A model for the complex between the helix destabilizing protein GP32 of bacteriophage T4 and single-stranded DNA

A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed. In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base. This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments. It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides.     

Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure

In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.     

Orientation of the bases of single-stranded DNA and polynucleotides in complexes formed with the gene 32 protein of bacteriophage T4. A linear dichroism study

Linear dichroism measurements were performed in the wavelength region 250 to 350 nm on complexes between the single-stranded DNA binding protein of bacteriophage T4 (gp32) and single-stranded DNA and a variety of homopolynucleotides in compressed polyacrylamide gels. The complexes appeared to orient well, giving rise to linear dichroism spectra that showed contributions from both the protein aromatic residues and the bases of the polynucleotides. In most cases the protein contribution appeared to be very similar, and the linear dichroism of the bases could be explained by similar orientations of the bases for most of the complexes. Assuming a similar, regular structure for most of the polynucleotides in complex, only a limited set of combinations of tilt and twist angles can explain the linear dichroism spectra. These values of tilt and twist are close to (-40 degrees, 30 degrees), (-40 degrees, 150 degrees), (40 degrees, -30 degrees) or (40 degrees, -150 degrees), with an uncertainty in both angles of about 15 degrees. Although the linear dichroism results do not allow a choice between these possible orientations, the latter two combinations are not in agreement with earlier circular dichroism calculations. For the complexes formed with poly(rC) and poly(rA), the linear dichroism spectra could not be explained by the same base orientations. In these two cases also the protein contribution to the linear dichroism appeared to be different, indicating that for some aromatic residues the orientations are not the same as those in the other complexes. The different structures of these complexes are possibly related to the relatively low binding affinity of gp32 to poly(rC), and to a lesser extent to poly(rA).     

A maximum of two tryptophan residues in gene-32 protein from phage T4 undergo stacking interactions with single-stranded polynucleotides

The effect of specific photochemical and radiochemical modification of tryptophyl and cysteinyl residues of the gene 32 protein (gp 32) of bacteriophage T4 on its affinity towards single-stranded polynucleotides has been investigated. Oxidation of Cys residues of gp 32 by the free-radical anion I-.2 induces a partial loss of the protein affinity, probably by affecting the metal-binding domain which includes three of the four cysteine residues of gp 32. Ultraviolet irradiation of gp 32 in the presence of trichloroethanol results in the modification of three of its five Trp residues and total loss of the protein binding. Analysis of the relative affinity of ultraviolet-irradiated gp 32 for single-stranded polynucleotides suggest that modification of a Trp of enhanced reactivity occurs first and has no effect on the protein binding. Radiochemical modification of three Trp residues of gp 32 by (SCN)-.2 results in total loss of activity. Complexation of gp 32 with denatured DNA prior to gamma-irradiation protects two Trp residues and prevents the protein inactivation. These results suggest that at most two Trp residues are involved in stacking interactions with nucleic acid bases. However, time-resolved spectroscopic methods which allow us to monitor selectively the stacked tryptophan residues have not yielded evidence of more than a single residue undergoing such interactions.     

Investigation of complexes formed between gene 32 protein from bacteriophage T4 and heavy-atom-modified single-stranded polynucleotides using optical detection of magnetic resonance

Optical detection of triplet-state magnetic resonance (ODMR) is employed to study the complexes formed between gene 32 protein (GP32), a single-stranded DNA-binding protein from bacteriophage T4, and the heavy-atom-derivatized polynucleotides poly(5-HgU) and poly(5-BrU). The triplet-state properties of some of the tryptophan (Trp) residues in the complexes are dramatically different from those in the free protein, in that they are subject to an external heavy-atom effect. Direct evidence for the presence of a heavy-atom effect, and hence a close-range interaction between mercurated or brominated nucleotide bases and Trp residues in the complex, is provided by the observation of the zero-field (D) + (E) ODMR transition of Trp, which is not normally observed in the absence of a heavy-atom perturbation. The amplitude-modulated phosphorescence-microwave double-resonance (AM-PMDR) technique is employed to selectively capture the phosphorescence spectrum originating from the heavy-atom-perturbed Trp residue(s) in the GP32-poly(5-HgU) complex. Arguments based on our experimental results lead to the conclusion that the heavy-atom perturbation arises from aromatic stacking interactions between Trp and mercurated bases. Wavelength-selected ODMR measurements reveal the existence of two environmentally distinct and spectrally different types of Trp in GP32. One of these types is perturbed selectively by the heavy atom and hence undergoes stacking interactions with the heavy-atom-derivatized bases of the polynucleotide while the second type of Trp residue is unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Models for the binary complex of bacteriophage T4 gp59 helicase loading protein: gp32 single-stranded DNA-BINDING protein and ternary complex with pseudo-Y junction DNA

Bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596-18607).     

Proteolytic removal of the COOH terminus of the T4 gene 32 helix-destabilizing protein alters the T4 in vitro replication complex

The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. 1. Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32I protein for this synthesis. 2. Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. 3. Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. 4. The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled inadequately, so that polymerizing enzymes tend to be displaced from the growing 3'-OH end of a polynucleotide chain and are thereby inhibited. Eukaryotic helix-destabilizing proteins may also have similar functional domains essential for the control of their activities.     

Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein

Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.     

Photoaffinity labeling of T4 bacteriophage 32 protein

With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.     

The characterization of a complex of three bacteriophage T4 recombination proteins, uvsX protein, uvsY protein, and gene 32 protein, on single-stranded DNA

Three recombination proteins of bacteriophage T4, uvsX, uvsY, and gene 32 proteins, were examined for the formation of a complex with short single-stranded DNA (ssDNA) molecules containing either 24 or 69 nucleotides. Gel-shift assays revealed that either the uvsX or uvsY protein, when present alone, formed a stable complex only with the 69-mer, while the gene 32 protein bound stably to both ssDNAs. However, a characteristic stable complex formed on the 24-mer when both the uvsX and uvsY proteins were present, and the uvsY protein bound to this DNA in the presence of the gene 32 protein. Isolation of the complexes by centrifugation through a glycerol gradient revealed their protein constituents and showed that the uvsX protein-uvsY protein-24-mer ssDNA complex formed even in the presence of excess gene 32 protein. The possible biological significance of these protein-DNA complexes is discussed.     

Regulation of the synthesis of bacteriophage T4 gene 32 protein

The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment.Mutations in genes required for late gene expression (genes 33 and 53) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not dependent on DNA synthesis.These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by its own function. Thus, in the absence of P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently in the maturation of replicated DNA, the increased P32 production is caused by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41, 56, 58, 60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.     

Kinetics and mechanism of the association of the bacteriophage T4 gene 32 (helix destabilizing) protein with single-stranded nucleic acids. Evidence for protein translocation

We have investigated the association kinetics of the co-operatively binding T4-coded gene 32 (helix destabilizing) protein with a variety of single-stranded homopolynucleotides (both RNA and DNA). Stopped-flow mixing experiments were performed by monitoring the partial quenching of the intrinsic tryptophan fluorescence of the protein upon binding to the nucleic acid under conditions where the nucleic acid concentration is in great excess over the protein concentration. Investigations of the association rate (and rate constants) as a function of solution variables has demonstrated quite different behavior at the extremes of “low” and “high” salt concentration. Under low salt (high binding constant) conditions the non-co-operative association is rate-limiting and we measure a bimolecular rate constant of 3 × 10⁶ to 4 × 10⁶ m^?1 (nucleotide)s^?1 (0·1 m-NaCl, 25·0 °C). However, at higher salt concentrations (lower binding constant) a pre-equilibrium involving non-co-operatively bound protein is established, followed by the rate-limiting formation of co-operatively bound protein clusters.Based on these observations we have proposed a mechanism for the formation of co-operatively bound T4 gene 32 protein clusters, under conditions of low binding density, which consists of three steps: (1) pre-equilibrium formation of non-co-operatively bound protein (nucleation); followed by (2) association of free protein to the singly contiguous sites established in the nucleation step, hence forming the first co-operative interactions (growth step); and (3) a redistribution of the growing protein clusters to form the final equilibrium distribution. From comparisons of our experimental values of the forward rate constant for the second step (growth of clusters) with theoretical estimates based on the work of Berg &; Blomberg (1976,1978) we infer that the T4 gene 32 protein is able to translocate along singlestranded polynucleotides. The implications of these results for the in vivo action of the T4 gene 32 protein are discussed.     

Nucleotide-dependent binding of the gene 4 protein of bacteriophage T7 to single-stranded DNA

The gene 4 protein of bacteriophage T7 is a multifunctional enzyme that catalyzes (i) the hydrolysis of nucleoside 5'-triphosphates, (ii) the synthesis of tetraribonucleotide primers at specific recognition sequences on a DNA template, and (iii) the unwinding of duplex DNA. All three activities depend on binding of gene 4 protein to single-stranded DNA followed by unidirectional 5' to 3' translocation of the protein (Tabor, S., and Richardson, C. C. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 205-209). Binding of gene 4 protein to single-stranded DNA, assayed by retention of DNA-protein complexes on nitrocellulose filters, is random with regard to DNA sequence. Although gene 4 protein does not bind to duplex DNAs, the presence of a 240-nucleotide-long single-stranded tail on a 7200-base pair duplex DNA molecule is sufficient for gene 4 protein to cause retention of the DNA on a filter. The binding reaction requires, in addition to MgCl2, the presence of a nucleoside 5'-triphosphate, but binding is not dependent on hydrolysis; nucleoside 5'-diphosphate will substitute for nucleoside 5'-triphosphate. Of the eight common nucleoside triphosphates, dTTP promotes optimal binding. The half-life of the gene 4 protein-DNA complex depends on both the secondary structure of the DNA and on whether or not the nucleoside 5'-triphosphate cofactor can be hydrolyzed. Using the nonhydrolyzable nucleoside 5'-triphosphate analog, beta,gamma-methylene dTTP, the half-life of the gene 4 protein-DNA complex is greater than 80 min. In the presence of the hydrolyzable nucleoside 5'-triphosphate, dTTP, the half-life of the gene 4 protein-DNA complex using circular M13 DNA is at least 4 times longer than that observed using linear M13 DNA.     

A single-stranded DNA-binding protein of bacteriophage T7 defective in DNA annealing

The annealing of complementary strands of DNA is a vital step during the process of DNA replication, recombination, and repair. In bacteriophage T7-infected cells, the product of viral gene 2.5, a single-stranded DNA-binding protein, performs this function. We have identified a single amino acid residue in gene 2.5 protein, arginine 82, that is critical for its DNA annealing activity. Expression of gene 2.5 harboring this mutation does not complement the growth of a T7 bacteriophage lacking gene 2.5. Purified gene 2.5 protein-R82C binds single-stranded DNA with a greater affinity than the wild-type protein but does not mediate annealing of complementary strands of DNA. A carboxyl-terminal-deleted protein, gene 2.5 protein-Delta26C, binds even more tightly to single-stranded DNA than does gene 2.5 protein-R82C, but it anneals homologous strands of DNA as well as does the wild-type protein. The altered protein forms dimers and interacts with T7 DNA polymerase comparable with the wild-type protein. Gene 2.5 protein-R82C condenses single-stranded M13 DNA in a manner similar to wild-type protein when viewed by electron microscopy.     

On the role of the single-stranded DNA binding protein of bacteriophage T4 in DNA metabolism. I. Isolation and genetic characterization of new mutations in gene 32 of bacteriophage T4

Summary The product of gene 32 of bacteriophage T4 is a single-stranded DNA binding protein involved in T4 DNA replication, recombination and repair. Functionally differentiated regions of the gene 32 protein have been described by protein chemistry. As a preliminary step in a genetic dissection of these functional domains, we have isolated a large number of missense mutants of gene 32. Mutant isolation was facilitated by directed mutagenesis and a mutant bacterial host which is unusually restrictive for missense mutations in gene 32. We have isolated over 100 mutants and identified 22 mutational sites. A physical map of these sites has been constructed and has shown that mutations are clustered within gene 32. The possible functional significance of this clustering is considered.     

The role of the bacteriophage T4 gene 32 protein in homologous pairing

The gene 32 protein of the bacteriophage T4 is required for efficient genetic recombination in infected Eschericia coli cells and strongly stimulates in vitro pairing catalyzed by the phage uvsX protein, a RecA-like strand transferase. This helix-destabilizing factor is known to bind tightly and cooperatively to single-stranded DNA and to interact specifically with the uvsX protein as well as other phage gene products. However, its detailed role in homologous pairing is not well understood. I show here that when the efficiency of uvsX protein-mediated pairing is examined at different gene 32 protein and duplex DNA concentrations, a correlation between the two is found, suggesting that the two interact in a functionally important manner during the reaction. These and other data are consistent with a model in which the gene 32 protein binds to the strand displaced from the recipient duplex during pairing, thereby stabilizing the heteroduplex product. An alternative model in which the gene 32 protein replaces UvsX on the invading strand, thereby freeing the strand transferase to bind to the displaced strand, is also considered.     

Crystallization of a tryptic core of the single-stranded DNA binding protein of bacteriophage T4

A tryptic core (residues 22 to 253) of the single-stranded DNA binding protein, or gene 32 protein, of bacteriophage T4 has been crystallized in four different crystal forms. One of these forms appears suitable for high-resolution X-ray crystallographic studies. It is triclinic, space group PI, with $\text{a = 67.7}\text{A}\text{?}\text{, b = 67.8}\text{A}\text{?}\text{, c = 66.0}\text{A}\text{?}\text{, α = 101.6 °, β = 107.0 °, γ = 105.2 °}$. There appear to be three protein protomers in a near-rhombohedral packing in the unit cell.