It seems like only yesterday.

I spent my childhood and adolescence in North and South Carolina, attended Duke University, and then entered Duke Medical School. One year in the laboratory of George Schwert in the biochemistry department kindled my interest in biochemistry. After one year of residency on the medical service of Duke Hospital, chaired by Eugene Stead, I joined the group of Arthur Kornberg at Stanford Medical School as a postdoctoral fellow. Two years later I accepted a faculty position at Harvard Medical School, where I remain today. During these 50 years, together with an outstanding group of students, postdoctoral fellows, and collaborators, I have pursued studies on DNA replication. I have experienced the excitement of discovering a number of important enzymes in DNA replication that, in turn, triggered an interest in the dynamics of a replisome. My associations with industry have been stimulating and fostered new friendships. I could not have chosen a better career.

The discovery of error-prone DNA polymerase V and its unique regulation by RecA and ATP.

My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.

Historical perspective: Arthur Kornberg, a giant of 20th century biochemistry.

For physics, the period from the beginning to the middle of the 20th century was one of great scientific excitement and revolutionary discovery. The analogous era for biochemistry, and its offspring, molecular biology, was the second half of the 20th century. One of the most important and influential leaders of this scientific revolution was Arthur Kornberg. The DNA polymerase, which he discovered in 1955 and showed to have the remarkable capacity to catalyze the template-directed synthesis of DNA, contributed in major ways to the present-day understanding of how DNA is replicated and repaired, and how it is transcribed. The discovery of DNA polymerase also permitted the development of PCR and DNA sequencing, upon which much of modern biotechnology is based. Kornberg's studies of DNA replication, which spanned a period of nearly 30 years, culminated in a detailed biochemical description of the mechanism by which a chromosome is replicated. The final years of Kornberg's life were devoted to the study of polyphosphate, which he was convinced had a crucial role in cellular function.

A passion for research.

During my postdoctoral training in Severo Ochoa's laboratory, I determined the direction of reading of the genetic message and I discovered two proteins in Escherichia coli involved in the initiation of protein synthesis. After my return to Spain, I have been working with the Bacillus subtilis phage varphi29. We discovered a protein covalently linked to the 5' DNA ends that is the primer for the initiation of varphi29 DNA replication. We also found that the phage-encoded DNA polymerase has unique properties such as processivity and strand displacement activity. These properties, in addition to its high fidelity, have made the varphi29 DNA polymerase the ideal enzyme for DNA amplification, both for rolling circle and whole-genome amplification. I am happy to say that the work carried out in my laboratory has been possible thanks to many brilliant students and collaborators, most of whom have become high quality independent scientists.

DNA polymerase gamma, the mitochondrial replicase.

DNA polymerase (pol) gamma is the sole DNA polymerase in animal mitochondria. Biochemical and genetic evidence document a key role for pol gamma in mitochondrial DNA replication, and whereas DNA repair and recombination were thought to be limited or absent in animal mitochondria, both have been demonstrated in recent years. Thus, the mitochondrial replicase is also apparently responsible for the relevant DNA synthetic reactions in these processes. Pol gamma comprises a catalytic core in a heterodimeric complex with an accessory subunit. The two-subunit holoenzyme is an efficient and processive polymerase, which exhibits high fidelity in nucleotide selection and incorporation while proofreading errors with its intrinsic 3' 5' exonuclease. Incorporation of nucleotide analogs followed by proofreading failure leads to mitochondrial toxicity in antiviral therapy, and misincorporation during DNA replication leads to mitochondrial mutagenesis and dysfunction. This review describes our current understanding of pol gamma biochemistry and biology, and it introduces other key proteins that function at the mitochondrial DNA replication fork.