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.

Motors, switches, and contacts in the replisome.

Replisomes are the protein assemblies that replicate DNA. They function as molecular motors to catalyze template-mediated polymerization of nucleotides, unwinding of DNA, the synthesis of RNA primers, and the assembly of proteins on DNA. The replisome of bacteriophage T7 contains a minimum of proteins, thus facilitating its study. This review describes the molecular motors and coordination of their activities, with emphasis on the T7 replisome. Nucleotide selection, movement of the polymerase, binding of the processivity factor, unwinding of DNA, and RNA primer synthesis all require conformational changes and protein contacts. Lagging-strand synthesis is mediated via a replication loop whose formation and resolution is dictated by switches to yield Okazaki fragments of discrete size. Both strands are synthesized at identical rates, controlled by a molecular brake that halts leading-strand synthesis during primer synthesis. The helicase serves as a reservoir for polymerases that can initiate DNA synthesis at the replication fork. We comment on the differences in other systems where applicable.

Simultaneous Real-Time Imaging of Leading and Lagging Strand Synthesis Reveals the Coordination Dynamics of Single Replisomes.

The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis. We show that loops in the lagging strand predominantly occur during priming and only infrequently support subsequent Okazaki-fragment synthesis. Fluorescence imaging reveals polymerases remaining bound to the lagging strand behind the replication fork, consistent with Okazaki-fragment synthesis behind and independent of the replication complex. Individual replisomes display both looping and pausing during priming, reconciling divergent models for the regulation of primer synthesis and revealing an underlying plasticity in replisome operation.

Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA.

The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide-nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.

Gp2.5, the multifunctional bacteriophage T7 single-stranded DNA binding protein.

The essential bacteriophage T7-encoded single-stranded DNA binding protein is the nexus of T7 DNA metabolism. Multiple layers of macromolecular interactions mediate its function in replication, recombination, repair, and the maturation of viral genomes. In addition to binding ssDNA, the protein binds to DNA polymerase and DNA helicase, regulating their activities. The protein displays potent homologous DNA annealing activity, underscoring its role in recombination.

Catalytically inactive T7 DNA polymerase imposes a lethal replication roadblock.

Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity depends on the presence of a functional E. coli trxA allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis.

We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading- and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading- and lagging-strand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.

Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers.

A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

Primer release is the rate-limiting event in lagging-strand synthesis mediated by the T7 replisome.

DNA replication occurs semidiscontinuously due to the antiparallel DNA strands and polarity of enzymatic DNA synthesis. Although the leading strand is synthesized continuously, the lagging strand is synthesized in small segments designated Okazaki fragments. Lagging-strand synthesis is a complex event requiring repeated cycles of RNA primer synthesis, transfer to the lagging-strand polymerase, and extension effected by cooperation between DNA primase and the lagging-strand polymerase. We examined events controlling Okazaki fragment initiation using the bacteriophage T7 replication system. Primer utilization by T7 DNA polymerase is slower than primer formation. Slow primer release from DNA primase allows the polymerase to engage the complex and is followed by a slow primer handoff step. The T7 single-stranded DNA binding protein increases primer formation and extension efficiency but promotes limited rounds of primer extension. We present a model describing Okazaki fragment initiation, the regulation of fragment length, and their implications for coordinated leading- and lagging-strand DNA synthesis.

Hydrophobic Residue in Escherichia coli Thioredoxin Critical for the Processivity of T7 DNA Polymerase.

Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 N-terminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutagenesis, identifies leucine 95 as a critical element. This residue may contribute to hydrophobic interactions with the thioredoxin-binding loop of the polymerase; levels of DNA binding and thus nucleotide polymerization are significantly decreased in the absence of this residue. The results suggest that the specific interactions at the interface of thioredoxin and DNA polymerase, rather than the overall structure, are important in the interactions that promote high processivity.

Binding Affinities among DNA Helicase-Primase, DNA Polymerase, and Replication Intermediates in the Replisome of Bacteriophage T7.

The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed, the loop is released, and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase, and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7, the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase · DNA complex are much faster at a nick DNA region than the release from a ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging-strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.

Synthesis of 2'-Fluoro RNA by Syn5 RNA polymerase.

The substitution of 2'-fluoro for 2'-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2'-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2'-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2'-fluoro-dNMPs by Syn5 RNA polymerase have been identified.

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery.

Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading- and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.

Syn5 RNA polymerase synthesizes precise run-off RNA products.

The enzyme predominantly used for in vitro run-off RNA synthesis is bacteriophage T7 RNA polymerase. T7 RNA polymerase synthesizes, in addition to run-off products of precise length, transcripts with an additional non-base-paired nucleotide at the 3'-terminus (N+1 product). This contaminating product is extremely difficult to remove. We recently characterized the single-subunit RNA polymerase from marine cyanophage Syn5 and identified its promoter sequence. This marine enzyme catalyses RNA synthesis over a wider range of temperature and salinity than does T7 RNA polymerase. Its processivity is >30,000 nt without significant intermediate products. The requirement for the initiating nucleotide at the promoter is less stringent for Syn5 RNA polymerase as compared to T7 RNA polymerase. A major difference is the precise run-off transcripts with homogeneous 3'-termini synthesized by Syn5 RNA polymerase. Therefore, the enzyme is advantageous for the production of RNAs that require precise 3'-termini, such as tRNAs and RNA fragments that are used for subsequent assembly.

Flap endonuclease activity of gene 6 exonuclease of bacteriophage T7.

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5'-3'-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5'-overhang. A 3'-single-stranded tail arising from the same end of the duplex as the 5'-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5'-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5'-terminal overhangs as well as to remove nucleotides from the 5'-termini enables it to effectively process the 5'-termini of Okazaki fragments before they are ligated.

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Molecular interactions in the priming complex of bacteriophage T7.

The lagging-strand DNA polymerase requires an oligoribonucleotide, synthesized by DNA primase, to initiate the synthesis of an Okazaki fragment. In the replication system of bacteriophage T7 both DNA primase and DNA helicase activities are contained within a single protein, the bifunctional gene 4 protein (gp4). Intermolecular interactions between gp4 and T7 DNA polymerase are crucial for the stabilization of the oligoribonucleotide, its transfer to the polymerase, and its extension by DNA polymerase. We have identified conditions necessary to assemble the T7 priming complex and characterized its biophysical properties using fluorescence anisotropy. In order to reveal molecular interactions that occur during delivery of the oligoribonucleotide to DNA polymerase, we have used four genetically altered gp4 to demonstrate that both the RNA polymerase and the zinc-finger domains of DNA primase are involved in the stabilization of the priming complex and in sequence recognition in the DNA template. We find that the helicase domain of gp4 contributes to the stability of the complex by binding to the ssDNA template. The C-terminal tail of gp4 is not required for complex formation.

Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication.

DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3'-terminal sequence (5'-ACCA-3') of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA-DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5-tRNA complex abolishes this inhibition.

Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.

The RNA polymerase of marine cyanophage Syn5.

A single subunit DNA-dependent RNA polymerase was identified and purified to apparent homogeneity from cyanophage Syn5 that infects the marine cyanobacteria Synechococcus. Syn5 is homologous to bacteriophage T7 that infects Escherichia coli. Using the purified enzyme its promoter has been identified by examining transcription of segments of Syn5 DNA and sequencing the 5'-termini of the transcripts. Only two Syn5 RNAP promoters, having the sequence 5'-ATTGGGCACCCGTAA-3', are found within the Syn5 genome. One promoter is located within the Syn5 RNA polymerase gene and the other is located close to the right genetic end of the genome. The purified enzyme and its promoter have enabled a determination of the requirements for transcription. Unlike the salt-sensitive bacteriophage T7 RNA polymerase, this marine RNA polymerase requires 160 mm potassium for maximal activity. The optimal temperature for Syn5 RNA polymerase is 24 °C, much lower than that for T7 RNA polymerase. Magnesium is required as a cofactor although some activity is observed with ferrous ions. Syn5 RNA polymerase is more efficient in utilizing low concentrations of ribonucleotides than T7 RNA polymerase.