bob) Lahue and Karin Au, both of whom were Paul’s former postdoctoral fellows, reconstituted E. coli MMR in vitro using highly-purified E. coli proteins [15]. These studies, which elucidated the mechanism of MMR in E. coli, were instrumental in subsequent studies of eukaryotic MMR. Paul’s lab was also responsible for ground-breaking studies in human MMR. Paul and Jude Holmes (a graduate student) made the initial discovery that human cells possess a strand-specific MMR that is similar to E. coli MutHLS-dependent methyl-directed MMR [16]. However, in contrast to the E. coli system, in which hemimethylated d(GATC) sites act as the strand discrimination signal, in eukaryotic MMR, repair specificity appeared to be conferred by single-strand breaks (i.e., nicks) in the newly synthesized DNA strand. This finding began a new chapter in studies of genome stability in human cells, including the discovery that MMR defects cause several types of human cancer (see below). These incredible discoveries prompted me to apply for a postdoctoral position in Paul’s laboratory. I was lucky enough to be offered a position, shortly after I visited Duke University and met Paul in person. I later found out that both Paul and Lou did postdoctoral studies in Charles Richardson’s laboratory at Harvard University, and this connection and Lou’s generous recommendation may have helped to earn me this honor and opportunity. I worked in Paul’s laboratory from February 1991 to August 1995. When I joined Paul’s lab, the lab had 5 graduate students (Deani Cooper, Woei-hrong Fang, Michelle Grilley, Jen-Chi Hsieh and Shawn Zinnen), 3 postdoctoral fellows (Dwayne Allen, Karin Au, and Leroy Worth), and a lab manager (Susana Clark). Woeihrong was the only one working on the human MMR project, Jen-Chih and Shawn were investigating the HIV reverse transcriptase [17,18], and everyone else was studying E. coli MMR. Paul assigned me to the human MMR project, and specifically asked me to isolate human MMR proteins. Although I was reasonably well-trained in Lou’s laboratory, I had little experience purifying and characterizing novel proteins. Fortunately, Paul provided a lot of excellent guidance and advice, and he also asked Deani and Michelle, both of whom had extensive experience with column chromatography and protein purification, to be my advisors on the project. Karin, Woei-hrong and Dwayne also helped me learn other important techniques, including how to prepare nuclear extracts and heteroduplex DNA substrates and how to perform the in vitro MMR assay. In the remainder of this short article, I will focus on the human MMR studies conducted in Paul’s laboratory from 1991 onward. I apologize to my fellow lab mates for not describing their work in greater detail here. Briefly, I would like to acknowledge Leroy Worth, who revealed that E. coli MMR proteins block recombination between diverged DNA sequences, a novel role for MMR at the time [19]; Karin Au, who, in addition to the work reconstituting the MMR reaction in collaboration with Bob Lahue [15], elucidated the initiation of methyl-directed MMR and identified MutY, which plays another disticnt role in mutation avoidance [20–23]; Dwayne and John Taylor (a postdoctoral fellow who joined the lab in late 1991), whose work provided an exciting model for how mismatch binding by MutS triggers mismatch-provoked excision at a distance [24]; Deani and Michelle, who demonstrated that E. coli MMR is bidirectional [25,26]; Vivian Dao (a postdoctoral fellow) and Miyuki Yamaguchi (a graduate student), who joined the lab later and discovered the critical role of helicase II (also called UvrD) in methyl-directed MMR [27,28]. In E. coli, MMR increases replication fidelity by approximately three orders of magnitude, making it absolutely critical for genome stability. Paul and others reasoned that human cells were likely to express a similar system, which he postulated would be likely to play a critical role in the viability of human cells, as well as in general

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health at the level of the individual. Later studies showed that Paul’s intuition was correct. The details of how this work evolved in the early 1990s are described below. I began working on the next phase of the human MMR project, along with Woei-hrong, Matthew Longley (who joined Paul’s lab in December 1991) and James Drummond (who joined Paul’s lab in 1994). We worked together to identify the protein components required for human MMR. Paul’s strategy was to target on biochemical “mutants” (e.g., by fractionating HeLa nuclear extracts) or mutant human cell lines that are defective in MMR, which were the basis of a series of complementation assays. Immediately after joining Paul’s lab, I began to execute Paul’s plan, using both approaches. I separated MMR-proficient HeLa nuclear extracts into three distinct fractions using ammonium sulfate precipitation. Six months after I joined Paul’s lab, I succeeded in purifying a mismatch binding activity from one of the fractions, using a mismatch-containing DNA affinity column and a gel-shift assay. The mismatch binding activity copurified with two polypeptides, 105 kDa and 160 kDa, respectively, but this was temporarily a ‘dead-end’ until its reidentification in 1995 (see the discovery of hMutS alpha below), because we did not have a good way and the resources to determine the nature of these two proteins in 1991. As an aside, it was highly instructive to me, as a young scientist, very proud of my accomplishment, to realize that as good as the data seemed to me at the time, it was not up to Paul’s standards, and he would not even consider trying to publish the result, until we knew more about the two protein components in this human mismatch-binding fraction. As disappointed as I was, I now realize, in hindsight, the value of the lesson that I learned from Paul at that time. Paul’s standards were (and are) high, and he always made sure that the data published by his lab was 100% correct, completely validated, and properly interpreted. Anything less was not good enough, no matter how great the reward might be, and no matter how great the risk of being ‘scooped’ by our skilled and motivated colleagues, as we waited for more or better data. As I continued in my work on human MMR proteins, I found that the biochemical “mutants” created by fractionation were complex, in that some components were separated into two “mutants” (e.g., 70% in one, and 30% in another), which gave background repair when used as a receptor in reconstitution assay. We therefore shifted our focus to mutant human cell lines. Paul was aware of a cell line (designated MT1) created by William Thilly at Massachusetts Institute of Technology, which was derived from human lymphoblastoid TK6 line by frameshift mutagenesis. This cell line displayed a mutator phenotype, and its spontaneous mutation rate was ∼60-fold higher than the spontaneous mutation rate of the parental cell line [29]. With the idea that MT1 could be defective in MMR, Paul obtained MT1 and the parental TK6 cell lines from Billy Thilly at the end of 1992. Unfortunately, I could not handle these cell lines, because they put me at risk of infection with the Epstein-barr virus, with which MT1 and TK6 were transformed. Instead, Woei-hrong, who had been previously exposed to the virus and was protected by his existing antibodies to the virus, stepped in to analyze these cell lines. As Paul predicted, Woei-hrong demonstrated that MT1 is defective in strand-specific MMR, and this led to characterization of the first known MMR-deficient human cell line [30]. In May 1993, nearly at the same time as we published our characterization of the MT1 cell line, a remarkable new discovery was reported in the back-to-back papers in Science by Bert Vogelstein [31] and Stephen Thibodeau [32]. These scientists reported a strong correlation between microsatellite instability (MSI) and hereditary non-polyposis colorectal cancer (HNPCC, also called Lynch syndrome), as well as a similar association in some sporadic colorectal cancers. A very similar observation was also reported in Nature by Manuel Perucho [33]. Given that these reports were reminiscent

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of the observation that simple repetitive sequences are highly unstable in MMR-deficient E. coli [34,35], Paul immediately linked the two observations and suspected that MSI-positive colorectal cancer cells carry defects in an MMR component. Paul obtained patient-derived MSI-positive cell lines from Bert Vogelstein, which we used to test his hypothesis. We received two colorectal cancer cell lines, H6 and S0 (we later knew these as HCT116 and SW480, respectively) from Ramon Parsons, a postdoctoral fellow in Bert Vogelstein’s laboratory. Myself, Matt and Woei-hrong then worked feverishly for the next several months, and were rewarded with the discovery, as Paul had predicted, that MSI-positive H6 cells, but not MSI-negative S0 cells, are defective in repair of both base-base mismatches and microsatellite mispairs (also called insertiondeletion mispairs). Furthermore, the defect was complemented by a partially purified activity from HeLa cells [36]. At the same time, Richard Kolodner and Bert Vogelstein demonstrated that HNPCC families are tightly linked to mutations in human MMR genes MSH2 [37,38]. These discoveries, together with those from others including Thomas Petes, Mike Liskay and Thomas Kunkel [39–44], showed that HNPCC and a subset of sporadic colorectal cancers are caused by defects in MMR. As a result, MSI has become a hallmark of MMR-deficiency and is now widely used to screen for MMR-deficient cancers in clinical settings. Collectively, these discoveries elevated the field of DNA repair, previously thought of as a collection of ‘housekeeping’ pathways, to a new level of significance. DNA repair, largely due to the discovery of MMR defects in HNPCC by Paul, Richard Kolodner and Bert Vogelstein, but complemented by the work of Aziz Sancar, Philip Hanawalt and others, was named Science magazine’s Molecule of the Year in December 1994 [45,46]. The link between MMR-deficiency and human colon cancer greatly facilitated our search for human MMR genes/proteins. However, purification of novel human proteins from cultured cells is never an easy task. Due to the high complexity of human cell extracts, it can require five to seven chromatography steps to reach even near homogeneity of any human protein of interest. In addition, human cells grow slowly and preparative cell culture is labor-intensive, time-consuming and very expensive. At the time, our budget was limited, and I remember that Paul had to specifically authorize any purchase for more than five dollars. HeLa cells cost ∼$25 per liter at the time, and we needed at least 1.5 g of nuclear extract protein, equivalent to 100 liters of HeLa cells to have a chance of a successful purification (if we started with less, there would not be activity to make it through all steps of the purification). Paul made sure that we had the necessary amount of starting material for each new purification attempt, although it took significant effort and resources to do that. Initially, Susana worked nearly full-time to grow the required amount of HeLa cells. Paul then hired Elisabeth Penland, who is an outstanding cell-culture specialist, to handle all cell cultures in the lab. After that, we could reliably harvest 20-30 liters of HeLa cells every week. Matt and I spent at least two days per week preparing nuclear extracts from HeLa and other cancer cell lines. I alone probably used more than 300 liters of HeLa cells in order to purify the human MMR activity, later identified as hMutL␣. I began to purify the H6-complementing activity from HeLa nuclear extracts in mid-December, 1993, just before the Christmas Holiday. I found that H6 cells were not defective in mismatch binding, and were not complemented by the mismatch binding activity I had already purified in 1991. This meant I needed basically to start from scratch, to purify the H6-complementing factor. Paul suggested that I first perform a pilot experiment applying small amounts of cell extract to small chromatography columns; in this manner, I could determine resins that would be most useful for purifying the H6-complementing activity. This strategy helped a great deal, and I identified five columns (P-11 phosphocellulose,

S sepharose, hydroxylapatite, Mono Q and Mono S) to which the H6 complementing activity bound with reasonable affinity. Nevertheless, a sixth step was needed to get a pure form of the activity, which, at Paul’s suggestion, was fractionation by sucrose gradient centrifugation. After these six steps, the activity remained associated with two polypeptides of 85 kDa and 110 kDa, which was not consistent with the single polypeptide nature of all known E. coli MMR proteins. We then performed peptide microsequence by Edman degradation [47], which showed that both polypeptides were homologs of bacterial MutL; the 85 kDa and 110 kDa polypeptides were the gene products of MLH1 and PMS2, respectively, two human MutL homolog genes identified by Bert Vogelstein, Richard Kolodner and Mike Liskay and their colleagues in 1994 [40,43,44]. Given the 1:1 ratio of these two proteins, the simplest explanation was that human MLH1 and PMS2 gene products form a heterodimer, which we designated hMutL␣ [48]. As it turned out, the heterodimeric nature of hMutL␣ was not unique, but it did set a precedent for other eukaryotic MMR proteins, as many of them are heterodimers. These include MutS␣ (MSH2-MSH6), MutS␤ (MSH2-MSH3), MutL␤ (MLH1-PMS1), and MutL␥ (MLH1MLH3) [49–51]. Purification of hMutL␣ protein was demanding and difficult. The purification procedure involved six steps, and each step, at a preparative scale, required at least 20 hours for chromatography and at least 7 additional hours to analyze MMR activity in more than 100 column fractions. The MMR assay included a 4-hour electrophoresis step, which was the only downtime in each step of the purification. When possible, I took that opportunity to grab a few hours of sleep on a couch in a break room close to the lab. Without Paul’s support, encouragement and timely advice, and the powerful functional in vitro MMR assay that Paul and A-Lien had established, it would not have been possible for me to succeed in purifying human hMutL␣. Our lab went on to purify and characterize hMutS␣, using its ability to complement the MMR defect in the colorectal cancer cell line, LoVo, which was defective in the MSH2 gene. Jim Drummond, who joined the lab in 1994, worked on this project. With Paul’s help, Jim developed a very efficient procedure to isolate the LoVocomplementing activity. He found that this activity binds tightly to the ssDNA cellulose column in the absence of ATP, but could be readily eluted from the column by adding ATP to the elution buffer. This characteristic of the LoVo-complementing activity allowed Jim to purify this factor essentially using a one-column procedure. Like hMutL␣, the LoVo-complementing activity copurified with two polypeptides in a 1:1 molar ratio, one 105 kDa and another 160 kDa in molecular size. Since LoVo cells are defective in MSH2 [42] and since the molecular size of these polypeptides matched the size of the mismatch binding activity I purified in 1991 (also published by Joseph Jiricny in 1992 [52]), Paul asked me to test if the LoVocomplementing factor had mismatch binding activity. The answer was ‘yes’, and the conclusion that the LoVo-complementing activity is a MutS homologue was also confirmed by peptide sequencing and Western blot. The 105-kDa polypeptide was identified as the MSH2 gene product, and the 160-kDa protein (known as a G-T binding protein and later renamed MSH6) is an essential part of the human mismatch binding activity [52,53]. Paul concluded that the LoVocomplementing activity is a human mismatch recognition protein that functions as a heterodimer, and we named it hMutS␣ [54]. To identify additional human MMR components, Paul obtained more than 20 MSI-positive colorectal cancer cell lines from commercial sources and our collaborators, including Vaco series colorectal cancer cell lines [55–57] established by Sandy Markowitz and colleagues at Case Western Reserve University. The three of us, Matt, Jim and I, analyzed their MMR activity and performed complementation assays. Our analysis showed that these MSIpositive human cancer cell lines belong to two complementation

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groups, corresponding to cell lines with defects in either hMutS␣ or hMutL␣. As a result, this set of cell lines did not help us in our search for additional factors that play critical roles in human MMR. So, Matt then went back to the alternative approach suggested by Paul, analyzing biochemical “mutants”. After significant effort, Matt obtained an MMR-deficient HeLa nuclear fraction that he could use to screen for complementing factor(s). Ultimately, Matt isolated a complementing activity that copurified with DNA polymerase ␦, revealing that replicative DNA polymerase ␦ is required for MMR in human cells [58]. This finding was in agreement with the notion that MMR couples with DNA replication [59–61]. Using a similar approach, Jochen Genschel, a postdoctoral fellow in Paul’s laboratory, isolated ExoI as an essential activity for in vitro human MMR [62], consistent with previous studies showing that ExoI is involved in MMR [63–65]. Subsequently, proliferating cellular nuclear antigen (PCNA), replication protein A (RPA) and replication factor C (RFC) were also shown to be involved in eukaryotic MMR [66–70]. MMR-deficient human cancer cell lines were also used to rapidly advance mechanistic studies of human MMR. Paul’s lab carefully characterized each of the human mutant cell lines for their substrate specificity and bi-directionality. My experiments demonstrated that nuclear extracts from MT1, HCT15 and DLD1 cells are defective in repairing all base-base mismatches, but remain proficient in repairing small insertion-deletion mispairs, implying two distinct MMR subpathways, each potentially with a unique mismatch recognition protein [54]. Indeed, hMutS␤, a heterodimer of MSH2 and MSH3 subunits that specifically recognizes small insertion-deletion mispairs, was identified later in human cells by Jochen [71]. MT1, HCT15 and DLD-1 cell lines were each found to be defective in the MSH6 subunit of hMutS␣ [72]. Since hMutS␣ and hMutS␤ both are MSH2-containing heterodimers, their cellular ratio depends on the ratio of MSH3 and MSH6 proteins in the cell. Jim found that under normal circumstances, cells express hMutS␣ and hMutS␤ at ∼10:1 molar ratio; when the ratio is pushed much higher, to 1:100, due to overproduction of MSH3, cells exhibit a mutator phenotype [73]. Thus, the ratio of hMutS␣ to hMutS␤ strongly influences mutation rate and genome stability in human cells. Overexpression of MSH3 also causes trinucleotide repeat expansion [74,75], the molecular basis of a number of neurodegenerative and neuromuscular disorders [76]. Identification of key MMR components allowed Paul and colleagues to elucidate the detailed mechanism of human MMR. Leonid Dzantiev, a postdoctoral fellow in Paul’s laboratory, who was also a former student of Lou Romano, demonstrated that a purified minimum MMR system containing hMutS␣, hMutL␣, PCNA, RFC, ExoI and RPA was sufficient to carry out mismatch-provoked mismatch removal, when the DNA substrate contains a single strand break 5 or 3 to the mismatch [77]. Subsequently, Nicoleta Constantin, another postdoctoral fellow in Paul’s lab, succeeded in reconstituting human MMR in vitro using purified MMR proteins [78], a milestone in the field of MMR. It is worth mentioning that despite the bidirectional nature of the human MMR reaction, the in vitro assay only contained an exonuclease (i.e., ExoI) capable of excising DNA from a 5 to 3 orientation, and questions remained: what enzyme in the in vitro system supports 3 nick-directed mismatch removal? This question was answered by experiments of Paul and Farid Kadyrov (a postdoctoral fellow in Paul’s lab), who demonstrated that hMutL␣ is a latent endonuclease that is active only in the presence of a mismatch, MutS␣, RFC, PCNA, and ATP. Under these conditions, the activated MutL␣ endonuclease makes an incision on the nicked strand 5 to the mismatch, thereby allowing mismatch removal by ExoI [79]. These observations solved the long-standing puzzle of how misincorporated nucleotides in the leading strand, which does not contain a 5 nick, are corrected by

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the MMR pathway. Tom Kunkel and Joe Jiricny showed recently that cleavage of misincorporated ribonucleotises by RNase H also plays an important role in in leading strand mismatch removal [80,81]. People often ask me about my experience in Paul’s laboratory. The time I spent in Paul’s lab was unquestionably the most exciting time in my life, leading to remarkable discoveries and in the process, laying the groundwork for my entire future scientific career. Although Paul never pushed us, we were under extreme pressure to produce, because of the importance and urgency of our studies, as well as our admiration for Paul, which motivated us to work extremely long hours. Everyone in Paul’s lab worked very hard, and the lab was regarded as a “sleepless lab,” because someone was in the lab working essentially at all hours of the day and night. Paul is a great mentor, at least in part because he makes himself available for questions at any time, weekdays and weekends, when he was in town, and when he was traveling around the country or the world. Paul announced lab meetings on a message board 1 or 2 days in advance, based on his availability and how urgently he needed data for meetings/seminars, grant applications and reports. The meetings could be 1-2 weeks apart or a month apart. Paul randomly called on individuals to present their data, and this was an excellent opportunity for us to practice/improve our presentation and question-answering skills. It was also a valuable opportunity to receive critical comments from Paul. Paul also liked to walk through the lab at least twice per day, once in the early morning and again in the late afternoon. He interacted with us casually, chatting briefly about news headlines, sports (especially Duke basketball and Coach K), jokes, and of course, our latest preliminary experimental results. Depending on the progress of a project, he would schedule daily or weekly meetings with individuals or teams for trouble-shooting and/or experiment-planning. Even when Paul was out of town, he kept in touch by phone, talking in turn with everyone in the lab. These interactions led to productive training for each of us. Under Paul’s mentorship, everyone contributed to understanding the mechanism of MMR. Paul rewarded students/postdocs by sending them to national and international meetings, representing him to give plenary talks. Each year, Paul received more invitations to speak in those meetings and institutes than he could handle, because he preferred to travel no more than once a month. Paul generously allowed his most productive students and postdoctoral fellows to represent him, thereby gaining invaluable experience in this aspect of being a scientist. I had the honor of filling in for Paul at the 1995 Nucleic Acids Gordon Research Conference in New Hampshire, and the 1995 United States-Japan Cooperative Cancer Research Conference in Hawaii. Paul’s lab was a family to us. Everyone’s birthday, including Paul’s, was celebrated, and the person whose birthday was next in line was usually the designated host of the party. Paul and his wife, Dr. Vickers Burdett, held several parties for the lab in their beautiful home to celebrate holidays and lab achievements, as well as a way to say goodbye to lab individuals who had completed their training with Paul (Figure 1). We enjoyed the delicious food (e.g., pig pickin and steaks) Paul and Vickers made for us! Paul and Jack Griffith (Professor at the University of North Carolina and a close friend of Paul’s) also hosted joint informal parties for distinguished seminar speakers. These parties were attended by many students and postdocs and many professors and lab heads from the larger DNA repair community in Research Triangle Park, Durham and Chapel Hill, North Carolina. At these remarkable and fun events, junior scientists such as myself, had the opportunity to interact with accomplished scientists from around the world. The list of names is long, but to name a few, we had the opportunity to meet Arthur Kornberg, Bob Lehman, Charles Richardson and Richard Kolodner, Aziz Sancar and Tom Kunkel. These social events

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Figure 1. Paul with students and postdoctoral fellows from the lab. The date is March 28, 1992. The party was at Paul’s house. The photo captures our enthusiastic response, just after the famous final seconds of the Regional Final of the 1992 NCAA men’s basketball tournament. Duke defeated Kentucky 104–103 on the prayer of a jump shot by Christian Laettner. (Sometimes, prayers are answered!).

made a large impression on me had a significant influence on how I interact socially with my colleagues and how I mentor the young scientists who now come to me to ‘learn the ropes.’ While the field of DNA repair is now celebrating the Nobel Prize awarded to Paul for his mechanistic studies of MMR, we should also acknowledge others who have made significant contributions to our field. These include Richard Kolodner, who is responsible for the mechanistic MMR studies in yeast [61,82–91] and for the initial identification of genetic and epigenetic defects of MMR genes in human cancers [37,41,92–95]; Bert Vogelstein, who identified several key MMR genes and their associations with colorectal cancers [38,43,72,96]; Thomas Petes, whose genetic studies in yeast revealed the link between loss of MMR function and microsatellite instability [39]; Thomas Kunkel, who also demonstrated MMR activity in human cells and its deficiency in cancer cells, as well as involvement of PCNA and RNAse H in MMR [42,66,80,97,98]; Wei Yang, Titia Sixma and Lorena Beese, who delivered mechanistic insights of MMR by resolving the crystal structures of key MMR proteins [99–105], Joseph Jiricny, who identified a human mismatch binding activity and RNAse H in MMR [52,53,81,106]; Mike Liskay, Hein te Riele, T. W. Mak and Raju Kucherlapati, who provided the initial MMR knockout mouse models for HNPCC [107–111]; and Richard Fishel, whose work provided some stimulating models for understanding the mechanism of MMR [112,113]. After getting off the phone with the Editor in China, I called Paul’s cell phone to congratulate him for winning the Nobel Prize. However, no one picked up the phone. I thought Paul may already

have been inundated by phone calls from media, colleagues, family and friends. Only later did I find out that Paul was on vacation in a remote place, so he was ‘off-the-grid’ at the time. Instead of calling repeatedly, I sent Paul a congratulatory note by email, knowing that his mailbox would soon be full with similar messages from others, and I did not expect his usual prompt reply. In fact, he never responded directly to my email. But, in the end, I was not disappointed; about a week later, Paul invited me by e-mail to attend the Nobel Prize Award Ceremony in Stockholm, Sweden on December 10, 2015. I was so thrilled! It was truly one of the highlights of my professional life so far, to see Paul, Aziz Sancar and Tomas Lindahl receive their awards from the King of Sweden, Carl XVI Gustaf, in Stockholm, Sweden (Figure 2). I was happy that I was able to congratulate them in person for their remarkable achievements. I am always grateful that I was given the opportunity to make my own small contribution to the historic work in Paul’s lab. I own my entire scientific career to Paul and his outstanding training. By the time I left Paul’s lab to start my own career as a junior professor at the University of Kentucky, I had acquired the skills I needed to mentor students and postdocs, not to mention the needed knowledge of DNA enzyme biochemistry and molecular biology. In subsequent years, my group went on to identify and characterize several MMR proteins. Ultimately, we succeeded in reconstituting human MMR in vitro [67,69,114,115] and identifying novel MMR regulatory factors such as H3K36me3 and PCNA phosphorylation [116,117].

Figure 2. Paul Modrich (left), Aziz Sancar (middle) and Tomas Lindahl (right) receive the Nobel Prize in Chemistry from the King of Sweden, Carl XVI Gustaf on December 10, 2015, at the Nobel Prize Award Ceremony, Stockholm, Sweden (Photos: Courtesy of the Nobel Foundation).

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