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entire generation of outstanding American TIMELINE biochemists. Following what Kornberg describes as perhaps the most memorable The eureka : the discovery of 18 months of his career, he returned to the NIH where Sebrell appointed him to a staff position and promoted the establishment of DNA an Enzyme Section in the Nutrition Division at the NIH under the direction of Kornberg, Errol C. Friedberg Horecker and Leon Heppel1,3.

Abstract | The identification and partial purification by and his Synthesizing nucleic acids in vitro colleagues in 1956 of an enzyme — DNA polymerase I of — that Watson and Crick’s model of the structure catalysed the stable incorporation of deoxyribonucleotides into DNA in vitro came of DNA and the celebrated concluding as a surprise. At the time, most scientists in the field believed that DNA synthesis sentence in their 1953 Nature paper, “It was too complicated to be accurately reflected outside the living cell. has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for The first DNA polymerase was discovered classmates systematically and measured their the genetic material,”4 did not immediately 50 years ago this year, just 3 years after the (and his own) bilirubin levels, performing take the scientific world by storm. Many elucidation of the DNA structure by Watson analyses “…on a borrowed bench, late at in the scientific community remained and Crick. This finding was remarkable night and at weekends”1. In due course, he unconvinced that DNA was the hereditary for many reasons, not least because many published his first contribution to the bio- material in cells, and at least as many viewed believed that it would not be possible to medical literature, documenting latent liver Watson and Crick’s model as nothing more duplicate the exquisite genetic specificity that disease in medical students2. than that — a model. But Kornberg was not is required for DNA replication outside the This initial foray into investigative medi- among the sceptics. He was aware of the intact cell. Here, I summarize events that led cine was to have far-reaching consequences experiments by the famous phage group led to the ‘eureka’ discovery of the enzyme DNA for Kornberg’s career. World War II was raging by Max Delbrück and Salvador Luria, and of polymerase (TIMELINE), and how this finding and jaundice among American troops who the challenging questions prompted by the opened the door for the discovery of many were inoculated against yellow fever was a observation that within a short time after more such . With this historical bias matter of concern to the US Armed Forces. infection of a bacterium with a single bacte- in mind, this article focuses primarily on the Shortly after he enlisted in the navy in August riophage, hundreds of new phages, replete initial detection, by Arthur Kornberg (FIG. 1) 1942, Kornberg’s small study came to the with new , were released. With his and his colleagues, of what became known attention of the Director of the National new-found appreciation of the importance of as DNA polymerase I of Escherichia coli, and Institute of Health (NIH) and he was sum- enzymes in biosynthetic processes, Kornberg on some of the seminal events in Kornberg’s marily transferred from sea duty to a research “…hoped eventually to find an enzyme that scientific career that led to this discovery. commission in Henry Sebrell’s Nutrition assembles the building blocks of DNA and Laboratory at the NIH. Now fully engaged as RNA into nucleic acid chains”1. In 1950, he Arthur Kornberg — the early years a bench scientist, Kornberg’s intellectual hori- began investigating the biosynthesis of the Few fundamental scientific discoveries zons broadened to the point that he switched precursors for DNA synthesis, his revolved so entirely around the efforts of to Bernard Horecker’s biochemistry labora- formal entrée into nucleic-acid biochemistry. a single investigative team than that of the tory to explore how ATP is generated during In 1954, when he was just 35 years old, discovery of DNA polymerase by Arthur the oxidation of pyruvic acid. It was here that Kornberg was appointed chairman of the Kornberg and his colleagues. When he he began to hone his skills as an enzymologist Department of Microbiology at Washington entered the University of Rochester Medical — and to seek a career as a biochemist. University in St Louis, Missouri, USA. It School in 1937, Kornberg had no explicit Kornberg soon became aware of the was here that he began a serious search for intention of becoming a biochemist. But, work of the young Spanish-born biochemist enzymes that catalyse the assembly of poly- similar to many successful scientists, Severo Ochoa at New York University, who nucleotide chains. Enzymatic biosynthesis Kornberg was endowed with an essential was “…doing the very kind of thing I hoped had been demonstrated for other polymeric characteristic that is fundamental to the pur- to do…”1 He organized a one-year period of molecules, such as starch and lipids. Despite suit of knowledge — a burning intellectual postdoctoral training with Ochoa, followed the prevailing view that the biosynthesis curiosity. When, during his introduction by an additional six months under the tute- of DNA was beyond the pale of in vitro to pathology, he noted that his eyes were lage of Carl and Gerty Cori at Washington biochemistry 5, Kornberg held unrelenting slightly jaundiced, Kornberg examined his University, the ‘father’ and ‘mother’ of an faith in the fidelity with which biochemical

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Timeline | DNA Thomas Kornberg and Malcolm Gefter19, Rolf Myron Goodman and his 31 Knippers20, and Robb Moses colleagues and Zvi Livneh 32 and Charles Richardson21 et al. independently independently discover a discover that UmuC protein of E. coli is a DNA Arthur Kornberg, second DNA polymerase (DNA polymerase II) in polymerase, designated together with DNA polymerase V. I. R. Lehman, extracts of the E. coli polA1 M. J. Bessman and G. F. Kalf and J. J. Ch’ih, . E. S. Simms, publish a and R. R. Meyer and Byrnes et al. report the Ulrich Hübscher and Robert Fuchs and his paper announcing M. V. Simpson, report David Baltimore27, and discovery of a third colleagues discover a colleagues discover the enzymatic the isolation of a DNA Howard Temin and Satashi DNA polymerase in fourth nuclear DNA another DNA synthesis of DNA in polymerase from Mizutoni28 independently mammalian cells polymerase in polymerase in E. coli extracts of mitochondria of discover RNA-directed DNA designated DNA mammalian cells called that is named DNA Escherichia coli 11. mammalian cells25,26. polymerase. polymerase-δ35. DNA polymerase-ε37. polymerase IV33.

1956 1957 1968 1969 1970 1971 1976 1977 1989 1996 1999

Fred Bollum and Van Paula De Lucia and Multiple laboratories discover a Arthur Weissbach and Chris Lawrence and his (1999–2004). Multiple Potter discover a DNA- John Cairns announce second DNA-polymerase activity his colleagues provide colleagues demonstrate groups discover polymerase activity in the isolation of a in mammalian cell extracts. This evidence that a DNA translesion DNA synthesis multiple specialized extracts of calf thymus, viable mutant strain enzyme is designated DNA polymerase from by a yeast DNA polymerase DNA polymerases in designated DNA of E. coli (polA1) that polymerase-β. mitochondria is a called DNA polymerase-ζ38. eukaryotic cells, many polymerase-α23. is defective for DNA fifth DNA polymerase These investigators also of which (including polymerase I (REF. 18). called DNA demonstrate a dCMP DNA polymerase-ζ and polymerase-γ36 activity in the Thomas Kornberg and . REV1 protein) belong yeast REV1 protein39 Malcolm Gefter discover . to the novel Y-family of 34 a third DNA-polymerase proteins . activity in E. coli 22. reactions in the living cells can be mirrored mere 50 scintillation counts above the back- observation that the radioactivity recovered in vitro. “It always seemed to me”, he wrote, ground) was transferred to acid-precipitable in the acid-insoluble fraction increased “that a biochemist devoted to enzymes could, material, presumably DNA. correspondingly. More reassuring was the if persistent, reconstitute any metabolic event finding that the radioactivity was lost as well as the cell does it. In fact, better!”1 The discovery of DNA polymerase following incubation of the acid-precipitable Together with postdoctoral fellow Uri While labouring to improve the robustness material with pancreatic DNase. Kornberg Littauer, Kornberg observed that following of this assay, Kornberg and Littauer learned later described how he felt. the addition of radiolabelled ATP to extracts that Marian Grunberg-Manago (then a post- of E. coli, significant amounts of radioactiv- doctoral fellow in Ochoa’s laboratory) had “Without the encouragement that the ity were incorporated into acid-precipitable stumbled onto the enzymatic synthesis of diagnostic action of DNase gave me, I material, presumably RNA. Excitement RNA in extracts of a nitrogen-fixing bacte- wonder whether I would have had the will reigned and efforts were quickly extended to rium. This reaction, catalysed by an enzyme to pursue such a feeble light.”1 include the biosynthesis of DNA. The latter that Ochoa dubbed “polynucleotide phos- studies were conspicuously promoted by phorylase”6, used ADP rather than ATP as a On learning about Kornberg’s return the availability of radiolabelled thymidine substrate. Spurred to redouble their efforts to in vitro DNA synthesis, I. Robert (Bob) from Morris Friedkin in the Department of on RNA synthesis in extracts of E. coli, Lehman, who had recently joined the Pharmacology at Washington University. Kornberg and Littauer switched to using Kornberg laboratory as a postdoctoral Friedkin had discovered the reverse reac- radiolabelled ADP instead of ATP. In so fellow, discovered that thymidine mono- tion of thymidine , which doing, they merely confirmed the existence phosphate was a better precursor for DNA catalyses the phosphorolysis of thymidine of polynucleotide phosphorylase in another synthesis than thymidine. Later, Lehman and other pyrimidine 2′ deoxyribosides, microorganism. Had they continued using showed that thymidine triphosphate therefore enabling the conversion of thymine ATP as a substrate, they almost certainly worked even better8. From the outset, to thymidine and inorganic phosphate in would have discovered RNA polymerase the in vitro assay included the addition the presence of deoxyribose-1-phosphate. — an enzyme not formally discovered until of E. coli DNA. Kornberg deployed this Friedkin was trying to demonstrate the 1959 (REF. 7). experimental nuance for several cogent incorporation of 14C-thymidine into DNA in This diversion postponed further attempts reasons. Primarily, he hoped that the DNA extracts of chick embryos, and was not at all at the enzymatic synthesis of DNA. But would function as a primer for extended motivated to donate his precious reagent for when these experiments were resurrected in DNA synthesis, much in the fashion that experiments with E. coli extracts. However, late 1955, the results were as inconclusive as carbohydrate chains had been shown by he offered Kornberg residual supernatants those of the original trials. Nonetheless, the Carl and Gerty Cori to be extended by from his studies. Initial results were disap- radiolabelled thymidine used in the second glycogen phosphorylase. Additionally, pointing. Only about 0.01% of the total round of experiments was about three times cognizant of the potent nucleases in radioactivity in the incubations (yielding a ‘hotter’, and Kornberg took faint heart at the E. coli extracts, Kornberg reasoned that

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adding exogenous DNA might alleviate the Two definitive papers on the enzyme degradation of newly synthesized DNA by DNA polymerase were published in the sequestering the nuclease activity. Assisted by Journal of Biological Chemistry in July 1958 Lehman, Maurice J. Tessman and Ernest S. (REFS 12,13). Sceptical that the enzymatic Simms, Kornberg began to enrich the protein reaction might not reflect bona fide DNA fraction that catalysed the synthesis of new synthesis, the reviewers suggested that the DNA. “Through this tiny crack we tried to authors use the term ‘polydeoxyribonu- drive a wedge, and the hammer was enzyme cleotide’ rather than ‘DNA’ to identify the purification.”5 product of the reaction. But reason prevailed The initial results of these studies were when the authors appealed to the Editor-in- announced at the 1956 annual meeting of Chief, John Edsall. the Federation of American Societies for Experimental Biology (FASEB) in Atlantic Cells have multiple DNA polymerases City, New Jersey, USA9, and at the 1956 Kornberg and his colleagues reasonably McCollum-Pratt Symposium entitled The assumed that the DNA polymerase they Chemical Basis of Heredity, in Baltimore, discovered supported semi-conservative Maryland, USA10. In the same year, Kornberg, replication of the E. coli , and that Lehmann, Bessman and Simms published a it was the only DNA polymerase in these 11 brief report (the 50th anniversary of which cells. But John Cairns (then Director of the Figure 1 | Arthur Kornberg, circa 1956 — the we celebrate this year), which clearly revealed Cold Spring Harbor Laboratory) was not year that he announced the discovery of Kornberg’s reservations that the DNA that convinced that the Kornberg enzyme could DNA polymerase. Photograph courtesy of was being synthesized in this in vitro reaction catalyse the copying of DNA rapidly enough A. Kornberg. was probably not derived from the template- to account for the in vivo rate of DNA directed process that determines the fidelity replication in E. coli, or that E. coli possessed of DNA replication in living cells. He and his enough enzyme to accommodate the rate of co-authors referred to the addition of E. coli DNA replication. from the efforts of Thomas Kornberg, one DNA as “…‘primer’ for the crude enzyme Cairns was also aware of studies on an of Arthur Kornberg’s three sons (FIG. 2). In fraction”, and did not entertain the notion of E. coli mutant that undergoes aberrant cell 1970, the younger Kornberg, then a graduate a template-directed process until later, when division near one , resulting in the student with Malcolm Gefter at Columbia key observations by Lehman prompted them generation of small enucleate cells known University, reported the identification of to do so. In fact, the authors were initially as minicells14. Minicells were known to what became known as DNA polymerase II aware of the possibility that the DNA that was contain RNA and protein, but were essen- of E. coli19. Similar observations were being synthesized in their reaction might not tially devoid of DNA, despite the fact that independently reported at about the same reflect the genetic fidelity of DNA replication the specific activity of DNA polymerase in time by Rolf Knippers20 and by Robb Moses in living cells. minicell extracts was essentially the same as and his mentor Charles Richardson21. The in normal cells15,16. “This said to me”, Cairns following year, Thomas Kornberg and Gefter “Further investigations with phage-infected later related, “that the 400 molecules of DNA reported the identification of what became E. coli and studies with biologically active polymerase were not busily replicating DNA. DNA polymerase III of E. coli22, the true rep- DNA may begin to clarify the question They were just floating around. Instantly the licative polymerase. The enzyme discovered of how genetically specific DNA is thought came to me that they were not there by Arthur Kornberg is now known as DNA assembled.”11 to replicate DNA …”17 polymerase I. Cairns decided to attempt the isolation In his own recent reflections on the of a mutant of E. coli defective in DNA Yet more DNA polymerases DNA-polymerase story, Lehman com- polymerase activity. If such a mutant The discovery of DNA polymerase in mented on the imperatives for considering a was viable, the Kornberg enzyme was eukaryotes did not lag far behind Kornberg’s template-directed process. clearly not the polymerase that replicated pioneering efforts. By 1957, radiolabelled thy- DNA semi-conservatively. Cairns set his midine was commercially available and Fred “The requirement for all four dNTPs was laboratory assistant Paula De Lucia the Bollum announced the identification of what puzzling. If the DNA that we added was daunting task of propagating thousands of eventually came to be called DNA polymer- simply serving as a primer, why would all individual E. coli colonies, making cell-free ase-α in rat liver homogenates23. It took years four dNTPs be needed? Was it possible that extracts of each, and assaying them for of effort by multiple investigators to sort out the DNA polymerase was performing the DNA-polymerase activity. De Lucia struck the plethora of DNA polymerases in mam- template-directed replication proposed gold with clone number 3478 and identi- malian cell extracts, to define the multiprotein by Watson and Crick for their double- fied the polA1 mutant18 (so-named because composition of many of these enzymes and stranded structure of DNA… To test this of the agreeable alliteration of ‘polA’ with to distinguish each as independent DNA idea we used with AT/GC ratios ‘Paula’ [De Lucia] whom Cairns wanted to polymerases24. We now recognize the exist- ranging from 0.5 to 1.9 as ‘primers.’ The credit.17) ence of four such enzymes, designated DNA result was stunning … Clearly, the added These studies naturally begged the ques- polymerase-α, -β, -δ and -ε (TABLE 1). In the DNA was serving as a template to direct tion, which DNA polymerase replicates the late 1960s, two groups reported the identifica- the polymerase as it synthesized new DNA E. coli ? Answers to this came tion of DNA-polymerase activity in extracts chains…”8 quickly. And, remarkably, they came first of rat liver mitochondria25,26, and additional

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replicative machinery, a process known as translesion DNA synthesis (TLS)29. It was initially speculated that TLS in E. coli was effected by the replicative enzyme DNA polymerase III, the fidelity of which was relaxed by interaction with the products of the umuC (and umuD) that are obligatory for UV-radiation-induced muta- genesis30. But several studies subsequently demonstrated that the UmuC protein is itself a DNA polymerase with novel properties31,32. At about the same time, it was shown that DinB, an E. coli protein that shares exten- sive structural homology with the UmuC protein, is also a DNA polymerase33. DinB is now designated DNA polymerase IV Figure 2 | Arthur Kornberg with his sons Roger (left), Kenneth (next to his mother Sylvia) and (TABLE 1) and UmuC acquired the formal Thomas (next to his father), circa 1959. Photograph courtesy of A. Kornberg. name of DNA polymerase V (TABLE 1). By the late 1990s, the availability of the nucleotide sequences of several eukaryotic studies in the 1970s led to the remarkable reaction (PCR), an innovation that prompted organisms facilitated the identification of observation that mitochondria contain a the discovery of thermostable archaeal DNA an astonishing array of DinB and UmuC dedicated DNA polymerase (DNA polymer- polymerases. homologues in eukaryotes34, leading to ase-γ; TABLE 1) encoded by the mitochondrial Still more DNA polymerases laid in the discovery of a plethora of new DNA genome. In 1970, David Baltimore27 and wait, and, once again, bacteria paved polymerases (TABLE 1). It is now established Howard Temin28 independently discovered the way. Genetic studies on that vertebrate cells contain no less than 14 a viral RNA-dependent DNA polymerase (UV)-radiation-induced mutagenesis in distinct DNA polymerases that belong to the (now known as ), efforts E. coli prompted the hypothesis that muta- A, B, X and Y families. that won them the Nobel Prize in 1975. Such tions in E. coli cells that were exposed to scientific recognition was also accorded to UV light were generated (at least in part) by Concluding remarks Kary Mullis in 1983 for his application of inaccurate DNA replication directly across Following his discovery of DNA polymerase I DNA polymerases to the polymerase chain photoproducts in the template strand by the of E. coli (for which Kornberg was acknowl- edged with a Nobel Prize in 1959), he devoted most of his career to deciphering the mecha- Table 1 | DNA polymerases in , lower eukaryotes and mammalian cells nism of DNA replication in E. coli, a challeng- E. coli S. cerevisiae H. sapiens Polymerase family ing problem that is far more complex than the simple deployment of DNA polymerases. Pol I – – A Indeed, many questions about DNA replica- Pol II – – B tion, especially in eukaryotes, remain unan- Pol III – – C swered to this day and conferences on DNA Pol V – – Y replication continue to abound. As for Arthur Kornberg, at the age of 87 he has returned full Pol IV – κ Y Pol cycle to an early love affair with polyphosphate –– Pol β X biochemistry and continues to manage an – Pol α Pol α B active laboratory at Stanford University with undiminished curiosity — and with a timeless – Pol δ Pol δ B ‘love of enzymes’. – Pol ε Pol ε B Errol C. Friedberg is at the Laboratory of Molecular – γ γ A Pol Pol Pathology, Department of Pathology, – Pol IV Pol λ X University of Texas Southwestern Medical Center, Dallas, Texas 79503-9072, USA. – Rev1 Rev1 Y e-mail: [email protected]

– Pol η Pol η Y doi:10.1038/nrm1787 – Pol ζ Pol ζ A Published online 21 December 2005 – – Pol I Y 1. Kornberg, A. For the Love of Enzymes: The Odyssey of a Biochemist. (Harvard University Press, Cambridge, –– Pol µ X Massachusetts, 1989). 2. Kornberg, A. Latent liver disease in persons recovered –– Pol θ A from catarrhal jaundice and in otherwise normal medical students, as revealed by the bilirubin –– Pol ν A excretion test. J. Clin. Invest. 21, 299–308 (1942). 3. Kornberg, A. Never a dull enzyme. Annu. Rev. E. coli, Escherichia coli; H. sapiens, Homo sapiens; S. cerevisiae, Saccharomyces cerevisiae; Pol, DNA polymerase. Biochem. 58, 1–30 (1989).

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4. Watson, J. D. & Crick, F. C. A structure for 30. Rajagopalan, M. et al. Activity of the purified mutagenesis, is predicted to encode a nonessential deoxyribose nucleic acid. Nature 171, 737–738 mutagenesis proteins UmuC, UmuD’, and RecA in DNA polymerase. J. Bacteriol. 171, 5659–5667 (1989). (1953). replicative bypass of an abasic DNA lesion by DNA 39. Nelson, J. R., Lawrence, C. W. & Hinkle, D. C. 5. Kornberg, A. Biological synthesis of polymerase III. Proc. Natl Acad. Sci. USA 89, Deoxynucleotidyl transferase activity of yeast REV1 deoxyribonucleic acid. Science 131, 1503–1508 10777–10781 (1992). protein. Nature 382, 729–731 (1996). (1960). 31. Tang, M. et al. UmuD’2C is an error-prone DNA 6. Grunberg-Manago, M. & Ochoa, S. J. Enzymatic polymerase, Escherichia coli pol V. Proc. Natl Acad. Acknowledgements synthesis and breakdown of polynucleotides; Sci. USA 96, 8919–8924 (1999). I apologize for the necessarily restricted listing of references concern- polynucleotide phosphorylase. J. Am. Chem. Soc. 77, 32. Reuven, N. B., Arad, G., Maor-Shoshani, A. & Livneh, Z. ing mammalian DNA polymerases. I thank L. Loeb, M. DePamphilis, 3165–3166 (1955). The mutagenesis protein UmuC is a DNA polymerase U. Hübscher, T. Kunkel, S. Wilson and D. Bogenhagen for helpful dis- 7. Weiss, S. B. & Gladstone, L. A mammalian system for activated by UmuD’, RecA, and SSB and specialized cussions, and L. McDaniel and N. Kosarek for careful reading of the the incorporation of cytidine triphosphate into for translesion synthesis. J. Biol. Chem. 274, manuscript. ribonucleic acid. J. Am. Chem. Soc. 81, 4118–4119 31763–31766 (1999). (1959). 33. Wagner, J. et al. The dinB gene encodes a novel E. coli Competing interests statement 8. Lehman, I. R. Discovery of DNA polymerase. J. Biol. DNA polymerase, DNA Pol IV, involved in mutagenesis. The author declares no competing financial interests. Chem. 278, 34743–34738 (2003). Mol. Cell 4, 281–286 (1999). 9. Kornberg, A., Lehman, I. R. & Simms, E. S. 34. Ohmori, H. et al. The Y-family of DNA polymerases. DATABASES Polydesoxyribonucleotide synthesis by enzymes from Mol. Cell 8, 7–8 (2001). The following terms in this article are linked online to: Escherichia coli. Fed. Proc. 15, 291 (1956). 35. Byrnes, J. J., Downey, K. M., Black, V. L. & So, A. G. Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. 10. Kornberg, A. in The Chemical Basis of Heredity. A new mammalian DNA polymerase with 3′ to 5′ fcgi?db=gene (eds McElroy, W. D. & Glass, B.) 579–608 (Johns activity: DNA polymerase δ. Biochemistry umuC | umuD Hopkins Press, Baltimore, 1957). 15, 2817–2823 (1976). 11. Kornberg, A., Lehman, I. R., Bessman, M. J & 36. Bolden, A., Noy, G. P. & Weissbach, A. DNA FURTHER INFORMATION γ Simms, E. S. Enzymatic synthesis of desoxyribonucleic polymerase of mitochondria is a polymerase. Errol Friedberg’s laboratory: acid. Biochim. Biophys. Acta 21, 197–198 J. Biol. Chem. 252, 3351–3356 (1977). http://webpath.swmed.edu/research/FMPro?-db=labname. δ (1956). 37. Focher, F. et al. Calf thymus DNA polymerase fp5&-format=rlab%5fmain.html&-lay=cgi&-sortfield=labnam 12. Lehman, I. R., Bessman, M. J., Simms, E., S. & independent of proliferating cell nuclear antigen. e%5ft&error=error.html&divisionID%5fc=1&-recid=8&- Kornberg, A. Enzymatic synthesis of deoxyribonucleic Nucleic Acids Res. 17, 1805–1821 (1989). token.0=1&-find= acid I. Preparation of substrates and partial 38. Morrison, A. et al. REV3, a Saccharomyces cerevisiae Access to this interactive links box is free online. purification of an enzyme from Escherichia coli. J. Biol. gene whose function is required for induced Chem. 233, 163–170 (1958). 13. Bessman, M., Lehman, I. R., Simms, E. S. & Kornberg, A. Enzymatic synthesis of deoxyribonucleic acid II. General properties of the reaction. J. Biol. Chem. 233, 171–177 (1958). 14. Adler, H. I., Fisher, W. D. & Stapleton, G. E. National OPINION Academy of Sciences. Abstracts of papers presented at the autumn meeting, Durham, North Carolina, 17–19 October 1966. Science 154, 417 (1966). 15. Adler, H. I., Fisher, W. D., Cohen, A. & Harigree, A. A. Miniature Escherichia coli cells deficient in DNA. Not so divided: the common basis of Proc. Natl Acad. Sci. USA 57, 321–326 (1967). 16. Cohen, A., Fisher, W. D., Curtiss, R. 3rd & Adler, H. I. The properties of DNA transferred to minicells during plant and animal cell division conjugation. Cold Spring Harbor Symp. Quant. Biol. 33, 635–641 (1968). 17. Friedberg, E. C. Correcting the Blueprint of Life: An Clive Lloyd and Jordi Chan Historical Account of the Discovery of DNA Repair Mechanisms. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1997). Abstract | Plant cells do not have centrioles and their mitosis is frequently likened 18. De Lucia, P. & Cairns, J. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature to the chromosome-based mechanism seen in acentriolar animal cells. However, 224, 1164–1166 (1969). this is a false analogy. Although plants can use this mechanism, they generally 19. Kornberg, T. & Gefter, M. L. DNA synthesis in cell-free extracts of a DNA polymerase-defective mutant. divide by a method that uses bipolar mitotic caps, which is more similar to the Biochem. Biophys. Res. Commun. 40, 1348–1355 (1970). canonical centrosome-based method of animals. 20. Knippers, R. DNA polymerase II. Nature 228, 1050–1053 (1970). 21. Moses, R. E. & Richardson, C. C. A new DNA polymerase activity of Escherichia coli. I. Purification As plants evolved from an aquatic to a land pair focuses the MTOC during interphase and properties of the activity present in E. coli PolA1. Biochem. Biophys. Res. Commun. 40, 1557–1564 environment, their gametes became trans- and, when duplicated, also defines the spin- (1970). mitted through the air by insects or wind dle poles of animal cells during mitosis. It is 22. Kornberg, T. & Gefter, M. L. Purification and DNA synthesis in cell-free extracts: properties of DNA and they lost the ability to make the flagella the loss of the centriole or basal body that is polymerase II. Proc. Natl Acad. Sci. USA 68, 761–764 that are used by animals and lower plants thought to be responsible for the dispersion (1971). 23. Bollum, F. J. & Potter, V. R. Thymidine incorporation to propel their gametes through water. As a of the MTOC and the more diffuse spindle into deoxyribonucleic acid of rat liver homogenates. consequence, higher land plants no longer poles in higher plant cells. J. Am. Chem. Soc. 79, 3603–3604 (1957). 24. Hübscher, U., Maga, G. & Spadari, S. Eukaryotic DNA possess the basal body from which flagellar However, here we argue that the loss of polymerases. Annu. Rev. Biochem. 71, 133–163 (or ciliary) microtubules grow1. However, the centriole as an organizing entity has (2002). 25. Kalf, G. F. & Ch’ih, J. J. Purification and properties of virtually all vertebrate cells contain basal been consistently overemphasized, and has deoxyribonucleic acid polymerase from rat liver bodies, which are composed of a ring of nine diverted attention away from the true man- mitochondria. J. Biol. Chem. 243, 4904–4916 (1968). specialized microtubules. Even those animal ner in which plant cells divide. In terms of 26. Meyer, R. R. & Simpson, M. V. DNA biosynthesis cells that do not form cilia or flagella retain mitosis, plants are usually lumped together in mitochondria: partial purification of a distinct DNA polymerase from isolated rat liver mitochondria. basal bodies in the form of structurally iden- with those animal cells that also lack cen- Proc. Natl Acad. Sci. USA 61, 130–137 (1968). tical centrioles that are found in the middle trioles (for example, germ-line cells), and 27. Baltimore, D. RNA-dependent DNA polymerase in virions of RNA tumor viruses. Nature 226, of the centrosome or microtubule-organizing it is commonly assumed that all such cells 1209–1211 (1970). centre (MTOC). Although they are vestigial, must divide by a non-canonical method that 28. Temin, H. M & Mizutani, S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature in the sense that they no longer function as lacks strong input from the spindle poles. 226, 1211–1213 (1970). flagellar templates, the centrioles do provide We stress that plants do possess a pole-based 29. Echols, H. & Goodman, M. F. Fidelity mechanisms in DNA replication. Annu. Rev. Biochem. 60, 477–551 a focal point for gathering the amorphous mitotic mechanism, which has been over- (1991). microtubule-nucleating material. A centriole looked in the discussions about centrioles.

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