Abortive bacteriophage T4 head assembly in mutants of Escherichia coli

We describe two mutants (tabB-212 and tabB-127) of Escherichia coli K12 in which T-even phage production is temperature-sensitive. Both mutants are linked to purA and may identify a single new bacterial gene tabB. The uninfected bacterium is indistinguishable from wild type at both 30 °C and 42.4 °C. Sodium dodecyl sulphate—polyacrylamide gel electrophoresis of labelled extracts of tabB mutants infected by T4 wild-type phage shows that the modification of viral head precursors (Laemmli, 1970) does not occur, indicating that capsid formation is blocked. The effect is reversible with at least one of the tabB mutants: a shift to 30 °C leads to the cleavage of a significant fraction of precursors synthesized at 42.4 °C.Two classes of T4 mutants are described: one (com^B) which grows on tabB even at 42.4 °C, the other (k^B) which fails to grow on tabB even at the permissive temperature. Both mutants map in T4 gene 31, suggesting an interaction between gene 31 and tabB products.Since gene 31 mutants lead to the random aggregation of head precursors (Laemmli, 1970), we argue that a host product is involved in the ordered polymerization of T4 proteins into capsids or capsid-related structures.     

Isolation and in vitro characterization of temperature-sensitive mutants of the bacteriophage f1 gene V protein

In vivo selections were used to isolate 43 temperature-sensitive gene V mutants of the bacteriophage f1 from a collection of mutants constructed by saturation mutagenesis of the gene. The sites of temperature-sensitive substitutions are found in both the beta-sheets and the turns of the protein, and some sites are exposed to the solvent while others are not. Thirteen of the variant proteins were purified and characterized to evaluate their free energy changes upon unfolding and their affinities for single-stranded DNA, and eight were tested for their tendencies to aggregate at 42 degrees C. Each of the three temperature-sensitive mutants at buried sites and six of ten at surface sites had free energy changes of unfolding substantially lower (less stabilizing) than the wild-type at 25 degrees C. A seventh mutant at a surface site had a substantially altered unfolding transition and its free energy of unfolding was not estimated. The affinities of the mutant proteins for single-stranded DNA varied considerably, but two mutants at a surface site, Lys69, had much weaker binding to single-stranded DNA than any of the other mutants, while two mutants at another surface site, Glu30, had the highest DNA-binding affinities. The wild-type gene V protein is stable at 42 degrees C, but six of the eight mutants tested aggregated within a few minutes and the remaining two aggregated within 30 minutes at this temperature. Overall, each of the temperature-sensitive proteins tested had a tendency to aggregate at 42 degrees C, and most also had either a low free energy of unfolding (at 25 degrees C), or weak DNA binding. We suggest that any of these properties can lead to a temperature-sensitive gene V phenotype.     

Site-directed mutagenesis of the T4 endonuclease V gene: the role of arginine-3 in the target search

Endonuclease V, a pyrimidine dimer specific endonuclease in T4 bacteriophage, is able to scan DNA, recognize pyrimidine dimer photoproducts produced by exposure to ultraviolet light, and effectively incise DNA through a two-step mechanism at the damaged bases. The interaction of endonuclease V with nontarget DNA is thought to occur via electrostatic interactions between basic amino acids and the acidic phosphate DNA backbone. Arginine-3 was chosen as a potential candidate for involvement in this protein-nontarget DNA interaction and was extensively mutated to assess its role. The mutations include changes to Asp, Glu, Leu, and Lys and deleting it from the enzyme. Deletion of Arg-3 resulted in an enzyme that retained marginal levels of AP specificity, but no other detectable activity. Charge reversal to Glu-3 and Asp-3 results in proteins that exhibit AP-specific nicking and low levels of dimer-specific nicking. These enzymes are incapable of affecting cellular survival of repair-deficient Escherichia coli after irradiation. Mutations of Arg-3 to Lys-3 or Leu-3 also are unable to complement repair-deficient E. coli. However, these two proteins do exhibit a substantial level of in vitro dimer- and AP-specific nicking. The mechanism by which the Leu-3 and Lys-3 mutant enzymes locate pyrimidine dimers within a population of heavily irradiated plasmid DNA molecules appears to be significantly different from that for the wild-type enzyme. The wild-type endonuclease V processively incises all dimers on an individual plasmid prior to dissociation from that plasmid and subsequent reassociation with other plasmids, yet neither of these mutants exhibits any of the characteristics of this processive nicking activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42°?C but not at 32°?C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32°?C but not at 42°?C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant.     

Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.     

Dark Repair of Ultraviolet-Irradiated Deoxyribonucleic Acid by Bacteriophage T4: Purification and Characterization of a Dimer-Specific Phage-Induced Endonuclease

The purification and properties of an ultraviolet (UV) repair endonuclease are described. The enzyme is induced by infection of cells of Escherichia coli with phage T4 and is missing from extracts of cells infected with the UV-sensitive and excision-defective mutant T4V(1). The enzyme attacks UV-irradiated deoxyribonucleic acid (DNA) containing either hydroxymethylcytosine or cytosine, but does not affect native DNA. The specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites. The purified enzyme alone does not excise pyrimidine dimers from UV-irradiated DNA. However, dimer excision does occur in the presence of the purified endonuclease plus crude extract of cells infected with the mutant T4V(1).     

Studies on the substrate specificity of the T 4 excision repair endonuclease

Previous studies have shown that the v gene of bacteriophage T4 codes for an endonuclease that specifically attacks pyrimidine dimer sites in UV-irradiated DNA. The present studies have examined the role of this endonuclease in the repair of DNA damaged by nitrogen mustard, N-methyl-N′-nitro-N-nitrosoguanidine (NTG), mitomycin C and 4-nitroquinoline-N-oxide. The observation by Harm that the v gene product of phage T4 facilitates repair of UV damage to the host DNA of excision-repair defective strains enabled us to test whether it does the same with other cellular DNA lesions. It was shown that infection of UV-irradiated E. coliB_s−1 with UV-inactivated phage T4v+ resulted in rescue of a certain fraction of the host cells. However no v gene mediated repair E. coli B_s−1 was observed following treatment with the chemical agents mentioned. Furthermore, though phage T4v₁ is more sensitive to UV-irradiation than phage T4, there was no observed difference in the sensitivity of these phages to nitrogen mustard or NTG. On the basis of these observations it was concluded that the v gene coded endonuclease of T4 is specific for the excision repair of pyrimidine dimers and does not participate in the repair of chemically damaged DNA. In vitro enzymatic degradation of DNA alkylated with nitrogen mustard was observed, but it is probable that this degradation is not part of a repair reaction in vivo.     

Action of T4 endonuclease V on DNA containing various photoproducts

The action of T4 endonuclease V on DNA containing various photoproducts was investigated. (1) The enzyme introduced strand breaks in DNA from ultraviolet-irradiated vegetative cells of Bacillus subtilis but not in DNA from irradiated spores of the same organism. DNA irradiated with long wavelength (360 nm peak) ultraviolet light in the presence of 4,5',8-trimethylpsoralen was not attacked by the enzyme. These results indicate that 5-thyminyl 5,6-dihydrothymine (spore photoproduct) and psoralen mediated cross-links in DNA are not recognized by T4 endonuclease V. (2) DNA of phage PBS1, containing uracil in place of thymine, and DNA of phage SPO1, containing hydroxymethyluracil in place of thymine, were fragmented by the enzyme when the DNA's had been irradiated with ultraviolet light. T4 endonuclease V seems to act on DNA with pyrimidine dimers whether the dimers contain thymine residues or not.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

Bacteriophage T4 ν mutants with a slow rate of thymine-dimer excision and a particular reactivation phenotype

Bacteriophage T4 uvs52 is a member of a class of UV-sensitive mutants with UV survival between T4 wild-type and v mutants. The mutation promotes recombination between extracellularly UV-irradiated phages. However, the location is adjacent to, or in, gene v. The question whether uvs52 is a v mutant with a particular type of v gene expression was investigated with acid solubilization of [¹⁴C]thymine dimers from DNA incubated with extracts from T4-infected cells. The dimer-removal activity of extracts from uvs52-infected cells was half that of wild-type T4, and similar to that of the v am5 and v op14 enzymes induced in the appropriate su⁺ hosts. The initial velocity of incision of UV-irradiated DNA by partially purified extracts from cells infected with uvs52 was 15% of that of the wild-type. Excision activity was not disturbed in such extracts. Further evidence of the location of uvs52 in gene v followed from the negative results from complementation assays with mixtures of extracts from cells infected with uvs52, uvs21 (another member of this class) or v1.

The relation between initial incision activity and substrate concentration (UV-irradiated ¹⁴C-DNA) suggested that the uvs52 endonuclease V is mutant with a high affinity and a slow rate of thymine-dimer incision. The reactivation phenotype was explained by assuming a slow rate of dimer excision in vivo as well, continuing throughout the reproductive cycle of the phage and leading to intermediate UV sensitivity and photoreactivability. The increased recombination frequencies were explained by assuming that the single-stranded regions of the DNA produced by incisions made at the end of the reproductive cycle are readily recombined into the growing DNA pool.     

den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a DNA glycosylase activity that produced alkali-labile sites in irradiated DNA and an apyrimidinic/apurinic endonuclease activity that converted these sites to nicks. Both activities could be detected in the presence of 10 mM EDTA. In experiments designed to determine which of the activities is coded by the denV gene, we found that the glycosylase was more heat labile in extracts of Escherichia coli infected with either of two thermosensitive denV mutants than in extracts of cells infected with wild-type T4. In contrast, apyrimidinic/apurinic endonuclease activity was no more heat labile in extracts of the former than in extracts of the latter. Our results indicate that the denV gene codes for a DNA glycosylase specific for pyrimidine dimers.     

Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activity in vitro and in vivo.

Endonuclease V of bacteriophage T4 possesses two enzymatic activities, a DNA N-glycosylase specific for cyclobutane pyrimidine dimers (CBPD) and an associated apurinic/apyrimidinic (AP) lyase. Extensive structural and functional studies of endonuclease V have revealed that specific amino acids are associated with these two activities. Controversy still exists regarding the role of the aromatic amino acid stretch close to the carboxyl terminus, in particular the tryptophan at position 128. We have expressed wild-type and mutant W128S endonuclease V in Escherichia coli from an inducible tac promoter. Purified W128S endonuclease V demonstrated substantially decreased N-glycosylase (approximately 5-fold) and AP lyase (10- to 20-fold) activities in vitro compared to the wild-type enzyme when a UV-irradiated poly(dA)-poly(dT) substrate was used. However, a much smaller difference in AP lyase activity between the two forms was observed with a site-specific abasic oligonucleotide. The difference in enzymatic activity was qualitatively, but not quantitatively, reflected in the survival of UV-irradiated bacteria, that is the W128S cells were slightly less UV resistant than wild-type cells. No difference was observed in the complementation of UV repair using UV-damaged denV- T4 phage. A more pronounced difference between the wild-type and W128S proteins was observed in human xeroderma pigmentosum group A cells by host-cell reactivation of a UV-irradiated reporter gene. The relatively large discrepancy between the in vitro and in vivo results observed with bacteria may be because saturated levels of DNA repair are obtained in vivo with relatively low levels of endonuclease V. However, under limiting in vitro conditions and in human cells in vivo a considerable difference between the W128S mutant and wild-type endonuclease V activities can be detected. Our results demonstrate that tryptophan-128 is important for endonuclease V activity.     

Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding

T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.     

Processive action of T4 endonuclease V on ultraviolet-irradiated DNA.

The action of the dimer-specific endonuclease V of bacteriophage T4 was studied on UV-irradiated, covalently-closed circular DNa. Form I ColE1 DNA preparations containing average dimer frequencies ranging from 2.5 to 35 pyrimidine dimers per molecule were treated with T4 endonuclease V and analysed by agarose gel electrophoresis. At all dimer frequencies examined, the production of form III DNA was linear with time and the double-strand scissions were made randomly on the ColE1 DNA genome. Since the observed fraction of form III DNA increased with increasing dimer frequency but the initial rate of loss of form I decreased with increasing dimer frequency, it was postulated that multiple single-strand scissions could be produced in a subset of the DNA population while some DNA molecules contained no scissions. When DNA containing an average of 25 dimers per circle was incubated with limiting enzyme concentrations, scissions appeared at most if not all dimmer sites in some molecules before additional strand scissions were produced in other DNA molecules. The results support a processive model for the interaction of T4 endonuclease V with UV-irradiated DNA.     

Host- and Phage-Mediated Repair of Radiation Damage in Bacteriophage T4

T4+ exhibits increased ultraviolet sensitivity on derivatives of Escherichia coli K12 or B lacking deoxyribonucleic acid (DNA) polymerase I. However, the sensitivity of T4v is not affected by the absence of host DNA polymerase. T4x and T4y also show increased sensitivity on DNA polymerase-deficient strains, but to a lesser extent than observed with wild-type T4. When T4x or T4y, but not T4+, are plated on a double mutant lacking both DNA polymerase and the uvrA gene product, a partial suppression of the polymerase effect is observed. Host ligase appears to be able to suppress to some extent the T4y phenotype but has no effect on wild-type T4 or other T4 mutants. T4xv incubated in E. coli B or B(s-1) in the presence of chloramphenicol (50 mug/ml) shows increased resistance over directly plated irradiated phage. Increased survival under the same conditions was not observed with T4+ or other T4 mutants. The repair of X-ray-damaged T4 was investigated by examining survival curves of T4+, T4x, T4y, T4ts43, and T4ts30. The repair processes were further defined by observing the effects of plating irradiated phage on various hosts including strains lacking DNA polymerase I or polynucleotide ligase. Two classes of effects were observed. Firstly, the x and y gene products seem to be involved in a repair system utilizing host ligase. Secondly, in the absence of host DNA polymerase, phage sensitivity is increased in an unknown manner which is enhanced by the presence of host uvrA gene product.     

Loss of thymine dimers from mammalian cell DNA. The kinetics for antibody-binding sites are not the same as that for T4 endonuclease V sites

Antiserum specific for thymine-containing dimers was used to assay DNA isolated from ultraviolet-irradiated cells following different repair periods. A 50% loss in antibody-binding sites was evident 1 h post-irradiation, and within 4 h 80% of the sites were removed. This result contrasts with data obtained with dimer-specific T4 endonuclease V and does not appear to be due to masking of the dimers by repair enzymes. T4 endonuclease V treatment of ultraviolet-irradiated DNA at 0 degree C resulted in conversion of the thymine dimers to apyrimidinic sites. This did not result in loss of antigenicity in either PM2 or CHO cell DNA. Likewise, treatment of ultraviolet-irradiated CHO cell DNA with T4 endonuclease at 37 degrees C did not change its antigenicity. These results suggest that aglycosylation of the dimers is not responsible for their inability to bind dimer-specific antibody 2-4 h post-irradiation. The possibility that T4 endonuclease V and the antiserum have different specificities for different dimers is discussed.     

Biochemistry of DNA-defective mutants of bacteriophage T4. VI. Biological functions of gene 42.

Bacteriophage T4 gene 1 and 42 amber mutants (defective in deoxynucleoside monophosphate kinase and deoxycytidylate hydroxymethylase, respectively) are able to synthesize DNA in cell-free lysates prepared as described by Barry and Alberts (1972), in contrast to their inabliity to do so in plasmolyzed and toluenized cell systems. Addition of extracts containing an active gene 1 or 42 product has no effect on synthesis in lysates defective in the respective gene. Thus, if these enzymes do play additional direct roles in replication, these roles are not manifest in the lysed-cell system. The gene 42 mutant am N122/m, a double mutant bearing an additional defect in DNA polymerase, is unable to synthesize DNA in these lysates. This inability is overcome by addition of extracts containing an active T4 DNA polymerase. m is a leaky amber mutation which reduces DNA polymerase activity to a very low level. However, this level is high enough to allow positive genetic complementation tests with gene 43 mutants. Two other gene 42 amber mutants contain additional defects: am 269 induces only half the normal level of DNA polymerase, and am C87 fails to induce a detectable level of thymidylate synthetase. These defects do not result from pleiotropic effects of the gene 42 mutations. In plasmolyzed cells, temperature-sensitive gene 42 mutants fail to synthesize DNA under conditions where replication forks and 5-hydroxymethyl-dCTP are present. This supports the idea that the gene 42 protein is directly involved in DNA synthesis.     

Suppression of gene 49 mutations of bacteriophage T4 by a second mutation in gene X: structure of pseudorevertant DNA.

Mutations in gene 49 of bacteriophage T4 were suppressed by a second mutation in gene X. Mapping studies located gene X between genes 41 and 42. Complementation results indicated that mutations in FdsA gene (a suppressor of gene 49 mutants) were in gene X. The intracellular pseudorevertant DNA was examined for unusual properties which could explain its successful encapsidation. After the in vivo inactivation of a temperature-sensitive gene 32 (DNA unwinding) protein, the intracellular pseudorevertant DNA was converted into DNA pieces of approximately genome size. A similar conversion was observed after in vitro digestion of pseudorevertant DNA with single-strand-specific S1 endonuclease. Appreciable quantities of oligomeric intermediates were not produced during this conversion process. These data indicate that pseudorevertant DNA contains sizable single-stranded gaps and has a conformation similar to that of wild-type DNA. The results further suggest that the suppression of gene 49 mutant abnormal DNA phenotype and the encapsidation defect by a second mutation in gene X is associated with the formation of sizable single-stranded gaps. These studies raise the possibility that single-stranded gaps may be involved directly in the DNA encapsidation process, or may act indirectly as a consequence of their effect on the organization of intracellular DNA.