Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.     

Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light.

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.     

Multiplicity reactivation of bacteriophage T7 inactivated by methyl methanesulfonate

Experiments were conducted to determine whether phage T7 treated with methyl methanesulfonate used multiplicity reactivation to repair alkylation lesions. This type of repair was found to be operative at high multiplicities in actively growing wild-type Escherichia coli B cells.     

Repair of topoisomerase-mediated DNA damage in bacteriophage T4

Type II topoisomerase inhibitors are used to treat both tumors and bacterial infections. These inhibitors stabilize covalent DNA-topoisomerase cleavage complexes that ultimately cause lethal DNA damage. A functional recombinational repair apparatus decreases sensitivity to these drugs, suggesting that topoisomerase-mediated DNA damage is amenable to such repair. Using a bacteriophage T4 model system, we have developed a novel in vivo plasmid-based assay that allows physical analysis of the repair products from one particular topoisomerase cleavage site. We show that the antitumor agent 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) stabilizes the T4 type II topoisomerase at the strong topoisomerase cleavage site on the plasmid, thereby stimulating recombinational repair. The resulting m-AMSA-dependent repair products do not form in the absence of functional topoisomerase and appear at lower drug concentrations with a drug-hypersensitive topoisomerase mutant. The appearance of repair products requires that the plasmid contain a T4 origin of replication. Finally, genetic analyses demonstrate that repair product formation is absolutely dependent on genes 32 and 46, largely dependent on genes uvsX and uvsY, and only partly dependent on gene 49. Very similar genetic requirements are observed for repair of endonuclease-generated double-strand breaks, suggesting mechanistic similarity between the two repair pathways.     

Recognition of chemically damaged DNA by the gene 32 protein from bacteriophage T4

We have used fluorescence spectroscopy to investigate the binding of gene 32 protein from bacteriophage T4 to DNA which has been chemically modified with carcinogens or antitumor drugs. This protein exhibits a high specificity for single-stranded nucleic acids and binds more efficiently to DNA modified either with cis-diaminodichloroplatinum(II) or with aminofluorene derivatives than to native DNA. This increased affinity is related to the formation of locally unpaired regions which are strong binding sites for the single-strand binding protein. In contrast, gene 32 protein has the same affinity for native DNA, DNA containing methylated purines and DNA that has reacted with trans-diaminodichloroplatinum(II) or with chlorodiethylenetriaminoplatinum(II) chloride. These types of damage do not induce a sufficient structural change to allow gene 32 protein binding. Depurination of DNA does not create binding sites for the T4 gene 32 protein but nicked apurinic sites are strong ligands for the protein. This T4 single-strand binding protein does not exhibit a significantly increased affinity for nicked DNA as compared with native DNA. These results are discussed with respect to the recognition of DNA damage by proteins involved in DNA repair and to the possible role of single-strand binding proteins in DNA repair mechanisms.     

In vitro recombination of bacteriophage T7 DNA damaged by UV radiation.

A system capable of in vitro packaging of exogenous bacteriophage T7 DNA has been used to monitor the biological activity of DNA replicated in vitro. This system has been used to follow the effects of UV radiation on in vitro replication and recombination. During the in vitro replication process, a considerable exchange of genetic information occurs between T7 DNA molecules present in the reaction mixture. This in vitro recombination is reflected in the genotype of the T7 phage produced after in vitro encapsulation; depending on the genetic markers selected, recombinants can comprise nearly 20% of the total phage production. When UV-irradiated DNA is incubated in this system, the amount of in vitro synthesis is reduced and the total amount of viable phage produced after in vitro packaging is diminished. In vitro recombination rates are also lower when the participating DNA molecules have been exposed to UV. However, biochemical and genetic measurements confirmed that there is little or no transfer of pyrimidine dimers from irradiated DNA into undamaged molecules.     

Clone size distributions of mutations induced by ethyl methanesulfonate in bacteriophage T4

Size distributions of mutant clones can reveal important aspects of the mutation process. Previously published data on mutant clones induced by ethyl methanesulfonate (EMS) in bacteriophage T4 generated a distribution that was essentially flat, implying a mutagenic mechanism involving only rare mispairing by reacted bases. Here, methods for estimating the spontaneous component of such a distribution are used to generate a corrected distribution. The corrected distribution is strongly peaked, implying frequent (but not obligatory) mispairing. Frequent mispairing is in accord with current views of the fates of DNA lesions believed to mediate EMS-induced mutagenesis.     

In vitro replication of bacteriophage T7 DNA damaged by ultraviolet radiation

The effect of ultraviolet radiation on DNA replication has been examined with an in vitro system capable of replicating intact chromosomes of T7 DNA from an exogenous template. Exposure of the template DNA to ultraviolet radiation resulted in a sharp drop in the amount of in vitro DNA synthesis. The residual replication detected when irradiated templates were used was found to proceed semiconservatively and to result in the production of pieces of duplex DNA approximately the same size as the average distance between pyrimidine dimers. It was also found that prior irradiation of the template inhibits formation of fast-sedimenting concatemer-like DNA structures normally synthesized in vitro. Hybridization studies demonstrated that the product synthesized in vitro from ultraviolet-irradiated templates includes DNA from both the left and right halves of the T7 chromosome. This may mean that after ultraviolet irradiation more than one origin of replication exists.     

Inhibition of DNA polymerase activity by methyl methanesulfonate

Methyl methanesulfonate (MMS) inhibits both thymidine incorporation into DNA in mitogen-activated human lymphocytes and deoxythymidine triphosphate incorporation into template DNA by DNA polymerase-alpha in a cell-free system. When MMS-modified DNA was used as the template for DNA synthesis utilizing unmodified DNA polymerase-alpha, nucleotide incorporation into template DNA was not inhibited. When unmodified DNA was used as the template for DNA synthesis utilizing MMS-modified DNA polymerase-alpha, nucleotide incorporation was differentially inhibited dependent on the MMS concentration. An analysis of the kinetics of DNA polymerase-alpha inhibition showed that incorporation of all 4 deoxynucleoside triphosphates into DNA template was noncompetitively inhibited by MMS, which is consistent with nonspecific MMS modification of the enzyme. These data indicate that MMS modification of DNA polymerase-alpha alone is sufficient to inhibit the incorporation of deoxynucleoside triphosphates into template DNA in vitro. The data further indicate that alkylation of both DNA polymerase-alpha and DNA template synergistically increases inhibition of DNA synthesis.     

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.     

Repair of benzo[a]pyrene diol epoxide damaged bacteriophage T7 DNA determined by survival of phage made by in vitro packaging

DNA from bacteriophage T7 was treated with benzo[a]pyrene diol epoxide (BPDE) and the number of covalently bound adducts per T7 genome was determined. BPDE treated T7 DNA was then incubated in an in vitro DNA packaging system so as to form infective T7 phage. The observed reduced survival of these phage measured with Escherichia coli uvrA- indicator bacteria showed that the BPDE treated DNA was in fact utilized by the in vitro packaging system and that the resulting phage contained DNA damage caused by in vitro exposure to BPDE. T7 DNA damage by BPDE was also incubated in an in vitro DNA repair system that used partially purified uvrABC proteins from E. coli. Alkaline sucrose gradient analysis demonstrated that nicks were introduced into the damaged DNA and that these incisions were repaired to yield nearly intact DNA molecules of about the size of a T7 genome. Encapsulation of the repaired DNA with the packaging system yielded phage that showed higher survival than the unrepaired control when plated on uvrA- indicator bacteria.     

Properties of condensed bacteriophage T4 DNA isolated from Escherichia coli infected with bacteriophage T4.

Methods developed for isolating bacterial nucleoids were applied to bacteria infected with phage T4. The replicating pool of T4 DNA was isolated as a particle composed of condensed T4 DNA and certain RNA and protein components of the cell. The particles have a narrow sedimentation profile (weight-average s=2,500S) and have, on average, a T4 DNA content similar to that of the infected cell. Their dimensions observed via electron and fluorescence microscopy are similar to the dimensions of the intracellular DNA pool. The DNA packaging density is less than that of the isolated bacterial nucleoid but appears to be roughly similar to its state in vivo. Host-cell proteins and T4-specific proteins bound to the DNA were characterized by electrophoresis on polyacrylamide gels. The major host proteins are the RNA polymerase subunits and two envelope proteins (molecular weights, 36,000 and 31,000). Other major proteins of the host cell were absent or barely detectable. Single-strand breaks can be introduced into the DNA with gamma radiation or DNase without affecting its sedimentation rate. This and other studies of the effects of intercalated ethidium molecules have suggested that the average superhelical density of the condensed DNA is small. However, these studies also indicated that there may be a few domains in the DNA that become positively supercoiled in the presence of high concentrations of ethidium bromide. In contrast to the Escherichia coli nucleoid, the T4 DNA structure remains condensed after the RNA and protein components have been removed (although there may be slight relaxation in the state of condensation under these conditions).     

Repair of damaged DNA by Arabidopsis cell extract

All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light- and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue-induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

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.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

9-Aminoacridine mutagenesis of bacteriophage T4 intracellular DNA.

Most of the intracellular T4 DNA made in the presence of 9-aminoacridine is of lower molecular weight than mature T4 DNA and does not get packaged into phage particles. Using a T4 DNA transformation assay, we have examined this intracellular T4 DNA for its content of 9-aminoacridine-induced revertants of certain rII gene frameshift mutations. The proportion of acridine-induced revertants in the intracellular DNA population is close to that found in the phage progency made in the presence of 9-aminoacridine. Thus, the generation of low molecular weight T4 DNA in the presence of 9-aminoacridine is not, in itself, also a mutagenic process.     

Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase

The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.