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.     

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.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex.     

A specific model for the conformation of single-stranded polynucleotides in complex with the helix-destabilizing protein GP32 of bacteriophage T4

The CD and absorption (OD) spectra of single-stranded nucleic acids in complex with the helix-destabilizing protein of either bacteriophage T4 (GP32) or bacteriophage fd (GP5) show similar and unusual features for all polynucleotides investigated. The change in the CD spectra between 310 and 240 nm is in all cases characterized by a considerable decrease in the positive band, while the negative band (if present) remains relatively intense. These changes are different from those due to temperature or solvent denaturation and, moreover, cannot be induced by the binding of simple oligopeptides. Absorption measurements show that all polynucleotides remain hypochromic in the complex. Both CD and OD spectra point to a specific and probably similar conformation in complex for all polynucleotides with substantial interactions between the bases. The spectral properties are almost temperature independent (0–40°C). Therefore, we conclude that the conformation must be regular and rigid. To investigate the relation between these optical properties and the specific polynucleotide structure, CD and OD spectra were calculated for an adenine hexamer over a wide range of the conformational parameters. It appears that the calculated CD intensity is not very sensitive to an increase in the axial increment and that many different conformations can give rise to more or less similar CD spectra. However, simulation of the very nonconservative experimental CD spectrum of the poly(rA)-GP32 complex requires that the conformation satisfies two criteria: (1) a considerable tilt of the bases (? – 10°) in combination with (2) a small rotation per base (?20°) and/or a position of the bases close to the helix axis (dx ? 0 Å). Such conformations can also explain the observed hyperchromism upon binding of GP32 to poly(rA)/(dA). Very similar structural characteristics also account for the optical properties of the complexes with GP5. These are discussed as an alternative to the structure suggested by Alma-Zeestraten for poly(dA) in the complex [N. C. M. Alma-Zeestraten (1982) Doctoral thesis Catholic University, Nijmegen, The Netherlands]. The secondary structure proposed in this work can be reconciled with the overall dimensions of the complex, assuming that the polynucleotide helix is further organized in a superhelix.     

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.     

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.     

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex.     

Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution

In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.     

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.     

Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.     

DNA binding site of the helix destabilizing protein gp32 from bacteriophage T4.

A helix destabilizing protein, the product of gene 32 (gp32) of bacteriophage T4, was subjected to limited proteolysis to produce three types of products with differing affinities for DNA. Previous work has suggested that the 18 amino acids at the N-terminus are required for tight binding to single-stranded DNA (Hosoda &; Moise, 1978). This paper reports the sequence of the N-terminal region and predicts the amino acid residues responsible for DNA binding.     

Autoregulation of gene expression: Quantitative evaluation of the expression and function of the bacteriophage T4 gene 32 (single-stranded DNA binding) protein system

The free concentration of bacteriophage T4-coded gene 32 (single-stranded DNA binding) protein in the cell is autoregulated at the translational level during T4 infection of Escherichia coli. The control of the synthesis of this protein reflects the following progression of net (co-operative) binding affinities for the various potential nucleic acid binding targets present: single-stranded DNA > gene 32 mRNA > other T4 mRNAs ? double-stranded DNA. In this paper we show that the free concentration of gene 32 protein is maintained at 2 to 3 μm, and use the measured binding parameters for gene 32 protein, extrapolated to intracellular conditions, to provide a quantitative molecular interpretation of this system of control of gene expression. These results are then further utilized to define the specific autoregulatory binding sequence (translational operator site) on the gene 32 mRNA as a uniquely unstructured finite binding lattice terminated by elements of secondary structure not subject to melting by gene 32 protein at the autoregulated concentration, and to predict how this site must differ from those found on other T4 messenger RNAs. It is shown that these predictions are fully consistent with available T4 DNA sequence data. The control of free protein concentration as a method of genome regulation is discussed in terms of other systems to which these approaches may apply.     

Mutations in a conserved motif inhibit single-stranded DNA binding and recombination mediator activities of bacteriophage T4 UvsY protein

The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4. UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange. UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsY-ssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors. A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis. Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised. These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY. Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNA-dependent ATP hydrolysis and DNA strand exchange. The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly. Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.     

The conformation of the complex of the helix destabilizing protein GP32 of bacteriophage T4 and single stranded DNA

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (less than or equal to -10 degrees) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.     

The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Delta26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Delta26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase.     

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).     

The carboxyl-terminal domain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta 26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-Delta 26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.     

Nucleic binding affinity of bacteriophage T4 gene 32 protein in the cooperative binding mode

This study reports on various parameters which affect the binding stoichiometry for complexes of bacteriophage T4 gene 32 protein (P32) and single stranded polynucleotides (determined by UV absorbance and fluorescence quenching) and presents results of a quantitative electron spin resonance assay to determine physiologically effective binding affinity differences of nucleic acid binding proteins. The assay employs macromolecular spin probes (spin-labeled nucleic acids) which are used to determine the fraction of saturation in competition experiments with unlabeled nucleic acids. It was found that the fraction of complexed spin-labeled polynucleotides can be directly monitored by ESR with a two-component analysis approach when ligands such as poly(L-lysine), gene 5 protein (P5) of filamentous bacteriophage fd, and gene 32 protein (P32) of bacteriophage T4 are used. The ESR data unequivocally show that: 1) the binding stoichiometry for poly(L-lysine), P5 and P32 is nucleotide/lysine, 4 nucleotides/P5 monomer, and 10 nucleotides/P32 monomer, respectively; and 2) under physiologically relevant buffer conditions the relative affinity of P32 in the cooperative binding mode for polythymidylic acid is about 4 times greater than for polydeoxyinosinic acid and about 12 times greater than for polyinosinic acid, and the relative affinity of P32 for polydeoxyinosinic acid is about 3 times greater than for polyinosinic acid.