Mutator mutations in bacteriophage T4 gene 32 (DNA unwinding protein).

Bacteriophage T4 gene 32 encodes a DNA unwinding protein required for DNA replication, repair, and recombination. Gene 32 temperature-sensitive mutations enhance virtually all base pair substitution mutation rates.     

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.     

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.     

Studies of the self-association of bacteriophage T4 gene 32 protein by equilibrium sedimentation.

The self-association of the bacteriophage T4 gene 32 protein has been examined in the analytical ultracentrifuge under varying conditions to determine the nature of the process. The process is not a simple indefinite association with one association constant (monomer

dimer

trimer

etc.). The complexity of the process is shown by (1) peculiarities in the molecular weight versus concentration curves, in the region of the dimer (observed with increasing ionic strength, at pH 10, in 0.04 m-MgCl₂, with aged preparations, at 19 °C and in the presence of the oligonucleotide d(pT)₁₀), (2) the increased sigmoidicity of the association curve in the presence of glycerol or oligo[d(pT)₄], and (3) the discontinuity in the association curve at the tetramer at a pH value of approximately 9.4. A model with two association constants which could vary independently (monomer

dimer

tetramer

etc.) explained many of the findings. However, a more complex model was required to explain curves which had a plateau at the dimer with increased association at higher protein concentrations. Thus, under all conditions examined there is evidence for more than one type of protein-protein interaction. These different interactions may be involved in a physiological function such as recombination.     

Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase

T4 phage polynucleotide kinase (PNK) displays 5′-hydroxyl kinase, 3′-phosphatase and 2′,3′-cyclic phosphodiesterase activities. The enzyme phosphorylates the 5′ hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5′ ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5′-GT DNA end than for a 5′-TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.     

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.     

Domain effects on the DNA-interactive properties of bacteriophage T4 gene 32 protein

Bacteriophage T4 gene 32 protein, a model for single-strand specific nucleic acid-binding proteins, consists of three structurally and functionally distinct domains. We have studied the effects of the N and C domains on the protein structure and its nucleic acid-interactive properties. Although the presence of the C domain decreases the proteolytic susceptibility of the core (central) domain, quenching of the core tryptophan fluorescence by iodide is unaltered by the presence of the terminal domains. These results suggest that the overall conformation of the core domain remains largely independent of the flanking domains. Removal of the N or the C terminus does not abolish the DNA renaturation activity of the protein. However, intact protein and its three truncated forms differ in DNA helix-destabilizing activity. The C domain alone is responsible for the kinetic barrier to natural DNA helix destabilization seen with intact protein. Intact protein and core domain potentiate the DNA helix-destabilizing activity of truncated protein lacking only the C domain (*I), enhancing the observed hyperchromicity while increasing the melting temperature. Proteolysis experiments suggest that the affinity of core domain for single-stranded DNA is increased in the presence of *I. We propose that *I can "mingle" with intact protein or core domain while bound to single-stranded DNA.     

Repair of DNA damaged by methyl methanesulfonate in bacteriophage T4.

A highly purified preparation of T4 endonuclease V does not degrade DNA alkylated with methyl methanesulfonate, and the methyl methanesulfonate sensitivity of T4 wild type and x mutant is not affected by the v mutation. Thus, T4 endonuclease V, the v gene product, does not seem to be involved in a repair or an abortive repair of methyl methanesulfonate-damaged T4 DNA. The x and y genes of T4 and the polA and the uvrD genes of Escherichia coli are concerned with the repair of methyl methanesulfonate-induced damages in T4 DNA. Since effects of the polA and the x or y mutations are additive, it is supposed that there are at least two pathways for the repair of methyl meth-anesulfonate-damaged T4 DNA, one controlled by the x and the y genes and the other in which E. coli DNA olymerase I is involved. The partial suppression of the x gene mutation at high temerature was also demonstrated.     

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.     

Crystallization of the gene 45 protein from the DNA replication fork of bacteriophage T4

The gene 45 protein from bacteriophage T4 has been purified and is crystallized. This protein is part of the T4 DNA replication complex. The crystallized protein is active in complementation assays. X-ray diffraction analysis is in progress; data are measured for the native and several heavy atom derivatives. The crystals diffract to about 3.5-A resolution.     

Photosensitized splitting of thymine dimers in DNA by gene 32 protein from phage T 4

The binding of denatured DNA to the protein coded by gene 32 of phage T 4 is accompanied by a quenching of the fluorescence of the protein tryptophyl residues. Gene 32 protein also binds to UV-irradiated DNA and photosensitizes the splitting of thymine dimers. Thymine bases are regenerated by this photosensitized reaction both in double stranded and in heat denatured DNA. No photosensitized splitting of thymine dimers is observed when the complex formed by gene 32 protein with UV-irradiated DNA is dissociated at high ionic strength. These results are discussed with respect to the possible stacking interaction of tryptophyl residues of gene 32 protein with bases in single stranded DNA.     

Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.     

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.     

Recognition of single-stranded DNA by the bacteriophage T4-induced type II topoisomerase

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.     

The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins

A strand exchange reaction between a single-stranded DNA circle and a homologous linear double-stranded DNA molecule is catalyzed by a mixture of two T4 bacteriophage proteins, the uvsX protein (a DNA-dependent ATPase that resembles the recA protein) and the gene 32 protein (a helix-destabilizing protein). The products are different from those formed in the corresponding recA protein-catalyzed reaction; rather than producing a linear single strand plus a nicked circular double-stranded (form II) DNA molecule as the final products, interlinked DNA networks are rapidly generated. Electron microscopy reveals that these networks form from multiple pairing reactions that involve the recombination intermediates. Since the uvsX protein is present in substoichiometric quantities, it presumably recycles to catalyze these successive pairing events. Recycling of the uvsX protein has been more directly examined in an assay that monitors the rate of uvsX protein-catalyzed branch migration. The branch migration reaction is rapidly inhibited by dilution of the uvsX protein or by the addition of a heterologous competitor DNA, showing that the uvsX protein-DNA filaments that catalyze strand exchange are dynamic structures. The evidence suggests that individual uvsX protein monomers are continuously entering and leaving the cooperatively formed filament in a cycle that is strongly affected by their ATP hydrolysis.     

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.     

Interaction between bacteriophage T4 coded gene 32 protein and poly(rA)

The cooperative binding of T4 gene 32 protein with polynucleotides, of which the quantitative aspects in the literature have not satisfied the requirements of thermodynamics, is studied by adopting a modified formula of the lattice theory. A moderate value is found for the cooperativity parameter (q approximately 200 at 0.2 M NaCl), which is weakly dependent on salt concentration. The cation effect on the binding suggests that the shielding of negative charges of the protein or a loose cation bridge between the bound protein molecules plays a role in the cooperative binding process.