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Graphical abstract, Flap endonuclease 1 (FEN1) is an important component of the intricate molecular machinery for DNA replication and repair. FEN1 is a structure-specific 5′ nuclease that cleaves nascent single-stranded 5′ flaps during the maturation of Okazaki fragments. Here, we review our research primarily applying single-molecule fluorescence to resolve important mechanistic aspects of human FEN1 enzymatic reaction. The methodology presented in this review is aimed as a guide for tackling other biomolecular enzymatic reactions by fluorescence enhancement, quenching, and FRET and their combinations. Using these methods, we followed in real-time the structures of the substrate and product and 5′ flap cleavage during catalysis. We illustrate that FEN1 actively bends the substrate to verify its features and continues to mold it to induce a protein disorder-to-order transitioning that controls active site assembly. This mechanism suppresses off-target cleavage of non-cognate substrates and promotes their dissociation with an accuracy that was underestimated from bulk assays. We determined that product release in FEN1 after the 5′ flap release occurs in two steps; a brief binding to the bent nicked-product followed by longer binding to the unbent nicked-product before dissociation. Based on our cryo-electron microscopy structure of the human lagging strand replicase bound to FEN1, we propose how this two-step product release mechanism may regulate the final steps during the maturation of Okazaki fragments.

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.

The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.

Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase–nuclease–RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary “driver” and “passenger” mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

Protein purification by affinity chromatography relies primarily on the interaction of a fused-tag to the protein of interest. Here, we describe a tag-free affinity method that employs functional selection interactions to a broad range of proteins. To achieve this, we coupled human DNA-clamp proliferating cell nuclear antigen (PCNA) that interacts with over one hundred proteins to an agarose resin. We demonstrate the versatility of our PCNA-Agarose column at various chromatographic steps by purifying PCNA-binding proteins that are involved in DNA Replication (DNA polymerase δ, flap endonuclease 1 and DNA ligase 1), translesion DNA synthesis (DNA polymerases eta, kappa and iota) and genome stability (p15). We also show the competence of the PCNA-Agarose column to purify non-PCNA binding proteins by fusing the PCNA-binding motif of human p21 as an affinity tag. Finally, we establish that our PCNA-Agarose column is a suitable analytical method for characterizing the binding strength of PCNA-binding proteins. The conservation and homology of PCNA-like clamps will allow for the immediate extension of our method to other species.

Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant molecular structures and develop possible mechanistic insights for the large dynamic TFIIH complex. TFIIH presents many puzzles involving how its two SF2 helicase family enzymes, XPB and XPD, function in transcription initiation and repair: how do they initiate transcription, detect and verify DNA damage, select the damaged strand for incision, coordinate repair with transcription and cell cycle through Cdk-activating-kinase (CAK) signaling, and result in very different specific human diseases associated with cancer, aging, and development from single missense mutations? By joining analyses of breakthrough cryo-electron microscopy (cryo-EM) structures and advanced computation with data from biochemistry and human genetics, we develop unified concepts and molecular level understanding for TFIIH functions with a focus on structural mechanisms. We provocatively consider that TFIIH may have first evolved from evolutionary pressure for TCR to resolve arrested transcription blocks to DNA replication and later added its key roles in transcription initiation and global DNA repair. We anticipate that this level of mechanistic information will have significant impact on thinking about TFIIH, laying a robust foundation suitable to develop new paradigms for DNA transcription initiation and repair along with insights into disease prevention, susceptibility, diagnosis and interventions.

The deep-sea brines of the Red Sea are remote and unexplored environments characterized by high temperatures, anoxic water, and elevated concentrations of salt and heavy metals. This environment provides a rare system to study the interplay between halophilic and thermophilic adaptation in biologic macromolecules. The present article reports the first DNA polymerase with halophilic and thermophilic features. Biochemical and structural analysis by Raman and circular dichroism spectroscopy showed that the charge distribution on the protein's surface mediates the structural balance between stability for thermal adaptation and flexibility for counteracting the salt-induced rigid and nonfunctional hydrophobic packing. Salt bridge interactions via increased negative and positive charges contribute to structural stability. Salt tolerance, conversely, is mediated by a dynamic structure that becomes more fixed and functional with increasing salt concentration. We propose that repulsive forces among excess negative charges, in addition to a high percentage of negatively charged random coils, mediate this structural dynamism. This knowledge enabled us to engineer a halophilic version of Thermococcus kodakarensis DNA polymerase.-Takahashi, M., Takahashi, E., Joudeh, L. I., Marini, M., Das, G., Elshenawy, M. M., Akal, A., Sakashita, K., Alam, I., Tehseen, M., Sobhy, M. A., Stingl, U., Merzaban, J. S., Di Fabrizio, E., Hamdan, S. M. Dynamic structure mediates halophilic adaptation of a DNA polymerase from the deep-sea brines of the Red Sea.

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

The strength of the biotin/avidin interaction makes it an ideal tool for the purification of biotin-labeled proteins via avidin-coupled resin with high specificity and selectivity. Nevertheless, this tight binding comes at an extra cost of performing the elution step under denaturing conditions. Weakening the biotin/avidin interaction improves the elution conditions, but only to mild or harsh denaturing buffers with the drawback of reducing the specificity and selectivity of this interaction. Here, we present two chromatographic protein purification schemes that are well-suited for application under native conditions thus preserving the strength of the biotin/avidin interaction. In the first scheme, we introduce a biotin-labeled SUMO-tag to each of human flap endonuclease 1 and Escherichia coli replication termination protein Tus, and elute both proteins by performing on-resin cleavage using SUMO protease. In the second scheme, we immobilize biotin-labeled human proliferating cell nuclear antigen (PCNA) on the avidin-coupled resin and use the resulting resin as a tag-free affinity method to purify the PCNA-binding protein human DNA Ligase 1. Furthermore, we streamlined the protein biotinylation protocol by constructing a single plasmid expression system that ensures high level of expression and solubility for each of the target protein bearing the biotin-tag and the enzyme responsible for the in vivo biotinylation reaction. Both chromatographic schemes resulted in a high yield of pure proteins in their native form.

Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca(2+) have suggested that base unpairing at the 5′-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps, from substrate binding to product release, during the hFEN1-catalyzed reaction in the presence of Mg(2+). We found that whereas hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg(2+)-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5′-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5′-flap:template junction, indicating that local DNA distortion (e.g. disruption of base stacking observed in structures), associated with positioning the 5′-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34 and Asp-181), steering the 5′-flap through the active site and binding the scissile phosphate (Lys-93 and Arg-100), and stacking against the base 5′ to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5′-flaps.

Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a “replication fork trap” – found within a chromosomal region where forks are allowed to enter but not leave – is set by the protein–DNA roadblock Tus–Ter. The exact sequence of events by which Tus–Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein–protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus–Ter interactions at the nonpermissive face – interactions that result in a highly stable “locked” complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus–Ter mechanism – leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA–protein system further.

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability. DOI: http://dx.doi.org/10.7554/eLife.21884.001, eLife digest When a cell divides it must copy its genetic information, which is found in the form of strands of DNA. Damage to the DNA may lead to cancer or a number of genetic diseases. However, every time a cell divides more than 10 million toxic “flaps” of excess DNA are generated. A protein called flap endonuclease 1 (FEN1) keeps the DNA in good repair by cutting off the flaps in a highly specific and selective manner. Many proteins that interact with DNA are attracted to specific genetic sequences within the DNA strands. However, this is not the case for FEN1 and several other “structure-specific” proteins that help to repair and replicate DNA strands. So how do these proteins select the correct regions of DNA to interact with? Rashid et al. used single-molecule fluorescence measurements to examine how purified FEN1 proteins interact with DNA flaps. The results show that FEN1 can perfectly recognize and correctly remove flaps through a process called “mutual-induced fit”, where the DNA and FEN1 are shaped by each other to produce a highly specific structure. Further work is now needed to examine whether other proteins that are related to FEN1 use a similar process to ensure that they always cut DNA in the same way. More detailed and direct examination of the structure of FEN1 through other experimental methods may also help to reveal how the mutual-induced fit process enables FEN1 to achieve such high levels of precision. This could increase our understanding of how problems with FEN1 and similar proteins lead to different genetic diseases. DOI: http://dx.doi.org/10.7554/eLife.21884.002

Summary Human flap endonuclease 1 (FEN1), one of the structure-specific 5′ nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5′ nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5′ end junctions. Using single-molecule Forster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5′ nuclease mechanisms. This is achieved by coordinating threading of the 5′ flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5′ nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection of DNA junctions from nonspecific bending and cleavage.

Processive DNA synthesis by the alpha epsilon theta core of the Escherichia coli Pol III replicase requires it to be bound to the beta(2) clamp via a site in the a polymerase subunit. How the epsilon proofreading exonuclease subunit influences DNA synthesis by alpha was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of epsilon. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel beta-binding site in e that, in conjunction with the site in a, maintains a closed state of the alpha epsilon theta-beta(2) replicase in the polymerization mode of DNA synthesis. The epsilon-beta interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this 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. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field.

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5'-TGGTC-3') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.

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.

Escherichia coli thioredoxin binds to a unique flexible loop of 71 amino acid residues, designated the thioredoxin binding domain (TBD), located in the thumb subdomain of bacteriophage T7 gene 5 DNA polymerase. The initial designation of thioredoxin as a processivity factor was premature. Rather it remodels the TBD for interaction with DNA and the other replication proteins. The binding of thioredoxin exposes a number of basic residues on the TBD that lie over the duplex region of the primer-template and increases the processivity of nucleotide polymerization. Two small solvent-exposed loops (loops A and B) located within TBD electrostatically interact with the acidic C-terminal tail of T7 gene 4 helicase-primase, an interaction that is enhanced by the binding of thioredoxin. Several basic residues on the surface of thioredoxin in the polymerase-thioredoxin complex lie in close proximity to the TBD. One of these residues, lysine 36, is located proximal to loop A. The substitution of glutamate for lysine has a dramatic effect on the binding of gene 4 helicase to a DNA polymerase-thioredoxin complex lacking charges on loop B; binding is decreased 15-fold relative to that observed with wild-type thioredoxin. This defective interaction impairs the ability of T7 DNA polymerase-thioredoxin together with T7 helicase to mediate strand displacement synthesis. This is the first demonstration that thioredoxin interacts with replication proteins other than T7 DNA polymerase.

Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5Delta22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5Delta22 with Trx, under oxidizing conditions, with an IC50 of approximately 10 microM.

T7 gene 5 DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin, together with the T7 gene 4 DNA helicase, catalyze strand displacement synthesis on duplex DNA processively (>17,000 nucleotides per binding event). The processive DNA synthesis is resistant to the addition of a DNA trap. However, when the polymerase–thioredoxin complex actively synthesizing DNA is challenged with excess DNA polymerase–thioredoxin exchange occurs readily. The exchange can be monitored by the use of a genetically altered T7 DNA polymerase (gp5-Y526F) in which tyrosine-526 is replaced with phenylalanine. DNA synthesis catalyzed by gp5-Y526F is resistant to inhibition by chain-terminating dideoxynucleotides because gp5-Y526F is deficient in the incorporation of these analogs relative to the wild-type enzyme. The exchange also occurs during coordinated DNA synthesis in which leading- and lagging-strand synthesis occur at the same rate. On ssDNA templates with the T7 DNA polymerase alone, such exchange is not evident, suggesting that free polymerase is first recruited to the replisome by means of T7 gene 4 helicase. The ability to exchange DNA polymerases within the replisome without affecting processivity provides advantages for fidelity as well as the cycling of the polymerase from a completed Okazaki fragment to a new primer on the lagging strand.

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow on- and off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling.

The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA (ssDNA)-binding protein (gp2.5) that is an essential component of the phage replisome. Similar to other prokaryotic ssDNA-binding proteins, gp2.5 has an acidic C terminus that is involved in protein-protein interactions at the replication fork and in modulation of the ssDNA binding properties of the molecule. We have used genetic and biochemical approaches to identify residues critical for the function of the C terminus of gp2.5. The presence of an aromatic residue in the C-terminal position is essential for gp2.5 function. Deletion of the C-terminal residue, phenylalanine, is detrimental to its function, as is the substitution of this residue with non-aromatic amino acids. Placing the C-terminal phenylalanine in the penultimate position also results in loss of function. Moderate shortening of the length of the acidic portion of the C terminus is tolerated when the aromatic nature of the C-terminal residue is preserved. Gradual removal of the acidic C terminus of gp2.5 results in a higher affinity for ssDNA and a decreased ability to interact with T7 DNA polymerase/thioredoxin. The replacement of the charged residues in the C terminus with neutral amino acids abolishes gp2.5 function. Our data show that both the C-terminal aromatic residue and the overall acidic charge of the C terminus of gp2.5 are critical for its function.

T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a two-fold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.

The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1∶1 complex with Escherichia coli thioredoxin. By a mechanism that is not fully understood, thioredoxin acts as a processivity factor and converts gp5 from a distributive polymerase into a highly processive one. We use a single-molecule imaging approach to visualize the interaction of fluorescently labeled T7 DNA polymerase with double-stranded DNA. We have observed T7 gp5, both with and without thioredoxin, binding nonspecifically to double-stranded DNA and diffusing along the duplex. The gp5/thioredoxin complex remains tightly bound to the DNA while diffusing, whereas gp5 without thioredoxin undergoes frequent dissociation from and rebinding to the DNA. These observations suggest that thioredoxin increases the processivity of T7 DNA polymerase by suppressing microscopic hopping on and off the DNA and keeping the complex tightly bound to the duplex.

This chapter reviews the various single molecule methods used and the type of information obtained by such studies of nucleic acid enzymes. Numerous different enzymes have evolved to catalyze the synthesis, digestion, unwinding, and unlinking of nucleic acids that are central to genomic maintenance. The development of single molecule techniques has allowed researchers to study the activity of nucleic acid enzymes, such as RNA polymerases, DNA polymerases, topoisomerases, exonucleases, andDNA helicases, at an unprecedented level of detail. The ability to observe the activity of RNA and DNA-binding proteins on the single molecule level provides tremendous opportunities in the field of nucleic acid enzymology. The chapter provides an overview of the various techniques and a number of nucleic acid enzyme systems where single molecule approaches have proven to be particularly powerful in unraveling the molecular details of enzymatic activity. The recent development of several methods, such as flow stretching, magnetic tweezers, and optical trapping to mechanically manipulate objects of microscopic scale has allowed researchers to stretch and twist individual DNA molecules. Advances in fluorescence spectroscopy and microscopy have made it possible to detect the fluorescence from a single fluorophore under biological tweezers, transcriptional elongation traces of individual E. coli RNA polymerases could be obtained with high spatial resolution, allowing for a detailed study of the statistics of transcriptional pausing. The technical improvements of optical trapping techniques allowed observation of movements of individual RNA polymerases along their template DNA with a resolution better than a single base pair.

Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3'-5' exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from the beta and gamma subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E. coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (k(cat)=100-650 min(-1), K(M)=0.4-2.0 mM, at pH 8.00 and 25 degrees C, with 1 mM Mn(2+)). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn(2+) being preferred over Mg(2+) as cofactor, and was inhibited by Ni(2+). The concentration dependency of Mn(2+) was examined, giving K(Mn) values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the α, ε, and θ subunits. The θ subunit is the smallest and least understood subunit. The three-dimensional structure of θ in a complex with the unlabeled N-terminal domain of the ε subunit, ε186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy 3+ , Er 3+ , or Ho 3+ ) at the active site of ε186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of θ in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed θ show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free θ (Keniry et al., Protein Sci. 9: 721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of θ in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of ε186 on θ was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of θ are located about 15 Å or farther from the lanthanide ion in the active site of ε186, in agreement with the extensive biochemical data for the θ-ε system.

Bacteriophage T7 DNA polymerase (gene 5 protein, gp5) interacts with its processivity factor, Escherichia coli thioredoxin, via a unique loop at the tip of the thumb subdomain. We find that this thioredoxin-binding domain is also the site of interaction of the phage-encoded helicase/primase (gp4) and ssDNA binding protein (gp2.5). Thioredoxin itself interacts only weakly with gp4 and gp2.5 but drastically enhances their binding to gp5. The acidic C termini of gp4 and gp2.5 are critical for this interaction in the absence of DNA. However, the C-terminal tail of gp4 is not required for binding to gp5 when the latter is bound to a primer/template. We propose that the thioredoxin-binding domain is a molecular switch that regulates the interaction of T7 DNA polymerase with other proteins of the replisome.

Electrospray ionization mass spectrometry (ESI-MS) has widespread routine use as a tool in proteomics for confirmation of primary structure determination and for characterization of purified proteins (Griffiths et al. 2001). Application of ESI-MS to detection and characterization of non-covalent complexes of biomolecules is not as well established, but there are now many examples of noncovalent interactions that have been studied in the gas phase, including those between protein subunits, proteins and nucleic acids, and enzymes and substrates (Veenstra 1999; Burkitt et al. 2003; Sanglier et al. 2003). The most obvious use of ESI-MS for study of these complexes is in determination of the stoichiometry of binding partners, and this has recently been extended to monitor subunit exchange between small heat shock proteins in real time (Sobott et al. 2002). The establishment of stoichiometry is a prelude to more detailed structural determination of biomolecules in complexes. The stability of the complex (dissociation constants), the types of noncovalent interactions (e.g., polar vs. nonpolar), and conformational changes in the binding partners upon complex formation are important when considering the mechanism of biological action of biomolecular complexes (e.g., protein–protein, protein–DNA). There is a suite of biophysical techniques that can be applied to study these properties. These range from monitoring of changes in fluorescence or surface plasmon resonance (SPR) for determination of dissociation constants and use of circular dichroism and NMR spectroscopy for following conformational changes, ultimately to determination of complete structures of complexes by NMR, X-ray crystallography, or cryo-electron microscopy. ESI-MS offers speed and sensitivity in monitoring components of equilibrium mixtures. Consequently, there are increasing numbers of reports of its use for determination of dissociation constants or relative binding affinities of non-covalent complexes (Jorgensen et al. 1998; Kapur et al. 2002; Bligh et al. 2003). Furthermore, there are ESI-MS studies where the stabilities of noncovalent complexes have been assessed by their resistance to dissociation in the mass spectrometer using CID (collision-induced dissociation) or thermal denaturation experiments (Gupta et al. 2001; de Brouwer et al. 2002; Benesch et al. 2003). Nevertheless, data obtained from ESI-MS studies need to be interpreted with caution. First, the ionization process itself might perturb equilibria (Wang and Agnes 1999). Second, there is a paucity of information about changes in the strength or specificity of noncovalent interactions that occur on transfer from the condensed to the gas phase during the ionization process. The stabilities of complexes between biological macromolecules involve contributions from ionic, hydrogen bonding, hydrophobic, and/or van der Waals interactions. Several ESI-MS studies support the proposal that electrostatic interactions are strengthened in vacuo, while hydrophobic interactions are unaffected or weakened through loss of water during desolvation and/or ionization (Loo 1997). For these reasons, it is important to study the behavior of noncovalent complexes that have been well characterized in solution to enable evaluation of data from ESI-MS experiments. Recently, as part of a study aimed at investigating the behavior of noncovalent complexes on transferal from solution to the gas phase, we used ESI-MS to study the well-characterized Tus-Ter (protein–DNA) complex that terminates DNA replication in Escherichia coli (Kapur et al. 2002). We showed that relative binding affinities of mutant Tus proteins for double-stranded TerB DNA were the same in the gas phase as in solution, and conversely, that the relative affinities of wild-type Tus for various double-stranded DNA sequences were unchanged on transferal to the gas phase. Both the X-ray structure (Kamada et al. 1996) and SPR studies of the ionic strength dependence of dissociation of the Tus-TerB complex (Neylon et al. 2000) show there are substantial polar and electrostatic contacts between the binding partners. Consistent with this, ESI mass spectra showed that dissociation of the complex required high concentrations of ammonium acetate, in the range of 1–2 M (Kapur et al. 2002). For the present work, we used ESI-MS to investigate the predominantly hydrophobic interactions between two protein subunits of E. coli DNA polymerase III: the θ subunit, and the N-terminal domain (residues 2–186) of the ɛ subunit (ɛ186). DNA polymerase III is a multisubunit enzyme that is the major replicative polymerase of E. coli (Kelman and O’Donnell 1995; McHenry 2003). Three of the 10 subunits, α, ɛ, and θ, comprise the catalytic core: the large α-subunit contains the polymerase active site, and ɛ contributes the proofreading 3′→5′ exonuclease activity, while the precise function of θ is not known (Studwell-Vaughan and O’Donnell 1993; Kunkel and Bebenek 2000). The ɛ subunit consists of two domains (Perrino et al. 1999; Taft-Benz and Schaaper 1999; Hamdan et al. 2000). The N-terminal domain (ɛ186) contains the exonuclease active site and forms a stable 1:1 complex with θ (Perrino et al. 1999; Hamdan et al. 2002a). The complex forms readily and essentially quantitatively on mixing of the two subunits, and is sufficiently stable that it can be isolated by ion-exchange chromatography (Hamdan et al. 2002a). It is stable for extended periods at 25°C in aqueous solution under conditions required for NMR studies (Pintacuda et al. 2004). Although there is no high-resolution structure yet available for the ɛ186–θ complex, we have reported the crystal structure of ɛ186 (Hamdan et al. 2002b) and the solution structure of θ (Keniry et al. 2000). NMR chemical shift mapping experiments in the latter study suggested that a series of small hydrophobic residues on the external face of the first helix of θ (residues 21–27, AAAGVAF) are involved in its association with ɛ186, and this has been confirmed in the more recent NMR structure of θ in the ɛ186–θ complex (M. Keniry, pers. comm.). Moreover, recent comparisons of NMR spectra of free ɛ186 and ɛ186–θ have identified hydrophobic residues in ɛ at the θ-binding interface, including Ile31, Val50, Val58, Ile68, Leu74, Ile154, Leu161, Leu165, and Leu166 (DeRose et al. 2003). It appears, therefore, that interaction between the two proteins is mediated largely via aliphatic side chains, and the forces that hold them together are largely hydrophobic in nature. In the present work, we aimed to use ESI-MS to supply “snapshots” of components of mixtures of ɛ186 and θ. The stability of the ɛ186–θ complex was studied under various solution and instrumental conditions. Its stability at high ionic strength is consistent with a dominant contribution of nonpolar interactions, in contrast with that of the Tus-Ter complexes, where interactions are primarily polar and electrostatic in nature and are disrupted at relatively low ionic strength (Kapur et al. 2002). In addition, ESI-MS experiments suggested that the ɛ subunit protects ɛ186 from aggregation in organic solvent/water mixtures. This is consistent with earlier experiments in which θ was shown to stabilize ɛ186 against thermal inactivation (Hamdan et al. 2002a).

The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation. For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM. Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP. Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+). Inhibition by TMP was formally competitive [K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple. K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH6.5 and 400 min(-1) at pH9.0. These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.

The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.

Controlled racemization of enantiopure phenyl-ring-containing secondary alcohols is achieved in this study using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TeSADH) and in the presence of the reduced and oxidized forms of its cofactor nicotinamide-adenine dinucleotide. Racemization of both enantiomers of alcohols accepted by W110A TeSADH, not only with low, but also with reasonably high, enantiomeric discrimination is achieved by this method. Furthermore, the high tolerance of TeSADH to organic solvents allows TeSADH-catalyzed racemization to be conducted in media containing up to 50% (v/v) of organic solvents.

The bacteriophage T7 DNA polymerase consists of a tight, 1:1 complex of T7 gp5, encoded by the phage, and thioredoxin, produced by the E. coli host. In the absence of thioredoxin, gp5 is capable of adding only a few nucleotides to the 3′ end of a primer before dissociating from the primer-template. But when complexed with thioredoxin, gp5 becomes highly processive, capable of polymerizing thousands of nucleotides complementary to the template strand. The mechanism by which thioredoxin acts as a processivity factor to gp5 is not fully understood. To understand the role of the thioredoxin in stabilizing polymerase-DNA interactions, we use a single-molecule imaging approach to observe individual, fluorescently labeled T7 DNA polymerase complexes diffusing along double-stranded DNA. Our results show that the average diffusion coefficient of T7 DNA polymerase complexes is insensitive to ionic strength and does not exceed the theoretical diffusion limit for a protein that tracks the helical pitch and rotates as it diffuses along the DNA helix. These results suggest that the T7 DNA polymerase slides along the DNA, remaining tightly bound to the DNA and tracking the helical pitch. However, the mean diffusion coefficients for fluorescently labeled T7 gp5 in the absence of thioredoxin increase with salt concentration, and exceed the theoretical limit for a protein tracking the DNA helix. Upon addition of unlabeled thioredoxin, the mean diffusion coefficient is restored to the value observed for the labeled T7 DNA polymerase, and becomes salt independent. These observations indicate that, in the absence of thioredoxin, T7 gp5 intermittently dissociates from the DNA as it diffuses, and that thioredoxin binding suppresses microscopic hopping on and off the DNA.