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DNA Repair 11 (2012) 637– 648

Contents lists available at SciVerse ScienceDirect

DNA Repair

jo u rn al hom epa ge: www.elsev ier .com/ locate /dnarepai r

utobiographical sketch

osing and finding myself in DNA repair

. Growing up in conservative east Texas

My father Henry L. Thompson, of whom I never had any memo-ies, came from a rural Texas family that ran Rosemont Nurseries, aholesale rose-growing business that shipped Tyler roses around

he country. The sandy soil of east Texas had made Tyler a pre-ier rose-growing region. I grew up not knowing much about my

ather, but he must have been mechanically minded and adven-uresome since he was flying airplanes when he was 15. Ratherurprisingly, he attended Cornell University in Ithica, New York,here he met and soon married my Mother who came from

well-educated New York family. They were both horticultureajors and upon graduation moved to Tyler to begin their own

andscaping and nursery business. Soon thereafter I was born in941. As World War II unfolded in Europe, my father enteredhe United States Army Air Forces and was stationed in Englandhere he was a P51 Mustang fighter pilot (http://www.yorkshire-

ircraft.co.uk/aircraft/planes/ryedale/106809.html; “On the 7th ofebruary 1945, three Mustangs took off for a training flight fromheir base at East Wretham, England, at 14:14 h. Two of the aircraftere new to the Unit and their pilots were to undertake tests on

he guns and their performance at altitude. The third aircraft was anlder aircraft and was in fact listed as “war weary” and was declaredot safe for combat flying; despite this it was mechanically sound.his third pilot was acting as an observer to the other two. . .”).n that flight my father’s plane lost its propeller and crashed nearcarborough on the eastern coast. His parachute was blown to seand he was never found. My younger brother Bryan was born fouronths later. These events thrust my Mother into the role of con-

inuing the nursery business while raising her two sons.From an early age, I seemed to enjoy school and took pleasure

n receiving the teachers’ admiration for making good grades atural Dixie School, which I attended through the ninth grade. Trans-erring to high school in downtown Tyler involved some culturalhock. There were many other good students, and I remember feel-ng disadvantaged by my Dixie education, whether that was true orot. I became a rather competitive student and took my homework,

ncluding Latin, very seriously. For my senior science-fair project Iuilt a Foucault pendulum (after the French physicist Léon Fou-ault), which can demonstrate the rotation of the earth. A woodenower was constructed to support a cable and iron cylinder, whichas energized from below with a huge coil of copper wire about a

oot in diameter. Each time the swinging iron bob approached the

oil a magnetic force provided enough acceleration to maintain itsotion.

568-7864/$ – see front matter

oi:10.1016/j.dnarep.2011.10.005

From an early age, I developed a deep curiosity about nature andthe universe, which I ascribe to spending a lot of time outdoors onour rural property, often by myself, as I was growing up. I do notrecall anyone in my pre-high school years nurturing me scientif-ically except an uncle in New York who sent me a chemistry setone Christmas. I began riding motorbikes at age 12, with almostno restrictions, and acquired a driver’s license two years later. Onememorable experience during high school was reading popular-ized science paperbacks about the universe and its possible origin,which were available in the drugstore. I remember spending hourswith my close friend Stephen Ramsey trying to understand Ein-stein’s “gedanken” experiments that described length contractionand time dilation in the Special Theory of Relativity.

In my senior year I was very fortunate to have abrilliant math and physics teacher named Bryant Saxon(http://www.branam.com/makingsense/justdoit.shtml), whomade physics and “�-Math” stimulating and challenging. Therewas something powerful about using the equations of probabilitytheory to solve problems. Undoubtedly because of this excellentmentoring, I had acquired a passion for science and graduatedvaledictorian; remarkably, my girlfriend in junior high school,Betty Neumeyer, was salutatorian. Our Dixie School education hadproved its worth. I instinctively developed a plan to attend theUniversity of Texas in Austin as a Physics major. That seemed likea cool thing to do and was made financially feasible by the WarOrphans’ Educational Assistance Act of 1956, which would pay me$110 a month. The registration fee was $50 a semester along witha requirement to sign a loyalty oath that I was not a member of theCommunist Party. I had little idea what a communist was, otherthan a dirty word for somebody innately evil.

After four years in Austin, I received a BS degree in Physics, butmuch of the math and physics I studied seemed terribly abstract,and nothing really captured my imagination and felt right for thefuture. I spent the summer of 1963 working at NASA Ames ResearchCenter in Mountain View, California. Perhaps the most importantthing that happened to me that summer was not working withNASA engineers who were preoccupied with space vehicle aerody-namics, but becoming very fond of classical music, made possibleby affordable reel-to-reel tape recorders. I returned to Austin withboxes of classical music tapes and enrolled in psychology and phi-losophy courses – in an attempt to get a broader perspective on lifeand grapple with my future. Being strongly influenced by two previ-ous girl friends, I took courses they had enjoyed and recommended.

As I continued metaphorically to “tread water”, the beginning of acareer path serendipitously unfolded.

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38 Autobiographical sketch /

. Finding a direction in science: biophysics and radiationiology at the M.D. Anderson Hospital in Houston

During my immersion into psychology and philosophy, I learnedbout a new Graduate School of Biomedical Sciences (GSBS) inouston from a fellow physics major, Dewitt Liles, who encour-ged me to investigate it. The GSBS was something of an experimenttself, since it had just been created, and I was immediately acceptednto the second-year class. Through the Physics Department ofhe M.D. Anderson Hospital, I was awarded a graduate fellow-hip stipend of $300 a month, which seemed like a lot of money,onsidering that my apartment rent was $115 monthly. In thehysics Department chaired by Robert Shalek, two young scien-ists, William Dewey and Ronald Humphrey (Fig. 1A and B), weretudying how ionizing and ultraviolet radiations killed mammalianells, within the framework of the cell division cycle. After spendingn introductory summer in Houston’s heat and humidity I returnedo UT Austin for remedial coursework. As an undergraduate I hadever taken a course in biology, which was largely a descriptivecience at that time. I had to delve into freshman biology, genetics,ell physiology, organic chemistry, and physical chemistry. Onceack in Houston, I learned biochemistry at Rice University as theSBS began developing its own courses.

In the Humphrey–Dewey labs, I was introduced to the “art” ofammalian cell culture and the X-ray machine, where a sign in the

ead-lined room warned not to point the beam out the window. learned how to weigh all the individual ingredients to prepareell culture medium. This was long before the days of laminar flowoods for sterility control, and cell culturing was an unpredictablendeavor. On good days the cells were growing, but sometimes aresumptive mycoplasma or “PPLO” episode would bring every-hing to a standstill. I was assigned Bill Dewey as my graduatedvisor, who introduced me to the cytogenetics of damaged chro-osomes in irradiated cells. Chromosome damage seemed to lie at

he heart of cell killing by radiation; damage to DNA as the criti-al target was a recurring theme during my graduate training. Billonceived the idea of using time-lapse photography to monitor theate of individual irradiated cells. This seemed like a pretty inter-sting project to me, especially since it had a mechanical aspecthat I could easily relate to, given my early experience repairing

otorbikes and lawnmowers. However, Bill soon decided to accept position in Fort Collins, Colorado, in 1965 before I could get myroject underway.

Most fortunately, Herman Suit, who had both medical and PhDegrees, became my new mentor (Fig. 1C) and strongly identi-ed with the goals of my project. Herman ran a very busy lab

n experimental radiotherapy in which he used mouse modelso treat tumors with radiation. We were soon thinking in detailow to best construct cell-division pedigrees of irradiated cells.ith the critical assistance of Ernest Libby in the photography

epartment, a World War II aerial reconnaissance camera (model-24) manufactured by Kodak was acquired to serve as the enginef the project. The camera was mounted at the top of a Fishericroscope-photographic stand and connected by a long exten-

ion tube to a Zeiss 63 mm Luminar objective (Fig. 2A). A T30lass cell culture flask (Fig. 2B) was mounted at the bottom ofhe illumination stand and contained a cylindrical insert in whichhe cells grew attached to the lower surface of a glass disc. Aooden incubator box enclosed the lower portion of the light stand

nd kept the cells at 37 ◦C (Fig. 2C). The camera was connectedo a timer that executed a photograph every 30 min with a loudcreech as the DC motor revved up and jarred everyone in the

oom.

The large-format camera took images that were 5 in. square. At0× magnification, each photo could easily accommodate thou-ands of cells. I could begin an experiment with several hundred

epair 11 (2012) 637– 648

cells monitored for several generations as controls, irradiate them,and continue monitoring through many generations. Thanks againto Kodak, we ordered custom made, high-resolution film in 56-foot rolls, long enough to take over 100 images. To develop thefilm Mr. Libby cleverly devised a simple apparatus with a motorthat would pull the film through the developing, fixing, and rins-ing stages (Fig. 2D). A hair dryer mounted at the upper end ofthe film track dried the film. It was a wonder that this deviceactually worked and never caused me to lose an experiment.After the photography came the hard part: manually analyzingthe images using a light box with a hand-crank to move thefilm (Fig. 2E). Looking through a dissection microscope, I couldeasily see each cell. By moving from one frame to the next, Ihad to track each cell and all its progeny for as many as 7 or 8generations. The classical definition of a “colony” is 50 cells, i.e.5–6 doublings. Thus, I could perform an analysis of cell genera-tion time and the probability of cell division that was congruentwith conventional survival curve data. By sitting for hundreds ofhours at the view box, I heard all the gossip in the lab while Iwas watching multipolar mitotic figures, cell disintegration, giantcells arising through, often repeated, cell fusion events follow-ing incomplete mitosis. I recall trying to imagine the complexityof life at the molecular level and feeling rather hopeless. Howcould we ever expect to understand the structure/function ofcomponents like genes and chromatin, which we had no way ofaccessing?

Before I completed my dissertation work, Herman accepted aposition at the Massachusetts General Hospital in Boston. SinceI associated a lot with Ron Humphrey while doing cell culturein his lab, he inspired me to perform an experiment with UV-Cradiation. Given the investment in building the time-lapse sys-tem, why not get the most out of it? Three publications on X-rays[1–3] and one on UV-C [4] resulted from the time-lapse stud-ies and other experiments. Although there was competition fromLeonard Tolmach, who was using HeLa cells to perform simi-lar studies [5,6], I was not greatly impacted since I was wellalong by the time he published. It is remarkable that Bill Deweyalso did extensive computerized video time-lapse studies in thelatter part of his career by investigating the details of divisiondelay, apoptosis, giant cell behavior, and “mitotic catastrophe”[7–12].

Herman Suit was an outstanding mentor in many respects:(a) He made sure my experiments were done properly; (b) hetaught me a lot about how to think and write critically; and (c)very importantly, he set me on a future path that would ensuresolid future training. We agreed that I would go to the OntarioCancer Institute for postdoctoral study with Gordon Whitmore(Fig. 1D), who was well known in the radiation biology commu-nity and had recently coauthored a textbook with Elkind [13].The move to Toronto in the summer of 1969 was very infor-mal. I had no idea what I would work on, but I had alreadydeveloped a fascination with the auxotrophic mutants of Chinesehamster ovary (CHO) cells that were being reported by Puck’s group[14–16]. For example, these studies showed by cell-hybridizationcomplementation analysis that glycine auxotroph strains werecaused by putative mutations in four different genes producedby mutagenic agents [17]. This approach captured my imagina-tion since it was reaching to the molecular level at specific geneloci.

3. Somatic cell genetics gets a foothold in Toronto

Before I arrived at the Ontario Cancer Institute in Toronto,Louis (Lou) Siminovitch (Fig. 1E) had convinced Gordon Whitmorethat there would be a big pay-off in the isolation of mammalian

Autobiographical sketch / DNA Repair 11 (2012) 637– 648 639

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hitmore, (E) Louis Siminovitch, and (F) Mortimer L. Mendelsohn.

ell mutants that displayed temperature-sensitive (TS) growth.t that time, it was unclear whether the auxotrophic and drug-esistance mutants of CHO cells actually involved mutations intructural genes as opposed to defective gene expression due toegulatory changes. In fact, much later Morgan Harris showed thathe native proline requirement of CHO cells could be reversed byxposure to 5-azacytidine, a DNA demethylating agent [18,19].

TS conditional lethal mutant would almost certainly reflect mutational change that increased the heat lability of a par-icular protein. Siminovitch was so enthusiastic that I did notorry too much about a mutant hunt not working. My risk-

aking genes had been activated during my youth while ridingotorbikes, and all I had to lose was a few months of my

ife.Gordon outlined a mutant selection approach that was based

n the incorporation into cycling cells of tritiated thymidine (3[H]-hd) of high specific activity, which kills by �-ray damage to DNA.e had already used short exposures of tritiated thymidine tobtain synchronous viable cell populations [20]. I proceeded toutagenize mouse L cells growing in suspension in spinner flasksith nitrosoguanidine and methodically went through multiple

nrichment cycles in which cells were shifted from 34 ◦C to 38.5 ◦C.fter allowing 24 h for any TS cells to stop cycling at the restric-

ive temperature, cultures were exposed to 3[H]-Thd for 48 h andhen retuned to 34 ◦C in its absence. This regimen required thatrrested TS cells remain viable for up to 72 h, which in retrospecteems like a naïve expectation. After five enrichment cycles overeveral months, I tested individual clones for their ability to grow

t 34 ◦C but not 38.5 ◦C. I was somewhat amazed to find that I hadsolated exactly what we were looking for, i.e. cells having a veryronounced TS phenotype [21]. Years later, in 2008 shortly before

received the EMS (Environmental Mutagen Society) Award, Lou

. (A) William C. Dewey, (B) Ronald M. Humphrey, (C) Herman D. Suit, (D) Gordon F.

told me on the phone that my chances of getting that first mutant(named tsA1) were 10−14. I replied that it was really more like 10−7

because the gene mutated gene was later found to reside on the X-chromosome, which conferred a dominant phenotype [22]. WhenI commented that our first publication must have helped him getthe EMS Award in 1986, he said this PNAS article was one of hisfour most important papers. Also gratifying to me was the factthat another laboratory eventually identified the defective gene intsA1 as UBE1, which encodes the ubiquitin activating enzyme E1[23].

We soon had many more independent TS mutants of mouseL cells [24], but after establishing this proof of principle theywere mostly left sitting in freezers for the duration of my career.Since immortalized mouse cells are notoriously heteroploid (vari-able chromosome number), we decided to switch to near-diploidCHO cells, which had yielded many auxotrophic mutants. Thiscell line also provided us with TS mutants, the first being dra-matically defective in protein synthesis because of altered leucyltRNA-synthetase [25]. Other members of the Toronto geneticsgroup were soon isolating auxotrophic and drug resistance mutantsof CHO cells [26–29]. These successes lead to an invitation byDavid Prescott to write a book chapter on mutant isolation [30],which Bud Baker and I undertook. This was a very difficult taskas my writing skills were weak, but Lou patiently gave us criticalguidance. In my fourth year in Toronto, I was invited by Mor-timer (Mort) Mendelsohn at the Lawrence Livermore NationalLaboratory (Fig. 1F) to interview for a position in the Biomed-ical Sciences Program where he had recently become director.

Mort, who also had a background in radiation biology and radio-therapy, was pretty keen to hire me, but Gordon Whitmore hadreservations. Deep down, my own biggest reservation was thatI really had little confidence that I could conduct a research

640 Autobiographical sketch / DNA Repair 11 (2012) 637– 648

Fig. 2. Equipment used during graduate school for time-lapse photography of irradiated cells. (A) Camera, supporting stand, and culture flask. (B) Close-up view of flaskand objective lens. (C) View showing incubator enclosing lower-portion of photographic stand, power supply, and timer. (D) Custom built Plexiglas film developer tanks. (E)L all pot

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ight box and stereomicroscope for viewing 5 in. × 5 in. images. (F) Example of a smo multipolar mitoses.

roject. Mort seemed to be impressed with our book chapter

nd other publications. His enthusiasm for my being a part ofis new program was a compelling factor in my accepting theosition.

rtion of field containing irradiated cells showing reference grid lines. Arrows point

4. From TS mutants to DNA repair mutants in CHO cells

For several years at LLNL, I continued work on TS mutants inCHO cells as the isolation of TS mutants became internationally

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opular. The main difficulty with most TS mutants was that iden-ifying their molecular defect was nearly impossible since theyreceded the advent of molecular biology. Therefore, we devised

strategy of using tritiated amino acids as the enriching agent forsolating additional protein synthesis mutants that proved to beefective in several other aminoacyl-tRNA synthetases [31–33].

In 1978 I became enthused about isolating DNA repair mutantsnd was committed to attempting it after attending the 1978 UCLANA repair conference at Keystone, Colorado. I had a certain naïveonfidence that was grounded in the TS mutant studies. Also, Stam-to and Waldren had just published a paper describing a modestlyV-sensitive CHO mutant identified using a replica plating tech-ique [34]. However, our strain of CHO cells, which grew well inuspension culture, was very loosely adherent, confounding thispproach. I was aware of a red dye that was specific for dead cellsnd also that UV-C would not penetrate through several layers ofells. Since CHO cells form “piled up” colonies, an irradiated UV-ensitive colony (given the appropriate UV fluence) would consistf many dead cells along with some surviving cells. This featureecame the basis of our identifying UV-sensitive colonies, whichere present at a frequency of ∼10−4 after chemical mutagenesis

35].Kerry Brookman had recently joined my lab in 1978 after work-

ng with Donald Glaser at U.C. Berkeley, who lost his NIH fundingefore completing a high-technology automated screening systemor mutants. Kerry graciously agreed to work with me on Satur-ays, allowing us to perform the mutant hunts with uninterruptedoncentration when the lab was quiet. I spent many hours at theicroscope scanning dishes for colonies that contained mostly

ead cells in response to prior UV-C, EMS, or MMC exposure. Weere off to a roaring start when we found that we could readily

dentify mutant colonies and recover live cells from them. However,s we began to characterize these new mutants, things soon begano take a very strange turn. Both my lab and others were initially hitith a wide range of bizarre events, which I documented in a previ-

us historical overview [36] and some of which received attentionn the local newspapers (Fig. 3, upper panel). As the months passedt became clear to me that our lab in particular was being deliber-tely sabotaged in ways much more devious than the lesser pranksuch as unplugged incubators. The overriding issue in the ensu-ng months was that the mutants repeatedly exhibited phenotypicnstability, which my collaborators in two other laboratories wereot observing with the mutant cultures we had sent them. Thepshot of all this was almost a year of lost effort before we discov-red that someone was introducing wild-type cells into the mediatock bottles, which were being stored in the cold room.

Once we finally overcame this impasse, there was a brightuture that included numerous UV-C mutants resulting from theollaboration with David Busch in Donald Glaser’s laboratoryt U.C. Berkeley [37]. There was also escalating competition asany other labs began to isolate and characterize hamster DNA

epair mutants sensitive to various mutagens [38–48]. Althoughumerous mutants were isolated in mouse lymphoma 5178Y cells49–52], these were little used in future molecular studies, likelyecause of their resistance to DNA transfection [53].

Our first EMS-sensitive mutant, designated EM9 [35], was foundo exhibit a very high baseline frequency of sister-chromatidxchange (SCE) [54], reminiscent of Bloom syndrome [55]. SCEsere receiving a lot of attention in the mutagenesis field because

hey were induced in varying degrees by virtually all DNA-amaging agents [56] and could be readily measured with highrecision. These features lead Tony Carrano and I to explore pos-

ible relationships between SCEs and single-gene mutations in anttempt to better understand the nature of SCEs [57].

When Errol Friedberg contacted me to write this article, I men-ioned to him that part of my early introduction to DNA repair was

epair 11 (2012) 637– 648 641

a book entitled “DNA Repair Processes” that he had edited for aworkshop 35 years ago. He said that would be an interesting intro-duction. As I began to write this article, I discovered that he was notthe editor, so my memory had slipped, and perhaps his too. How-ever, he did coauthor a chapter on excision repair and xerodermapigmentosum. As I was writing this biosketch, I decided to takea closer look at this 1977 publication to see what was being saidabout some of the “hot topics” at that time, before I had isolatedany DNA repair mutants. Sheila Galloway has a chapter entitled“What are SCEs?”, a question with which I later became preoccu-pied when we isolated the xrcc1 EM9 mutant of CHO cells [54]. SinceX-rays were known to be a poor inducer of SCEs, DNA single-strandbreaks were not considered to be a likely cause. From a considera-tion of the types of agents that efficiently induce SCEs, it seemed toDr. Galloway that SCEs were “. . . a consequence of lesions such ascrosslinks that cause deformation or kinks in the DNA backbone”.Then from considerations of the requirement for DNA synthesisshe states “. . . some initiating event at the replication fork, seemslikely” and suggested that single-stranded DNA during replicationmay be a vulnerable target. A salient observation she discussedis that lymphocytes of Fanconi anemia cells, which were puta-tively defective in crosslink repair at that time, were reported tobe defective for SCEs induced by mitomycin C (a DNA inter-strandcrosslinking agent) [58], thus presenting the concept that SCEs arethe manifestation of a DNA repair event. She also described exper-iments by H. Kato using BrdU plus fluorescent light that led him toinfer “. . .that strand breaks at replication forks were the predomi-nant cause of SCEs. . .”. She concluded by saying “They may involvea recombination event, often instituted at the replication fork, butalso provoked by any unrepaired damage in the replicated DNA”.This inference has stood the test of time even though it was madewell before it became apparent that mammalian cells possess themolecular machinery for homologous recombination [59–61]. Wenow know that most spontaneous and induced SCEs result fromthe repair of broken replication forks by HRR [62], but they arealso induced in G2 cells by a small fraction (∼7%) of the DSBs fromionizing radiation (IR) [63].

5. Using hamster mutants to clone and characterize humanDNA repair genes

From our extended collaborative gene mapping studies withMichael Siciliano at the M.D. Anderson Cancer Center, we knewthat human genes efficiently complemented the hamster mutantlines [64–68]. Over the next decade in my laboratory, characteri-zation of various hamster mutants and complementing genes ledus into multiple DNA repair pathways, including nucleotide exci-sion repair through the many ERCC2-defective cell lines. Humancells had generally proved to be recalcitrant for DNA transfection[69]. This shortcoming conferred a critical advantage to using UV-sensitive CHO mutants to clone the complementing human genes.ERCC2, which we found to encode a DNA helicase homologous toRad3 in yeast [70], was one of the most commonly mutated genes inthe collection of mutants identified in our collaborative effort withDavid Busch in Don Glaser’s laboratory at U.C. Berkeley [37,71,72]and in other laboratories [73]. This finding was especially surprisingin light of the fact that, as a member of the TFIIH transcription-repaircomplex, ERCC2 has an essential function in transcription [74,75];cells cannot tolerate large deletion mutations that disrupt the pro-tein. NER-inactivating ERCC2 mutations typically abolish the DNAhelicase activity [76,77]. Our initial efforts to test whether any of

the CHO mutants was equivalent to one of the genes defective inxeroderma pigmentosum, had proved technically challenging andnot particularly informative [78]. Once Christine Weber had clonedERCC2 and reported at a Noordwijkerhout DNA Repair meeting that

642 Autobiographical sketch / DNA Repair 11 (2012) 637– 648

Fig. 3. Newspaper articles about bad and good times in our research. Upper panel: article relating to our lab and others in our biology program that experienced strangei wereC

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nterference with experiments. The spinner flasks that were turned off on October 18hronicle (June 16, 1988) about our cloning of XRCC1 and ERCC2.

ts transfection conferred UV-C resistance to XP-D fibroblasts, Errolriedberg’s lab rushed to identify ERCC2 mutations in XP-D cellsnd published [79] before we had completed a systematic study80].

Kerry Brookman in my laboratory had the lead role in cloning the

uman XRCC1 gene, which complemented the EM9 mutant [81,82].s detailed previously [36], this proved to be a multi-year effort

hat was facilitated by the Alu-family sequences associated with theene. We were simply lucky that the intact gene was small enough

CHO suspension cultures in my laboratory. Lower panel: article in the San Francisco

(∼33 kb) to fit into a cosmid vector. It was especially gratifying tofind that cosmid clones containing the gene could fully correct theSCE level, the defect in single-strand break repair, and the �-raysensitivity to killing [82]. During his postdoctoral work with meKeith Caldecott obtained a full-length cDNA for the XRCC1 pro-

tein [83] and identified the functional association with LIG3 [84].Unlike the NER genes, XRCC1 has no counterpart in lower eukary-otes and proved to function by coordinating the action of otherproteins involved in BER and single-strand break repair [85–88].

Autobiographical sketch / DNA Repair 11 (2012) 637– 648 643

Fig. 4. Photograph taken in our laboratory during a meeting with newspaperreporters. I was attempting to explain how the XRCC1 gene controls the rate ofsister chromatid exchange and sensitivity of cells to killing by ionizing radiation.T

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isolation in my lab [99,100]. Nigel was happy with our continuing

he chromosome image, showing SCEs in color, was prepared by Pinkel et al. [120].

erry also cloned the mouse XRCC1 gene [89] with the motivationhat we would construct a mouse that was null for this gene. Thisdea eventually came to fruition in the work of Robert Tebbs, withhe frustration that the xrcc1 null mouse embryos experienced rel-tively early embryonic lethality that was not overcome by Tp53eficiency [90].

The cloning of ERCC2 and XRCC1, both of which were mapped tohromosome 19 [65,67,91], was a notable milestone for my labo-atory and for the Biology and Biotechnology Research Program atLNL [70,82,92]. At that time only the cloning of human ERCC1 hadeen accomplished for DNA repair genes – by Dirk Bootsma’s group

n Rotterdam [93]. The excitement of our success lead to an LLNLress release in which newspaper reporters were invited into our

aboratory to meet with us face to face (after we had been coachedy the public relations office on how to deal with them). I remem-er the room being packed as I held up an image of EM9 cells andointed to the SCEs while attempting to explain in simple languagehat we had accomplished (Fig. 4). The gene-cloning story was

overed in various regional newspapers, including the San Franciscohronicle (Fig. 3, lower panel). As the work progressed, it was high-

ighted in a special issue of LLNL’s Energy and Technology Reviewith Kerry’s photo on the cover (Fig. 5). Of course isolating the genes

old us little about protein function, but in principle we could nowroceed to identify the encoded proteins and their biological roles.

One of the interesting CHO mutants, UV40, from David Busch’sutant hunts had a complex phenotype of sensitivity to UV-C,-rays, and MMC [72]. I was intrigued by this mutant, which rep-esented a new complementation group for MMC sensitivity, andaw the possibility of using MMC as the selecting agent since UV40as ∼10-fold sensitive to this agent [94]. The characterization ofV40 by Busch and his many collaborators had led to the con-lusion: “Thus, its defect apparently does not involve nucleotidexcision repair but rather another process, possibly in replicatingast lesions” [94]. When Nan Liu joined my laboratory as a postdoc,loning the complementing human gene (named XRCC9) for UV40as one of her projects, and she rapidly succeeded [95]. Analysis

f the nucleotide sequence of the complementing cDNA, which weecovered from a transfectant, revealed no known motifs or homol-gy with known proteins. Thus, we were at an impasse for a while.owever, the next year Hans Joenje sent me e-mail stating that

hey had cloned the Fanconi anemia (FA) group G gene (FANCG) andound it to be identical to XRCC9 [96]. Thus, we now had a foothold

or future studies but were faced with the competition from thearge consortium that was working on the fascinating FANC genes,

hich seemed to be ever increasing in number.

LLNL. While Tony Carrano was director of the Biology and Biotechnology ResearchProgram at LLNL, this publication highlighted recent advances and featured KerryBrookman on the cover.

While Nan Liu was cloning XRCC9/FANCG, she was also cloningthe human cDNA that complements the irs1 X-ray sensitive mutantof V79 hamster cells [45]. Nigel Jones isolated irs1 during his PhDwork with John Thacker and had brought the mutant to LLNL in1987 when he did a postdoctoral stint with me. Like UV40, irs1is highly sensitive to MMC and was complemented by transfec-tion with a cDNA library [97]. As was my habit with many criticalexperiments, I was frequently in the lab sitting at the invertedmicroscope, watching the behavior of the transfectants. I noticedthat when these cells were taken off MMC selection, MMC-sensitivecells rapidly appeared in the cultures. This time, I had no reason tosuspect that the phenotypic instability was an artifact caused bycontaminating cells. I suggested to Nan that she make a Hirt extractand use it to transform Escherichia coli. Within a few days she haddone this and found that she had the cloned XRCC2 cDNA in herhands, without the need for laboriously constructing and screeninga genomic library. Rescuing an episomal cDNA was at odds with areport that V79 cells could not harbor an episomal plasmid that car-ries oriP and the EBNA-1 gene [98]. Dans les champs de l’observationle hasard ne favorise que les esprits préparés, i.e. In the fields of obser-vation, chance favors only the prepared mind – Louis Pasteur, 1854lecture.

The cloning of XRCC2 had strong political ramifications becauseJohn Thacker was also aggressively working on it. He sent a Faxstrongly advising us not to pursue the cloning, presumably becauseour success might cause him to lose his funding. So, who “owned”the mutant? Nigel Jones (back in the UK by that time) had char-acterized irs1 and conducted initial experiments towards the gene

to work on the cloning, which was also consistent with the con-cept that published materials, including cell lines, be available tothe scientific community. However, I well understood that isolating

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44 Autobiographical sketch /

igh-quality mutants was not a simple task and that making themoo freely available could be costly for one’s career. I tried to walk

middle ground by communicating with John Thacker about ouresults, and we published about the same time [97,101]. There wasurther controversy between us about what to name these Rad51-ike genes because he did not like the term “paralogs” for reasons

never understood. We had submitted our cloning paper to Cell,ut Benjamin Lewin thought there was insufficient insight into theolecular function of XRCC2 and XRCC3 to warrant acceptance.t the wise suggestion of my collaborator David Schild, I wrote aebuttal letter, and Lewin agreed to accept the manuscript for therst issue of his new journal Molecular Cell. Now, 14 years later, wetill have little understanding about how these paralogs assist thessembly of RAD51 onto single-stranded DNA (reviewed in [102]).

. From genes back to (isogenic) mutants by gene targeting

In a “Roots” article in 1998, I recounted the historical con-ributions of Chinese hamster cell mutants to the field of DNAepair [36]. At that time, we were just beginning a new endeavoro use gene-targeting methodology to construct isogenic mutantsn CHO cells. I felt that CHO cells were still the most attractiveystem in which to construct mutants. Near-diploid immortal-zed mouse cells do not exist (as far as I know), and human cellsre slower growing and much more difficult to work with thanodent cells. My motivation for starting this work was stronglynspired by the success of Rhonda Rolig and Rodney Nairn athe Science Park in Smithville, Texas, in knocking out the sin-le active allele of ERCC1 in CHO cells [103]. Their manuscriptoncluded with the statement: “Our results support the notionhat mammalian somatic cells, such as CHO cells, are suitableene targeting recipients, and should encourage the use of geneargeting for knock-out mutagenesis in cultured somatic cellines”.

Greatly encouraged and excited by this work, and also influ-nced by Gerry Adair’s extensive gene targeting work in CHO cells104,105], I undertook this new direction, thinking it had greatotential. During the writing of the proposal to construct knockoututants for the DOE, we emphasized making mutants for RAD51

aralogs RAD51B/C/D. We had only a short time frame to preparehe proposal. I was on a flight to a meeting when I realized thate did not even know whether these other paralogs were even

xpressed in CHO cells, and a reviewer might object to this defi-iency. I quickly made an $80 phone call to Kerry Brookman, urginger to obtain as soon as possible a northern blot showing the tran-cripts for these three RAD51 paralogs. This experiment provedo be successful, and we received funding to make new mutantsith a focus on DSB repair and IR sensitivity. This was a majorndertaking because it first required having a CHO genomic libraryrom which we could clone isogenic gene fragments for construct-ng targeting vectors. Fortunately, Pieter de Jong, who had workedn the Human Genomic Project at LLNL [106–108], had the tech-ical expertise to construct excellent arrayed BAC-clone libraries.ieter provided a library that proved to contain each of the genes weought. Kerry Brookman was in charge of performing experimentsoward knocking out the APE1 gene, which we had chosen becausef David Wilson III’s central interest in this gene. After a series ofegative experiments using increasingly larger numbers of trans-

ected cells with various targeting vectors, our initial frustrationsurned into major disappointments. We had made little progress

nd were facing a competing renewal of the project. Understand-bly but regrettably, Kerry decided to leave LLNL after 23 years ofur working together since isolating the initial CHO DNA repairutants. This was a big blow to me psychologically since I had

epair 11 (2012) 637– 648

relied on her technical competence and expertise for many yearsto keep projects moving and our lab well organized.

In 2001, John Hinz had just arrived (returned) from the UK tojoin the group as a postdoc, and I able to convince him to resumethe APE1 targeting project. However, after investing another year’sworth of effort in trying to construct a conditional knockout in cellsthat expressed tetracycline-dependent APE1 expression [109], wedid not succeed and finally had to admit defeat, with the view thatperhaps the targeting efficiency for some genes is impossibly low.

During this period, Robert Tebbs and I had decided to makea knockout for Fancg because I thought that an isogenic mutantwould be a uniquely valuable system to study the FANC pathway inCHO cells, at a time when there was no generally accepted molec-ular model for the function of the FANC proteins. Although NanLiu had cloned human FANCG using the UV40 CHO mutant, thismutant was patently unsatisfactory for further studies. I wantedto know, for example, whether the IR and UV-C sensitivity (andother features such as elevated SCE) seen with the UV40 mutantwould be present in an isogenic mutant. The Fancg locus is hem-izygous in CHO cells, as shown by the work of Jane Lamerdin, whocharacterized the hamster fancg mutants for her PhD thesis [110].In the face of a very low targeting efficiency of ∼10−4, Tebbs suc-cessfully identified targeting events in large pools of transfectedcells, followed by repeated subpooling and subcloning. John Hinzimmediately began to help characterize this new mutant (KO40). Avariety of studies on KO40 lead us to the view that, as a member ofthe FANC core complex, FANCG’s function is “. . . to maintain chro-mosomal continuity by stabilizing replication forks that encounternicks, gaps, or replication-blocking lesions” [111]. It was now eightyears since the ERCC1 knockout study grabbed my attention [103].To what extent did these new insights into the nature of the FANC“pathway”, other than the satisfaction of success, justify the con-siderable effort and expense of constructing this genetically “clean”mutant?

At that time in the Fanconi anemia field, the central defectin FA cells had not been ascribed to a failure to properly repli-cate damaged DNA. However, once I began attending the annualFARF (Fanconi anemia research fund) meetings, it occurred tome that a replication-associated repair defect was the likelyexplanation. Robert Tebbs and I presented a simple concep-tual model at the Houston FARF meeting in 2003, at whichthe projector stopped working during my talk (I was able toresume the next day). John Hinz and I detailed this modelin a review article [112] in which we stated “. . . the criticalmonoubiquitination of FANCD2, an event that somehow helpsstabilize blocked and broken replication forks. This stabiliza-tion facilitates two kinds of processes: translesion synthesis atsites of blocking lesions . . ., which produces point mutationsby error-prone polymerases, and homologous recombination-mediated restart of broken forks . . .” I was later pleased whenone review article formally acknowledged the new perspec-tive we had articulated for the function of the FANC proteins[113].

Robert Tebbs turned his attention and vector expertise to theRAD51D gene as the next knockout project. A cytogenetics exper-iment, which was performed by a collaborator putatively usingour hamster BAC clone containing RAD51D as a probe for FISH(fluorescence in situ hybridization), seemed to show clearly thatthere was only one copy of this gene in the wild-type CHO cells.This result seemed a little surprising, but we nevertheless begana targeting strategy based on this information. Once we had iden-tified a clone that appeared to be correctly targeted based on PCR

analysis, we were baffled because the cells showed no sensitivityto MMC. This result forced us to repeat the FISH analysis (with-out that collaborator’s help). This time we observed two copiesof the gene, which was consistent with our first-allele targeting

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Autobiographical sketch /

esults. There was no obvious way to account for the initiallyrroneous FISH result. Moreover, we had to devise a new tar-eting strategy, which eventually proved successful. Robert hadeveloped state-of-the-art Cre-Lox conditional targeting vectors heamed TnT (Tebbs “n” Thompson), which really proved their worth.hen we eventually transfected the doubly targeted cells with

re recombinase and screened the transfectants, in some cells wechieved simultaneous deletion in both alleles of RAD51D withinegions flanked by Lox sites, rendering the cells highly sensitiveo MMC. We felt we deserved this bit of luck. More important,hen we transfected these mutant cells with the hamster RAD51DAC clone, they were fully complemented for all end points tested114]. This result meant that we had constructed a quantitativelyobust CHO mutant with a substantial and well-defined deficiencyn HRR.

It was intellectually satisfying to use the rad51d mutant cellso measure radiosensitivity during the cell cycle in highly syn-hronous populations obtained by centrifugal elutriation, whichohn Hinz perfected [115]. We answered some of the questionshat were being asked during my graduate school days, such ashe basis of S-phase resistance. We found that, after reaching aeak in S-phase, HRR capacity declined as cells moved from S

nto G2 phase [115], giving a more refined picture versus whatas previously presented by one of my collaborators [116]. These

tudies lead me to the view that the purpose of G2 phase iso effect repair by HRR of a portion of the DSBs that arise dur-ng DNA replication since HRR occurs much more slowly thanHEJ.

. A human-cell DNA repair venture: Department of Energyow-dose Radiation Research

Beginning with the Atomic Energy Commission in 1973, myesearch was supported continuously (and generously), creatinghe financial stability that is needed for doing productive science.he DOE Low-dose Radiation Research Program had funded ourHO gene knockout work for six years, but there was increas-

ng pressure to perform more “relevant” research. I was muchmpressed by work describing the use of �H2AX foci as a veryensitive, quantitative assay to assess DNA DSB repair in humanbroblasts in the mGy dose range [117]. With Irene Jones as a col-

aborator, I was funded to assess the degree of variability in DSBepair capacity in the human population using non-immortalizeduman fibroblasts. We proposed to assess repair in 100 cell strains,ut this number proved to be quite unfeasible. Once again the ini-ial excitement about a new project was superseded by the realityf its difficulty. We soon discovered that the background foci lev-ls were typically 20-fold higher in our cell strains than what hadeen reported [117]. This difference imposed major constraints onhe lowest feasible doses and meant that much larger numbers ofells had to be scored to obtain statistically robust measurements.efore completing this study, we showed that the very low back-round of 0.05 foci/cell in one published strain [117] was 4-foldigher in our experiments. After four years of effort, we published

study of 25 normal cell strains and 10 mutant strains exposed tooses of 5, 10, and 25 mGy [118]. We did, in fact, document a sub-tantial degree of variability among the normal strains. Since theata collection in this project was very tedious and the work wasot a satisfying way of ending my research career, I undertook anal literature review, which I felt would be rewarding.

. The complexity of DSB repair in mammalian cells

One of the frustrations of every researcher is the overwhelm-ng volume of literature that one needs to read in order to feel

epair 11 (2012) 637– 648 645

abreast relevant developments. When I was compelled by LLNLmanagement to close my office in 2011, piles of old unread papershad to be discarded. Writing a literature review on DSB repairwas the last “opportunity” I would have to read numerous papersthat I had neglected. I began working on the review in early 2009with little idea at first how vast the literature had become. It wasfinally submitted in July of 2011 [102]. During this period I spentmost of my time reading, evaluating, and writing with a focuson IR-induced DSBs. I tried to keep it “simple” by sidesteppingreplication-associated DSBs. I encountered scores, if not hundreds,of new proteins and levels of complexity that I could never haveanticipated. Since I am a highly visual person, I spent a great dealof time preparing figures to help organize the information in acoherent temporal framework. I often reflected on my monthsspent with the time-lapse photos in graduate school when I hadbecome familiar with cells, pondered how they function, andcontemplated how radiation kills them.

In the 1960s it was impossible to imagine the birth of molec-ular biology and the deep insights it would bring within my ownlifetime. As I was writing 40 years later, I was acquiring a muchgreater appreciation of how typical DSB repair proteins interactwith multiple partners and undergo numerous posttranslationalmodifications that guide their spatiotemporal choreography. Mak-ing sense out this complexity in a way I could convey to readers wasa continuing struggle throughout the writing. I finished with somesense of despair akin to what I had experienced during the time-lapse studies, but the limitations of my understanding now seemedto lie more in my own ability to comprehend the vast quantities ofdata, rather than in the experimental tools being used to addressthe scientific questions. With mixed feelings of bewilderment, awe,and accomplishment, I “leave” the complexity of the field with thehope that younger DNA repair molecular biologists will be smarterand highly motivated [119].

Funding source

There was no involvement by the funding sources in the study.

Conflict of interest statement

The author declares that there are no conflicts of interest.

Acknowledgments

Besides the individuals specifically mentioned, I sincerely thankall of my other mentors, collaborators, and coworkers who con-tributed to our publications. This work was performed under theauspices of the U.S. Department of Energy by Lawrence Liver-more National Laboratory under Contract DE-AC52-07NA27344.This work was supported by the DOE Low Dose Radiation ResearchProgram and by Award Number R01CA112566 from the NationalCancer Institute. The content is solely the responsibility of theauthor and does not necessarily represent the official views of theNational Cancer Institute or the National Institutes of Health.

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[81] L.H. Thompson, K.W. Brookman, J.L. Minkler, J.C. Fuscoe, K.A. Henning, A.V.Carrano, DNA-mediated transfer of a human DNA repair gene that controlssister chromatid exchange, Mol. Cell. Biol. 5 (1985) 881–884.

[82] L.H. Thompson, K.W. Brookman, N.J. Jones, S.A. Allen, A.V. Carrano, Molecularcloning of the human XRCC1 gene, which corrects defective DNA strand-breakrepair and sister chromatid exchange, Mol. Cell. Biol. 10 (1990) 6160–6171.

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[84] K.W. Caldecott, C.K. McKeown, J.D. Tucker, S. Ljungquist, L.H. Thompson, Aninteraction between the mammalian DNA repair protein XRCC1 and DNAligase III, Mol. Cell. Biol. 14 (1994) 68–76.

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Lamerdin, A.V. Carrano, L.H. Thompson, Isolation and characterization ofmouse Xrcc1, a DNA repair gene affecting ligation, Genomics 22 (1994)180–188.

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[92] C.A Weber, E.P. Salazar, S.A. Stewart, L.H. Thompson, Molecular cloningand biological characterization of a human gene, ERCC2, that corrects thenucleotide excision repair defect in CHO UV5 cells, Mol. Cell. Biol. 8 (1988)1137–1146.

[93] A. Westerveld, J.H.J. Hoeijmakers, M. van Duin, J. de Wit, H. Odijk, A. Pastink,R.D. Wood, D. Bootsma, Molecular cloning of a human DNA repair gene, Nature310 (1984) 425–429.

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[96] J.P. de Winter, Q. Waisfisz, M.A. Rooimans, C.G. van Berkel, L. Bosnoyan-Collins, N. Alon, M. Carreau, O. Bender, I. Demuth, D. Schindler, J.C. Pronk,F. Arwert, H. Hoehn, M. Digweed, M. Buchwald, H. Joenje, The Fanconianaemia group G gene FANCG is identical with XRCC9, Nat. Genet. 20 (1998)281–283.

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[98] J.L. Yates, N. Warren, B. Sugden, Stable replication of plasmids derivedfrom Epstein–Barr virus in various mammalian cells, Nature 313 (1985)812–815.

[99] N.J. Jones, S.A. Stewart, L.H. Thompson, Biochemical and genetic analysis ofthe Chinese hamster mutants irs1 and irs2 and their comparison to culturedataxia telangiectasia cells, Mutagenesis 5 (1990) 15–23.

[100] N.J. Jones, Y. Zhao, M.J. Siciliano, L.H. Thompson, Assignment of the XRCC2human DNA repair gene to chromosome 7q36 by complementation analysis,Genomics 26 (1995) 619–622.

[101] R. Cartwright, C.E. Tambini, P.J. Simpson, J. Thacker, The XRCC2 DNA repairgene from human and mouse encodes a novel member of the recA/RAD51family, Nucleic Acids Res. 26 (1998) 3084–3089.

[102] L.H. Thompson, Recognition, signaling, and repair of DNA double-strandbreaks produced by ionizing radiation in mammalian cells: the molecularchoreography, Mutat. Res. Rev. (2011), Submitted.

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[104] G.M. Adair, J.B. Scheerer, A. Brotherman, S. McConville, J.H. Wilson, R.S.Nairn, Targeted recombination at the Chinese hamster APRT locus usinginsertion versus replacement vectors, Somat. Cell Mol. Genet. 24 (1998)91–105.

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[106] P.A. Ioannou, C.T. Amemiya, J. Garnes, P.M. Kroisel, H. Shizuya, C. Chen,M.A. Batzer, P.J. de Jong, A new bacteriophage P1-derived vector forthe propagation of large human DNA fragments, Nat. Genet. 6 (1994)84–89.

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[109] L.J. Schild, K.W. Brookman, L.H. Thompson, D.M.Iii. Wilson, Ape1 as a factorin cellular resistance to DNA-damaging and anti-cancer agents, Somat. CellMol. Genet. 25 (2002) 253–262.

[110] J.E. Lamerdin, N.A. Yamada, J.W. George, B. Souza, A.T. Christian, N.J. Jones, L.H.Thompson, Characterization of the hamster FancG/Xrcc9 gene and mutationsin CHO UV40 and NM3, Mutagenesis 19 (2004) 237–244.

[111] R.S. Tebbs, J.M. Hinz, N.A. Yamada, J.B. Wilson, E.P. Salazar, C.B. Thomas,I.M. Jones, N.J. Jones, L.H. Thompson, New insights into the Fanconi anemiapathway from an isogenic FancG hamster CHO mutant, DNA Repair 4 (2005)11–22.

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Environ. Mol. Mutagen. 45 (2005) 128–142.

[113] M. Levitus, H. Joenje, J.P. de Winter, The Fanconi anemia pathway of genomicmaintenance, Cell. Oncol. 28 (2006) 3–29.

[114] J.M. Hinz, R.S. Tebbs, P.F. Wilson, P.B. Nham, E.P. Salazar, H. Nagasawa,S.S. Urbin, J.S. Bedford, L.H. Thompson, Repression of mutagenesis by

6 DNA R

of Great Britain in 1945.E-mail address: thompson14ster@Gmail.com

48 Autobiographical sketch /

Rad51D-mediated homologous recombination, Nucleic Acids Res. 34 (2006)1358–1368.

[115] P.F. Wilson, J.M. Hinz, S.S. Urbin, P.B. Nham, L.H. Thompson, Influence ofhomologous recombination repair on cell survival and chromosomal aberra-tion induction throughout the cell cycle in �-irradiated CHO cells, DNA Repair9 (2010) 737–744.

[116] K. Rothkamm, I. Kruger, L.H. Thompson, M. Löbrich, Pathways of DNA double-strand break repair during the mammalian cell cycle, Mol. Cell. Biol. 23 (2003)5706–5715.

[117] K. Rothkamm, M. Löbrich, Evidence for a lack of DNA double-strand breakrepair in human cells exposed to very low X-ray doses, Proc. Natl. Acad. Sci.U.S.A. 100 (2003) 5057–5062.

[118] P.F. Wilson, P.B. Nham, S.S. Urbin, J.M. Hinz, I.M. Jones, L.H. Thompson, Inter-individual variation in DNA double-strand break repair in human fibroblastsbefore and after exposure to low doses of ionizing radiation, Mutat. Res. 683(2010) 91–97.

[119] B. Alberts, A grand challenge in biology, Science 333 (2011) 1200.[120] D. Pinkel, L.H. Thompson, J.W. Gray, M. Vanderlaan, Measurement of sis-

ter chromatid exchanges at very low bromodeoxyuridine substitution levelsusing a monoclonal antibody in Chinese hamster ovary cells, Cancer Res. 45(1985) 5795–5798.

epair 11 (2012) 637– 648

Larry H. Thompson ∗

Biology & Biotechnology Division, L452, LawrenceLivermore National Laboratory, P.O. Box 808,

Livermore, CA 94551-0808, United States

∗ Tel.: +1 925 455 9473.This article is dedicated to the memory of my

Father, Henry L. Thompon, a WW II pilot who wasmissing in action at the age of 26 in the North Sea