My scientific life

KEYWORDS:

• Bacteriophage ø29
• DNA replication
• DNA polymerase
• terminal protein

I was born in Canero, Spain, a very small village in the north coast of Asturias. When I was one year old, my parents moved to Gijón, also in Asturias, where I spent my childhood and early youth. There, I attended the school and obtained the baccalaureate title in 1954. Since I wanted to pursue a university career, I had to spend one year doing the so-called pre-university studies and to decide whether I wanted to follow a scientific or a humanistic career. I choose to go into science. At the end of the year I had to decide which specific career I wanted to follow. I doubted between medicine and chemistry. Since medicine was not available at Oviedo University, close to Gijón, I decided to go to Madrid University to follow a course common for both careers. Finally, I decided to study chemistry. In the third year we studied organic chemistry, and I enjoyed very much the long hours we spent in the laboratory. I thought that, in the future, I would like to do research in organic chemistry. But that was not the case.

In the summer of 1958, when I had finished my third year of chemistry, I went to Gijón to spend the holidays, and I was very lucky to meet Severo Ochoa, which had a decisive influence on my future. I attended a conference he gave about his work and I was fascinated by his talk. Since my father was a good friend of Ochoa, besides being cousins in law, I had the chance to talk to him about my future. I had not yet studied biochemistry and he promised to send me a biochemistry book. I was very excited when I received the book General Biochemistry by Fruton and Simmonds, dedicated by Severo Ochoa. When I finished my chemistry studies I had decided to dedicate myself to biochemistry. Ochoa advised me to do the Ph.D. Thesis in Madrid with Alberto Sols, an excellent biochemist, who had been trained with Carl and Gerty Cori at the Washington University School of Medicine in St. Louis. Then, I could go to Ochoa's laboratory at New York University (NYU) School of Medicine for a postdoctoral training. Ochoa wrote me a reference letter for Alberto Sols who accepted me, even if I was a woman, since he could not refuse a request made by Severo Ochoa who had already obtained the Nobel Prize. Thus, in January 1961, I started my Ph.D. Thesis working on carbohydrate metabolism, mainly on glucose-phosphate isomerase from yeast. I found that the enzyme has an anomerase-like activity producing the open form of glucose-6-phosphate. This was the first finding in my scientific career, something that was very exciting for me. The work was published in the Journal of Biological Chemistry.²⁶ At the end of my studies in chemistry, I became the fiancé of Eladio Viñuela, a very brilliant student, who also performed his Ph.D. Thesis in Sols’ laboratory. Eladio discovered a new enzyme, the liver glucokinase, that converts glucose into glucose- 6-phosphate, and disappears in fasted and in diabetic rats. I joined Eladio in this work, and we found that the enzyme reappears by refeeding and insulin administration, respectively. This work was also published in the Journal of Biological Chemistry.^30,25 Publishing at that time from Spain in this journal was quite an accomplishment for us.

Postdoctoral training

Thanks to a fellowship that I obtained from the Juan March Foundation, Eladio and I married in 1963. In August of 1964, after finishing our Ph.D. Theses, we went to Severo Ochoa's laboratory at the NYU School of Medicine in time to attend the International Congress of Biochemistry, where Philip Leder and Marshall Nirenberg presented their latest results on the use of trinucleotides of specific sequence for the binding of the different aminoacyl-tRNAs. This resulted in the final unravelling of the genetic code that completed the work performed in Ochoa's, Nirenberg's and Khorana's laboratories.

My initial research project in Ochoa's laboratory was to determine whether the direction of reading of the genetic message was in the 5’ to 3’ or in the 3’ to 5’ direction. We used a cell-free protein synthesis system that consisted in a high- speed supernatant of Lactobacillus arabinosus, with low nuclease activity, and ribosomes from Escherichia coli that had been washed with 0.5 M NH₄Cl. As mRNA we used synthetic polynucleotides that contained the AAC triplet at the 3’ or 5’ end. When we used the polynucleotide 5’(A)24AAC 3’, the amino acids lysine and asparagine were incorporated, the latter being at the carboxyl end.²⁴ When the AAC triplet was located at the 5’end, the asparagine was incorporated at the amino end.²⁸ Taking into account that the direction of protein synthesis takes place from the amino to the carboxyl end, the results obtained indicated that the direction of reading of the genetic message is from the 5’ to the 3’ end.

Afterwards, I worked on the translation of the E. coli phage MS2-RNA using a high-speed supernatant from E. coli and the ribosomes washed with 0.5 M NH₄Cl. This system was active when I used polyA as messenger but, to my surprise, it was inactive with MS2-RNA. When I precipitated with ammonium sulfate the 0.5 M NH4Cl ribosomal wash and added the fraction to the system, I recovered the activity with MS2-RNA. Then, I used the polynucleotide 5’AUG(A)24 3’ prepared by Wendell Stanley, Jr. and found that, as with MS2-RNA, this messenger was not active with the washed ribosomes but the activity was recovered when I added the ammonium sulfate fraction.²⁹ Since the triplet AUG at the 5’ end of a messenger codes for formyl-methionine, this suggested that the fraction I was adding was involved in the initiation of protein synthesis. I purified two proteins from the ribosomal wash, which I called F1 and F2 (later on called iF1 and iF2) and found that the two proteins were needed for the binding of formyl- methionyl-tRNA to the ribosomes in the presence of the AUG triplet.This result demonstrated that proteins F1 and F2 were required for the initiation of protein synthesis.²¹ The work on initiation factors for protein synthesis became the future focus in Ochoa's laboratory. Bacteriophage ø29 work.

After three exciting and fruitful years in Severo Ochoa's laboratory, Eladio and I decided to go back to Spain to develop in our country the molecular biology that we had learnt. An important decision was the project we wanted to carry out in Spain, taking into account that to do research in our country was going to be difficult. In the summer of 1966 we had taken a Phage Course at Cold Spring Harbor Laboratory and we thought that a phage could be a good model system to carry out our research. We choose the Bacillus subtilis phage ø29, shown in Dwight Anderson's laboratory to have a small size and a relatively complex structure.¹ It seemed to be a good model system to study the mechanisms of transfer of the genetic information and the morphogenesis of the phage particle. Thanks to a Grant we obtained from the Jane Coffin Childs Memorial Fund for Medical Research we could start our work in Madrid since at that time there was no funding in Spain to do research. We started our work in Madrid in September 1967 at the Center of Biological Research of the Spanish National Research Council (CSIC) of which we had been appointed research scientists. Very little was known about the phage except the size of its DNA (a molecular mass of ∼12 million Da) and its morphology from an electron micrograph obtained in Anderson's laboratory.¹ Thus, we started with the very basic knowledge of the phage: to do the genetics with the isolation of conditional lethal mutants (temperature-sensitive and suppressor-sensitive), and to characterize the structural proteins of the phage particle as steps previous to the study of the morphogenesis, to isolate the viral DNA, and to study its transcription and replication.

Fortunately, soon after our arrival to Spain, the first predoctoral fellowships were available and we could start the work with several brilliant and enthusiastic students that made important progress in the knowledge of the molecular biology of the phage. The Chairman of the Department of Biochemistry of the Faculty of Chemistry of Madrid Complutense University asked Eladio and me to teach a course on Molecular Genetics that was taught for the first time at a Spanish university. This allowed us to select the very best students to carry out the Ph.D. work in our laboratory.

One of the projects I would like to mention was the study of the control of the transcription of ø29 DNA. With that aim, we isolated the B. subtilis RNA polymerase showing that it had the subunits corresponding to those of the E. coli enzyme, β, β', σ, and α, results that we published in Nature.² I was very excited when I received a letter from James Watson inviting me to attend the Cold Spring Harbor Symposium on Transcription. There, I learned that Richard Losick had obtained results similar to ours. Later on, we showed that the early genes are transcribed by the host σA RNA polymerase that recognizes the early promoters named A1, A2b, A2c, and C2 that contain the -10 and -35 hexamers. The late genes are transcribed from the A3 promoter that lacks the -35 hexamer and requires, in addition to the σA RNA polymerase, the product of the viral gene 4. This protein, p4, was shown to be a transcriptional activator of the late A3 promoter stabilizing the σA RNA polymerase as a close complex. In addition, protein p4 represses promoter A2b displacing the RNA polymerase from it, as well as promoter A2c through a mechanism that implies the simultaneous binding of p4 and RNA polymerase to the promoter, preventing the escape of the RNA polymerase from it.¹⁹ Later on, we found that the phage double-stranded DNA binding protein p6, that is involved in the initiation of ø29 DNA replication (see below), promotes p4-mediated repression of promoter A2b and activation of promoter A3 by enhancing binding of p4 to its recognition site at promoter A3. On the other hand, p4 promotes p6-mediated repression of promoter A2c by favoring the formation of a p6-nucleoprotein complex that interferes with the binding of the RNA polymerase.⁹

An unexpected and very important finding was the fact that ø29 has a protein covalently linked to the 5’ends of the DNA, the so-called terminal protein (TP). This finding led us to the study of the replication of ø29 DNA as one of the main topics of the laboratory. When we isolated the DNA from the phage particles, to our surprise, the DNA was not obtained in a linear form, as it was expected, but as circular DNA and concatemers that were converted into unit-length linear DNA by treatment with a proteolytic enzyme. This indicated that protein was involved in the formation of circular and concatemeric DNAs.¹⁵ Interestingly, two years after our publication, a similar result was published in the case of adenovirus DNA.¹⁷ Later on, we characterized a protein of 31,000 Da, the product of the viral gene 3, covalently linked to the 5’ends of ø29 DNA,²² and we later showed that it was involved in the initiation of ø29 DNA replication.

We found that the TP is the primer for the initiation of ø29 TP-DNA replication catalyzed by the viral DNA polymerase, giving rise to the TP-dAMP covalent initiation product that is further elongated by the same DNA polymerase to produce full-length ø29 DNA. The product of the viral gene 6 stimulates the in vitro initiation reaction under conditions in which the DNA ends are closed (high salt and/or low temperature). The ø29 DNA polymerase is unique since it is able to catalize, not only the polymerization step of replication, but also the initiation step in which the hydroxyl group is provided by a specific amino acid (Ser232) in the TP. In addition, it has proofreading 3’ to 5’ exonuclease activity, as well as two intrinsic properties such as very high processivity (>70 kb) and strand displacement capacity, which made it an ideal enzyme for DNA amplification.⁴ Indeed, by using the ø29 proteins TP, DNA polymerase, protein p6 and the single-strand DNA binding (SSB) protein p5, we were able to amplify in vitro small amounts (0.5 ng) of the 19,285 bp-long ø29 TP-DNA by three orders of magnitude (0.5 μg) after 1 h of incubation at 30°C. Transfection experiments showed that the infectivity of the in vitro amplified DNA was the same to that of the natural DNA obtained from virions.⁵

Phage ø29 DNA has an inverted terminal repeat (ITR) of 6 nucleotides (3’TTTCAT5’). To our surprise, we found that the initiation of replication does not take place at the 3’terminal nucleotide, but at the second nucleotide from the 3’end. The DNA ends are recovered by a mechanism that we called sliding-back. The TP-dAMP complex formed, directed by the second T at the 3’ends slides backward, locating the dAMP in front of the first 3’terminal T of the template. Then, the next nucleotide, dAMP, is incorporated into the TP-dAMP initiation complex, directed again by the second T of the template.¹³

Internal initiation also occurs in the ø29-related phages Nf and GA-1, in the Streptococus pneumoniae phage Cp-1, in the E. coli phage PRD1 and in adenoviruses.²³ Structure-function studies on the ø29 DNA polymerase and TP were also performed. Using mutants obtained by site-directed mutagenesis in the DNA polymerase gene we identified amino acids involved in the 3’-5’ exonuclease activity as well as those involved in polymerization and protein-primed initiation. In addition, we identified amino acids involved in metal biding and catalysis, DNA binding, TP binding and dNTP interaction (reviewed in.^3,20). Later on, the crystal structure of the ø29 DNA polymerase was determined in collaboration with Tom Steitz's laboratory.¹⁰ The structure provided a topological basis for its intrinsic processivity and strand- displacement capacity. Ø29 DNA polymerase has two insertions, that we called TPR1 and TPR2, which are not present in other family B DNA polymerases. We made a deletion in the TPR2 subdomain and found that the resulting DNA polymerase had a reduced DNA binding and had lost its processivity and strand- displacement capacity.¹⁸ The crystal structure of the ø29 DNA polymerase/TP heterodimer was also obtained in collaboration with Tom Steitz's laboratory¹¹ showing three domains in the TP: the N-terminal domain that has sequence- independent DNA binding capacity and does not interact with the DNA polymerase; the intermediate domain that interacts with the TPR1 subdomain of the polymerase; and the priming domain, which contains Ser232 and occupies the same binding cleft in the polymerase as duplex DNA does during elongation. The N-terminal domain is responsible for the nucleoid association of the TP. In addition, the TP recruits the ø29 DNA polymerase to the bacterial nucleoid, and both proteins are later redistributed to helix-like structures in an MreB cytoskeleton-dependent way.¹⁴ Interestingly, the TPs of bacteriophages from diverse families and hosts such as those of ø29, Nf, PRD1, Bam35 and Cp-1 were found to localize in the nucleus when expressed in mammalian cells. The nuclear localization signal was localized within residues 1–37 of the ø29 TP. It is important to stress the fact that gene delivery into the eukaryotic nucleus was enhanced by the presence of ø29 TP attached at the 5’ ends.¹⁶

I would also like to mention some phage-host interactions in ø29 development. The early gen e56 encodes p56, a protein of 56 amino acids. Protein p56 inhibits the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair (BER) pathway. Inhibition of cellular UDG by p56 is a defense mechanism developed by ø29 to prevent the action of the BER pathway if uracil residues arise in their single-stranded replication intermediates, which might be unnecessarily degraded.²⁷ On the other hand, as mentioned above, the MreB cytoskeleton plays a crucial role in organizing ø29 DNA replication at the membrane. For ø29 development we used B. subtilis SpoOA⁻ since the phage does not develop efficiently in B. subtilis that is able to sporulate. The SpoOA protein is the master regulator for the initiation of sporulation and, interestingly, we found that it has several binding sites in ø29 DNA. SpoOA suppresses ø29 development by repressing the early promoters A2b, A2c and C2 and preventing activation of the late promoter A3. In addition, protein SpoOA inhibits ø29 DNA replication by binding near the ø29 DNA ends, preventing the formation of the protein p6-nucleoprotein complex that is required for the initiation of ø29 DNA replication.

From Molecular Biology to Biotechnology

As already mentioned, using four ø29 DNA replication proteins, TP, DNA polymerase, protein p6 and SSB, we were able to amplify TP-DNA 1000-fold. More recently, we have obtained TP-primed amplification of heterologous DNA inserted between the ø29 DNA replication origins containing 191 and 194 bp from the left and right ends, respectively, using the 4 replication proteins indicated above.¹² On the other hand, the capacity of the ø29 DNA polymerase to use circular ssDNA as template allowed asymmetric rolling-circle amplification, producing single-stranded concatemeric DNA containing more than ten copies of the initial template.⁴ This led to the development of a procedure by Amersham Biosciences/Molecular Staging in which ø29 DNA polymerase is combined with random hexamer primers to achieve isothermal and faithful 104- 106-fold amplification of either circular⁸ or linear⁷ genomes, yielding high-quality amplification products. More recently, to enhance the amplification efficiency of ø29 DNA polymerase we constructed chimeric DNA polymerases by fusing DNA-binding domains to the C-terminus of the ø29 DNA polymerase.⁶ The addition of (helix-hairpin-helix)₂ domains increases DNA binding of the chimeric DNA polymerases without affecting their replication rate. The chimeric DNA polymerases display an improved and faithful multiply-primed DNA amplification efficiency on both circular plasmids and genomic linear DNA and are unique ø29 DNA polymerase variants with enhanced amplification performance. Our research on ø29 DNA polymerase, performed over 30 y, has allowed us to exploit the potential of this small viral enzyme, a good example of basic research applied to biotechnology.

Concluding comments

I have dedicated 55 y of my life to research, 49 of them as an independent investigator working on phage ø29. We have made relevant contributions to unravelling the mechanism of protein-primed ø29 DNA replication as well as the control of ø29 DNA transcription. Although the aim of my work has been basic research, this led to an important biotechnological application: the use of ø29 DNA polymerase for DNA amplification. This is a good example of how basic research can lead to applications that were not foreseen. The 23 y of teaching Molecular Genetics at Madrid Complutense University were also very rewarding. I had very brilliant students, some of whom did the Ph.D. work in my laboratory and are now group leaders doing excellent research. The work performed in my laboratory is the result of the dedication and ideas of the pre- and postdoctoral fellows during these exciting 48 y. I am very grateful to all of them, especially to those that have helped me in the supervision of the work. Many thanks to José M. Lázaro, who has been working with me since 1972 and who has maintained the laboratory along all those years, and to my secretary, Angeles M. Villarraso, who has been with me for the past 20 y and helps me always with great efficiency. I would like to mention that every four years, from 1980 till 1996 I organized in Salamanca, Spain, the International Workshop on Bacteriophages, funded by the European Molecular Biology Organization (EMBO). The best phage workers over the world came to these Workshops. I also would like to acknowledge the funding agencies that supported my work: The Jane Coffin Childs Memorial Fund for Medical Research, that enabled us to start our ø29 work in Spain; the National Institutes of Health that funded our research for 24 y; the European Community; the Spanish Ministry of Education and Science; the Spanish Ministry of Science and Innovation; the Madrid Autonomous Community; and the Fundación Ramón Areces and Banco de Santander, which supplied institutional grants to the Centro de Biología Molecular “Severo Ochoa.”

I thank my teachers: Alberto Sols, for his teaching of Enzymology; Severo Ochoa, to whom I owe my dedication to biochemistry and who taught us (Eladio and myself) the molecular biology that we could teach and develop in Spain; and especially Eladio Viñuela, husband, friend, colleague, and always a teacher to me. Eladio, who is no longer with us, has been the most important person in my life, both from the personal and scientific points of view.

Dedication

I dedicate this Life in Science article to Eladio.