DNA injection and genetic recombination of alkylated bacteriophage T7 in the presence of nalidixic acid.

Marker rescue experiments with alkylated T7 bacteriophage carried out in the presence and in the absence of nalidixic acid suggest that the gradient in rescue is due to two alkylation-induced causes: a DNA injection defect and an interference with DNA synthesis.     

Difference in the action of ethyl and methyl methane sulfonates on DNA template activity for RNA synthesis in vitro.

RNA produced in vitro from alkylated T7 DNA has been characterized by polyacrylamide gel electrophoresis. Methylation of T7 DNA by methyl methane sulfonate reduces RNA chain length. In contrast, ethylation of T7 DNA by ethyl methane sulfonate, while reducing RNA synthesis to the same extent, does not alter chain length.     

Effect of DNA delay mutations of bacteriophage T4 on genetic recombination.

Studies have been made of the effect of the DNA delay mutations of bacteriophage T4 on growth and genetic recombination in a number of Escherichia coli hosts. DNA delay mutations in genes 39, 52, 58 (61), and 60 result in abnormally high recombination frequencies. These high recombination frequencies are discussed in the context of other observations.     

Defective DNA injection by alkylated and nonalkylated bacteriophage T7.

DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA. Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells. This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome. An essentially identical injection defect was observed when alkylated phage infected E. coli mutant cells unable to repair 3-methyladenine. Furthermore, untreated phage particles were discovered to be naturally injection-defective. Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all. In the presence of rifampicin, untreated phages injected only segments from the left end of the genome. These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome. The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E. coli cells. Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.     

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.     

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.     

Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

When Escherichia coli cells were infected with ³²P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental.To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the ³²P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated.In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the ³²P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear ³²P/BrdUrd-labeled DNA shifted the density of [³²P]DNA to almost fully light density. It was also found that approximately 50% of [³²P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.     

Deletion mutagenesis independent of recombination in bacteriophage T7.

Deletion between directly repeated DNA sequences in bacteriophage T7-infected Escherichia coli was examined. The phage ligase gene was interrupted by insertion of synthetic DNA designed so that the inserts were bracketed by 10-bp direct repeats. Deletion between the direct repeats eliminated the insert and restored the ability of the phage to make its own ligase. The deletion frequency of inserts of 85 bp or less was of the order of 10(-6) deletions per replication. The deletion frequency dropped sharply in the range between 85 and 94 bp and then decreased at a much lower rate over the range from 94 to 900 bp. To see whether a deletion was predominantly caused by intermolecular recombination between the leftmost direct repeat on one chromosome and the rightmost direct repeat on a distinct chromosome, genetic markers were introduced to the left and right of the insert in the ligase gene. Short deletions of 29 bp and longer deletions of approximately 350 bp were examined in this way. Phage which underwent deletion between the direct repeats had the same frequency of recombination between the left and right flanking markers as was found in controls in which no deletion events took place. These data argue against intermolecular recombination between direct repeats as a major factor in deletion in T7-infected E. coli.     

In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA.

A double-strand break in a bacteriophage T7 genome significantly reduced the ability of that DNA to produce viable phage when the DNA was incubated in an in vitro DNA replication and packaging system. When a homologous piece of T7 DNA (either a restriction fragment or T7 DNA cloned into a plasmid) that was by itself unable to form a complete phage was included in the reaction, the break was repaired to the extent that many more viable phage were produced. Moreover, repair could be completed even when a gap of about 900 nucleotides was put in the genome by two nearby restriction cuts. The repair was accompanied by acquisition of a genetic marker that was present only on the restriction fragment or on the T7 DNA cloned into a plasmid. These data are interpreted in light of the double-strand gap repair mode of recombination.     

Early intermediates in bacteriophage T4 DNA replication and recombination.

We investigated, by density gradients and subsequent electron microscopy, vegetative T4 DNA after single or multiple infection of Escherichia coli with wild-type T4. Our results can be summarized as follows. (i) After single infection (i.e., when early intermolecular recombination could not occur), most, if not all, T4 DNA molecules initiated the first round of replication with a single loop. (ii) After multiple infection, recombinational intermediates containing label from both parents first appeared as early as 1 min after the onset of replication, long before all parental DNA molecules had finished their first round and before secondary replication was detectable. (iii) At the same time, in multiple infections only, complex, highly branched concatemeric T4 DNA first appeared. (iv) Molecules in which two loops or several branches were arranged in tandem were only found after multiple infections. (v) Secondary loops within primary loops were seen after both single and multiple infections, but they were rare and many appeared off center. Thus, recombination in wild-type T4-infected cells occurred very early, and the generation of multiple tandem loops or branches in vegetative T4 DNA depended on recombination. These results are consistent with the previous finding (A. Luder and G. Mosig, Proc. Natl. Acad. Sci. U.S.A. 79:1101-1105, 1982) that most secondary growing points of T4 are not initiated from origin sequences but from recombinational intermediates. By these and previous results, the various DNA molecules that we observed are most readily explained as intermediates in DNA replication and recombination according to a model proposed earlier to explain various other aspects of T4 DNA metabolism (Mosig et al., p. 277-295, in D. Ray, ed., The Initiation of DNA Replication, Academic Press, Inc., New York, 1981).     

Gene rIIB from bacteriophage T4 in genetic recombination

The effect of the rIIB gene on genetic recombination in bacteriophage T4 was studied. Relationships between recombination frequency and the physical distance were determined in three series of isomarker two-factor crosses between rII mutants. In the first series of intergenic crosses (rIIa x rIIb), the rII gene function was restored owing to complementation. In the second series of crosses, identical to the first one, the rIIB gene function was suppressed, because the rIIa parent carried an additional amberlike mutation in the rIIB gene. The recombinants were scored by plating lysates on the amber-suppressor Escherichia coli strain, on which an amberlike mutation was not expressed phenotypically. In the third series, all crosses were intragenic (rIIb x rIIb). In two series of crosses in the absence of the rIIB function, the relationships between recombination frequency and the physical distance were identical, whereas enhanced recombination frequencies were observed in the rIIB+ background. The magnitude of the rIIB-related effect depended on distance, reaching the maximum in the region located 100 to 200 bp from the beginning of the rIIB gene. The possible role of the rIIB protein in genetic recombination is discussed.     

Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.     

In vitro recombination of bacteriophage T7 DNA detected by a direct physical assay

We developed a simple, direct, physical assay to detect genetic recombination of bacteriophage T7 DNA in vitro. In this assay two mature T7 DNA molecules, each having a unique restriction enzyme site, are incubated in the presence of a cell-free extract from T7-infected Escherichia coli cells. After extraction of the DNA, restriction enzyme digestion, and agarose gel electrophoresis, genetic recombination is detected by the appearance of a novel recombinant DNA band. Recombination frequencies as high as 13% have been observed. We used this assay to determine the genetic requirements for in vitro recombination. In agreement with results obtained previously with a biological assay, T7 recombination in vitro appears to proceed via two distinct pathways.     

Double-strand breaks increase the incidence of genetic deletion associated with intermolecular recombination in bacteriophage T7

An in vitro DNA replication system based on extracts prepared from Escherichia coli cells infected with bacteriophage T7 was used to study deletion associated with the repair of double-strand breaks. The gene for T7 ligase was interrupted by a DNA insert which included 17-bp direct repeats. Deletion between the repeats restored the reading frame of the gene, and these DNA molecules could be detected by their ability to give rise to ligase-positive phage after in vitro packaging. T7 genomes that had a pre-existing double-strand break located between the direct repeats were incubated together with intact genomes which had the same direct repeats. Genetic markers placed on either side of the insert in the ligase gene allowed identification of the source of DNA molecules that underwent deletion between the direct repeats. This allowed an assessment of the participation of the molecules with strand breaks in the deletion process, under conditions where any mechanism could contribute to deletion. Approximately three-quarters of the T7 molecules that had lost the region between the direct repeats contained one or both of the partial genomes originally introduced into the reactions. About 50% of the genomes which had undergone deletion had recombined markers between the partial and intact genomes. The data demonstrate that double-strand breaks substantially enhance the contribution of intermolecular recombination to deletion. Received: 19 November 1996 / Accepted: 26 February 1997     

Involvement of DNA gyrase in bacteriophage T7 growth.

We have found that the burst size of bacteriophage T7 was decreased in two Escherichia coli temperature-sensitive gyrase mutants incubated at the restrictive temperature. This reduction in burst size indicates that gyrase may be required for T7 growth.     

Intracellular organization of bacteriophage T7 DNA: analysis of parenteral bacteriophage T7 DNA-membrane and DNA-protein complexes.

After infection of Escherichia coli with bacteriophage T7, the parenteral DNA forms a stable association with host cell membranes. The DNA-membrane complex isolated in cesium chloride gradients is free of host DNA and the bulk of T7 RNA. The complex purified through two cesium chloride gradients contains a reproducible set of proteins which are enriched in polypeptides having molecular weights of 54,000, 34,000, and 32,000. All proteins present in the complex are derived from host membranes. Treatment of the complex with Bruij-58 removes 95% of the membrane lipid and selectively releases certain protein components. The Brij-treated complex has an S value of about 1,000 and the sedimentation rate of this material is not altered by treatment with Pronase or RNase.     

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.