T4 Single-stranded DNA Research Articles

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions.

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.

Control of Helicase Loading in the Coupled DNA Replication and Recombination Systems of Bacteriophage T4

The Gp59 protein of bacteriophage T4 promotes DNA replication by loading the replicative helicase, Gp41, onto replication forks and recombination intermediates. Gp59 also blocks DNA synthesis by Gp43 polymerase until Gp41 is loaded, ensuring that synthesis is tightly coupled to unwinding. The distinct polymerase blocking and helicase loading activities of Gp59 likely involve different binding interactions with DNA and protein partners. Here, we investigate how interactions of Gp59 with DNA and Gp32, the T4 single-stranded DNA (ssDNA)-binding protein, are related to these activities. A previously characterized mutant, Gp59-I87A, exhibits markedly reduced affinity for ssDNA and pseudo-fork DNA substrates. We demonstrate that on Gp32-covered ssDNA, the DNA binding defect of Gp59-I87A is not detrimental to helicase loading and translocation. In contrast, on pseudo-fork DNA the I87A mutation is detrimental to helicase loading and unwinding in the presence or absence of Gp32. Other results indicate that Gp32 binding to lagging strand ssDNA relieves the blockage of Gp43 polymerase activity by Gp59, whereas the inhibition of Gp43 exonuclease activity is maintained. Our findings suggest that Gp59-Gp32 and Gp59-DNA interactions perform separate but complementary roles in T4 DNA metabolism; Gp59-Gp32 interactions are needed to load Gp41 onto D-loops, and other nucleoprotein structures containing clusters of Gp32. Gp59-DNA interactions are needed to load Gp41 onto nascent or collapsed replication forks lacking clusters of Gp32 and to coordinate bidirectional replication from T4 origins. The dual functionalities of Gp59 allow it to promote the initiation or re-start of DNA replication from a wide variety of recombination and replication intermediates.

Interaction of T4 UvsW Helicase and Single-Stranded DNA Binding Protein gp32 through Its Carboxy-Terminal Acidic Tail

Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA (ssDNA) binding protein gp32 is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW-catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy-terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions.

Assembly and Dynamics of Gp59-Gp32-Single-stranded DNA (ssDNA), a DNA Helicase Loading Complex Required for Recombination-dependent Replication in Bacteriophage T4

The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks.

Dual Functions of Single-stranded DNA-binding Protein in Helicase Loading at the Bacteriophage T4 DNA Replication Fork

Semi-conservative DNA synthesis reactions catalyzed by the bacteriophage T4 DNA polymerase holoenzyme are initiated by a strand displacement mechanism requiring gp32, the T4 single-stranded DNA (ssDNA)-binding protein, to sequester the displaced strand. After initiation, DNA helicase acquisition by the nascent replication fork leads to a dramatic increase in the rate and processivity of leading strand DNA synthesis. In vitro studies have established that either of two T4-encoded DNA helicases, gp41 or dda, is capable of stimulating strand displacement synthesis. The acquisition of either helicase by the nascent replication fork is modulated by other protein components of the fork including gp32 and, in the case of the gp41 helicase, its mediator/loading protein gp59. Here, we examine the relationships between gp32 and the gp41/gp59 and dda helicase systems, respectively, during T4 replication using altered forms of gp32 defective in either protein-protein or protein-ssDNA interactions. We show that optimal stimulation of DNA synthesis by gp41/gp59 helicase requires gp32-gp59 interactions and is strongly dependent on the stability of ssDNA binding by gp32. Fluorescence assays demonstrate that gp59 binds stoichiometrically to forked DNA molecules; however, gp59-forked DNA complexes are destabilized via protein-protein interactions with the C-terminal "A-domain" fragment of gp32. These and previously published results suggest a model in which a mobile gp59-gp32 cluster bound to lagging strand ssDNA is the target for gp41 helicase assembly. In contrast, stimulation of DNA synthesis by dda helicase requires direct gp32-dda protein-protein interactions and is relatively unaffected by mutations in gp32 that destabilize its ssDNA binding activity. The latter data support a model in which protein-protein interactions with gp32 maintain dda in a proper active state for translocation at the replication fork. The relationship between dda and gp32 proteins in T4 replication appears similar to the relationship observed between the UL9 helicase and ICP8 ssDNA-binding protein in herpesvirus replication.

A Novel Mechanism of Virus–Virus Interactions: Bacteriophage P2 Tin Protein Inhibits Phage T4 DNA Synthesis by Poisoning the T4 Single-Stranded DNA Binding Protein, gp32

P2 prophages have been known to inhibit DNA replication and growth of T-even phages. We show here that this inhibition is due to poisoning of the T-even single-stranded DNA binding protein gp32 by the product of the nonessential P2tingene. Synthesis of Tin protein from a gene cloned in a multicopy plasmid is necessary and sufficient to completely preventde novoDNA replication and growth of wild-type T2 or T4 phage. We isolated more than 20 independent mutants that render T-even phages resistant to poisoning by the P2 Tin protein. In all of these mutants, which we callasp,Asp codon 163 of gene32is changed to a Gly or Asn codon. The mutant alleles are recessive; i.e., when wild-type andaspmutants coinfect the same host cells, most DNA replication is poisoned by P2 Tin protein. To explain our results, we propose that the P2 Tin protein interacts with T-even gp32 at position 163 and distorts the helical filament of gene32protein on single-stranded DNA. Thereby Tin protein inhibits either assembly or function, or both, of the T4 replisome. The inhibition of late gene expression by P2 Tin protein may be an indirect consequence of inhibition of DNA replication.

Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase.

The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of DNA replication. Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32). The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex. We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex. The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32. A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction. These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase. A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43.

“Macromolecular crowding”: thermodynamic consequences for protein-protein interactions within the T4 DNA replication complex.

In vitro biochemical assays are typically performed using very dilute solutions of macromolecular components. On the other hand, total intracellular concentrations of macromolecular solutes are very high, resulting in an in vivo environment that is significantly "volume-occupied." In vitro studies with the DNA replication proteins of bacteriophage T4 have revealed anomalously weak binding of T4 gene 45 protein to the rest of the replication complex. We have used inert macromolecular solutes to mimic typical intracellular solution conditions of high volume occupancy to investigate the effects of "macromolecular crowding" on the binding equilibria involved in the assembly of the T4 polymerase accessory proteins complex. The same approach was also used to study the assembly of this complex with T4 DNA polymerase (gene 43 protein) and T4 single-stranded DNA binding protein (gene 32 protein) to form the five protein "holoenzyme". We find that the apparent association constant (Ka) of gene 45 for gene 44/62 proteins in forming both the accessory protein complex and the holoenzyme increases markedly (from approximately 7 x 10(6) to approximately 3.5 x 10(8) M-1) as a consequence of adding polymers such as polyethylene glycol and dextran. Although the processivity of the polymerase alone is not directly effected by the addition of such polymers to the solution, macromolecular crowding does significantly stabilize the holoenzyme and thus indirectly increases the observed processivity of the holoenzyme complex. The use of macromolecular crowding to increase the stability of multienzyme complexes in general is discussed, as is the relevance of these results to DNA replication in vivo.

Lactate dehydrogenase and glyceraldehyde-phosphate dehydrogenase are single-stranded DNA-binding proteins that affect the DNA-polymerase-alpha-primase complex.

Affinity chromatography on double-stranded (ds) and single-stranded (ss) DNA-cellulose columns was employed to find analogs of the Escherichia coli and T4 single-stranded DNA binding proteins (SSB proteins) in calf thymus. The interaction of several purified SSB proteins with the pure DNA-polymerase-alpha--primase complex on DNA synthesis on activated DNA and on primase-initiated M13 DNA served as a criterion for a possible involvement of one of these proteins in the process of DNA replication. Two SSB proteins were purified to essential homogeneity. These most abundant proteins exhibited apparent relative molecular masses of 35,000 (SSB-35) and 37,000 (SSB-37) for the protomers and 140,000 and 80,000 for the native enzymes. Both proteins resisted elution with 0.5 mg/ml dextran sulfate and were eluted from the ssDNA-cellulose with 0.2 M and 1 M NaCl, respectively. SSB-35 stimulated the DNA-polymerase-alpha--primase complex from the same organism up to fivefold over a broad range of DNA covering. By contrast, SSB-37 inhibited the primase-initiated replication of M13 DNA. Like most eukaryotic SSB proteins, these proteins showed a 300-fold preference for binding to ssDNA over dsDNA in a nitrocellulose filter binding assay, as well as strong binding to several DNA and RNA homopolymers. Furthermore, we provide evidence for a cooperative mode of binding for SSB-37. Although SSB-35 and SSB-37 behave as typically eukaryotic SSB proteins in all assays employed, we tested these SSB proteins for dehydrogenase activities as well. SSB-35 was found to be identical with lactate dehydrogenase and SSB-37 was identical with a dimeric form of glyceraldehyde-3-phosphate dehydrogenase. These results imply that further studies are mandatory in order to prove the authenticity of eukaryotic SSB proteins.

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.

In vivo effects of recBC DNase, exonuclease I, and DNA polymerases of Escherichia coli on the infectivity of native and single-stranded DNA of bacteriophage T7

The effect of several enzymes of the DNA metabolism of Escherichia coli on the biological activity of native and single-stranded T7 DNA was studied by transfection of lysozyme-EDTA spheroplasts prepared from various E. coli mutants. It is shown that the presence of the recBC DNase in the recipient cells decreases the infectivity of native and denatured DNA by about 100- and 10-fold, respectively. Lack of exonuclease I did not stimulate transfection by single-stranded DNA. Separated light (l) and heavy (r) strands of T7 DNA are fully infective, with a linear dependence on DNA concentrations, whereas heat-denatured DNA shows a two-hit kinetics. Single-stranded DNA was observed to depend on a functional DNA polymerase III for infectivity in polAB cells, whereas transfection with native T7 DNA was independent of the host DNA polymerases. The results are discussed with respect to the mode of T7 DNA replication.

Purification and properties of an endonuclease from nuclei of uninfected and polyoma-infected 3T3 cells

An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells.

A Deoxyribonucleic Acid-Binding Protein Induced by Bacteriophage T7: PURIFICATION AND PROPERTIES OF THE PROTEIN

Abstract A protein which binds strongly and preferentially to single-stranded DNA has been purified by DNA-cellulose chromatography from Escherichia coli infected with bacteriophage T7. The protein, synthesized between 5 and 18 min following infection, comprises more than 0.1% of soluble protein in T7-infected cells. In vitro studies show that this protein can specifically stimulate the rate of polymerization catalyzed by the T7-induced DNA polymerase. At low temperatures, where the T7-induced DNA polymerase utilizes single-stranded DNA poorly as template primer, the binding protein stimulates the rate of synthesis 10- to 15-fold. The stimulatory effect is also observed employing single-stranded DNA at 37°; however, the stimulation is not as great (2- to 3-fold) at the higher temperature. Similarly, when a double-stranded DNA containing internal single-stranded regions is used as template, stimulation is observed, but is not as pronounced (approximately 5-fold). T7 DNA polymerase activity can be stimulated 20- to 50-fold by salt when a gapped DNA is used as template but only 1.5-fold when a single-stranded DNA is used as template. On the latter template, stimulation by binding protein is relatively constant over a range of salt concentrations, whereas on the former template, stimulation occurs only at salt concentrations less than optimal for polymerase activity. The stimulatory reaction is relatively insensitive to pH. The protein has a molecular weight of 31,000 and forms aggregates at high protein concentrations in the presence of magnesium ions. It is capable of binding to single-stranded DNA in quantities which exceed the weight of the DNA by a factor of 20. The protein does not bind to native DNA. The T7 DNA-binding protein dissociates from single-stranded T7 DNA with a rate constant of about 1 x 10-4 s-1 and a half-life of dissociation between 97 and 120 min under the conditions studied.

Stimulation of T7 DNA polymerase by a new phage-coded protein.

A bacteriophage-induced DNA-binding protein was purified from T7 infected E. coli. The protein has a molecular weight of about 25000, as judged by SDS-polyacrylamide gel electrophoresis. The purified protein binds to single-stranded but not to native T7 DNA. Like the T4 gene-32 protein and the 22000-dalton “unwinding protein” of E. coli, the T7 25000 protein lowers the melting temperature of poly d(A-T). Using partially single-stranded T7 DNA as template-primer, the protein stimulates in vitro DNA synthesis by T7 DNA polymerase about five-fold. It was also found that the DNA-unwinding protein of E. coli stimulates T7 DNA polymerase to approximately the same extent. However, neither of the unwinding proteins stimulate DNA polymerase I of E. coli.

Intrinsic viscosity of native and single‐stranded T7 DNA and its relationship to sedimentation coefficient

The intrinsic viscosity and sedimentation coefficient of native and single-stranded T7 DNA have been determined at 25⁰C as a function of ionic strength in neutral and alkaline NaCl. The relationship between [η] and S⁰(20,w), is well represented by the Mandelkern-Flory equation over the entire range of conditions between 0.0013 and 1M Na+. An apparent discrepancy between the two methods at moderate to high ionic strengths is probably due to a change in V with ionic strength. It appears that [η] is a more sensitive and reliable measure of molecular expansion for native DNA,but S⁰(20,w) is a better index of conformational change in single strands, since [η] becomes too small to measure conveniently at high ionic strengths. At moderate to high ionic strengths, denaturation leads to a decrease in [η], although unfolded single strands retain considerable viscosity. At sufficiently low ionic strength, the intrinsic viscosity of the single strands becomes higher than that of native DNA, and the effective volume of a single strand approaches that of the native molecule.

Conformational changes of single-stranded DNA

The conformation of intact, essentially monodisperse, single-stranded T7 DNA was studied in solutions between 0.001 and 1 m-NaCl at neutral and alkaline pH and temperatures between 5 and 35 °C. Over-all shape is assessed by measurement of S 20,w 0, and base stacking by absorbance at 260 mμ. The results are interpreted in terms of intrastrand interactions between the bases: stacking which occurs in the absence of H-bonding is referred to as free stacking; associations which involve H-bonding (as well as stacking) are referred to as folding. Folding has large effects on both absorbance and S 20,w 0; free stacking has a much smaller effect on absorbance and essentially no effect on S 20,w 0. Folding is sensitive to change in ionic strength, temperature, and pH; free stacking depends mainly on temperature and is affected to a lesser extent, if at all, by alkaline titration or by changes in ionic strength. The transition between unfolded and folded forms can be measured by changes in either absorbance or sedimentation coefficient; the two measurements give broadly similar results but do not agree exactly. The differences are interpreted in terms of short-range and long-range folding, the former involving associations between bases which are near each other in the chain, the latter involving associations between bases which are widely separated along the contour length of the chain. Knowledge of the conformational state of single-stranded T7 DNA over the above range of conditions allows study of the effects of conformation on reactions which might have biological significance. The succeeding paper (Studier, 1969) shows that folding is a major influence on the renaturation and aggregation of single-stranded DNA.

Single-strand breaks in duplex DNA of coliphage T7 as demonstrated by electron microscopy

Previous work based on ultracentrifugal studies had led to the hypothesis that there are either gaps or exceptional bonds, not of the phosphodiester type, in the single polynucleotide strands of the DNA of coliphage T7. To investigate this further, the length distribution of the single strands derived from native T7 DNA by alkaline denaturation has been obtained. An improved technique for electron microscopic visualization of single strands of DNA in a cytochrome c monolayer was used for this measurement. In contrast with the narrow length distribution for native, double-stranded DNA, the length distribution of the single strands was found to be very broad (about eightfold greater than for the native material). Single-stranded T7 DNA was then fractionated according to sedimentation coefficient by sucrose gradient centrifugation, and these various fractions were examined with the electron microscope. It was found that the more rapidly and more slowly moving fractions consist of longer and shorter filaments, respectively, from which it has been concluded that sedimentation heterogeneity of single-stranded DNA is a reflection of molecular weight heterogeneity. Hence, the electron microscopic studies confirm the original hypothesis that T7 DNA contains single-strand breaks or exceptional bonds.