On the repair mechanism responsible for multiplicity reactivation in bacteriophage T4

Summary Multiplicity reactivation has been studied using two T4 phages carrying a large number, twenty six, of genetic markers covering the whole viral genome. It has been found that in the progeny of irradiated phages the observed increase of recombination frequencies is not limited to the vicinity of radiation-induced lethal lesions, it can occur all along the phage genome. Most of the single bursts produced by multiplicity reactivation contain phages of entirely parental genotype. This fact may reveal the existence of repair by recombination between replication products of a single viral genome.     

Absence of interparental recombination in multiplicity reconstitution from incomplete bacteriophage T4 genomes.

Interparental recombination between injected T4 DNA molecules is indetectable for incomplete petite phages (carrying a terminally deficient genome and therefore unable to circularize) as well as for genetically complete phages. The nonvialbe petite phages can individually replicate their DNA repeatedly, and they aso undergo multiplicity reconstitution, producing complete phages, provided that a host bacterium is infected by several petite particles that carry genetically complementary segments of DNA. The formation of complete phages in multiplicity reconstitution must be due to recombination among incomplete progeny fragments, i.e., partial replicas of the T4 genomes. It evidently does not result from interparental recombination. To test for interparental recombination, light bacteria (containing no bromouracil) were simultaneously infected in light medium with light radioactive phage in minority (usually less than one per cell) and heavy (bromouracil-labeled) phage in majority (usually about nine per cell). Any interparental recombination should, under these circumstances of infection, head to movement of the radioactive label of the minority light phage DNA to a position of higher density. That possibility was not observed.     

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.     

Excision repair and patch size in UV-irradiated bacteriophage T4.

We determined the average size of excision repair patches in repair of UV lesions in bacteriophage T4 by measuring the photolysis of bromodeoxyuridine incorporated during repair. The average patch was small, approximately four nucleotides long. In control experiments with the denV1 excision-deficient mutant, we encountered an artifact, a protein(s) which remained bound to phenol-extracted DNA and prevented nicking by the UV-specific endonucleases of Micrococcus luteus and bacteriophage T4.     

Gene 49 endonuclease VII is not essential for multiplicity reactivation of bacteriophage T4

Summary Endonuclease VII, the product of phage T4 gene 49, has been shown previously to resolve Holliday structures in vitro. Two different processes, genetic recombination and multiplicity reactivation are presumed to have Holliday structure intermediates. Other workers have shown that genetic recombination is reduced in a gene 49 mutant infection. However, in the present study, multiplicity reactivation of UV-irradiated ts or amber mutant phage defective in gene 49 was nearly identical to that of UV-irradiated wild-type phage T4. Thus endonuclease VII is not thought to be essential for multiplicity reactivation of phage T4.     

T 4 -mediated v-gene reactivation of UV-irradiated phage Ti, and its comparison with host-cell reactivation

Soaked seeds of Vicia faba were irradiated with 800 or 400 R, or two 400-R exposures, both with VAC (vacuum) but one with a concomitant treatment of CHM (cycloheximide) (I μg/ml). The chromosome aberration yields from each regimen varied with fixation time such that a unique and characteristic aberration yield for each regimen relative to the others was not obtained. Were single fixations employed one could obtain yields which would indicate no, some, or maximum repair. A single fixation would lead to an incorrect estimation of chromosome damage repair.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

Multiplicity reactivation and repair of nitrous acid-induced lesions in bacteriophage T4

Nitrous acid-induced lesions in phage T4 were shown to be efficiently repaired by multiplicity reactivation. Mutants defective in genes 32, 46, 47, x and y showed substantially less MR ² of these lesions than wild type. The gene 47 mutant, which showed the least MR of nitrous acid lesions, also showed virtually no MR of ultraviolet lesions. Mutations in genes 30, 44 and v did not affect MR of nitrous acid-induced lesions. Each of the mutations which lowered MR was shown previously to reduce recombination. Our results suggest that the gene functions employed in MR are the same functions used in genetic recombination, and, based on this, we propose a tentative recombinational model for MR.The mutants defective in genes 32, 46 and 47, which are deficient in recombination, were shown to be more sensitive to nitrous acid inactivation than wild-type phage upon single infection. Our results indicate that, in wild-type single infections, about 20% of nitrous acid-induced lethal lesions may be repaired by a recombinational post-replication form of repair.     

Partial exclusion of genes of bacteriophage T2 with T4-glucosylated DNA in crosses with bacteriophage T4

A recombinant strain (D41) between phage T2 and T4 was isolated which possessed the T2 region of the genome between genes 32 and 39 and both the T4 genesgt ⁺ andgt ⁺ for glucosyltransferase. D41 was crossed with T4amber mutants in the genes for early functions and in some genes for late funcitions. The progeny of the crosses was examined for the frequency of theam ⁺ markers from D41. Genes 32, 60 and 39 in the T2 region of the recombinant strain were as sensitive to exclusion as those in standard-type T2. The T4 glucosylation of the DNA of these T2 genes did not protect them against partial exclusion by T4. However, genes in the region from gene 56 to 55 in the recombinant were resistent to exclusion. In standard-type T2 this region of the genome is sensitive to partial exclusion by T4. There are at least four exclusion sensitive sites in T2: one near gene 32, one near gene 60, one linked to gene 56 and one between genes 42 and 55.This investigation was carried out partially within the frame of the Association between Euratom and the University of Leiden, contract nr. 052-64-1-BIAN.     

Role of gene 59 of bacteriophage T4 in repair of UV-irradiated and alkylated DNA in vivo.

Nonsense mutants in gene 59 (amC5, amHL628) were used to study the role of this gene in the repair of UV-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to UV irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after UV irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to UV irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that UV repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B (su minus), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of UV lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules.     

Solution structure of bacteriophage T4D and icosahedral capsid geometry visualized in freeze-fractured, deep-etched replicas

The prolate icosahedral capsid geometry of wild type bacteriophage T4D has been determined by direct visualization of the triangular faces in stereoimages of transmission electron micrographs of phage particles. Bacteriophage T4 was prepared for transmission electron microscopy (TEM) following a protocol of freeze-fracturing, deep-etching (FDET) and replication by vertical deposition (80 degrees angle) of a thin platinum-carbon (Pt-C) metal layer of 1.01 nm. From direct statistical measurements of the ratio of the head length to width and of stereometric angles on T4 heads, we have estimated a Q number of 21. This confirms previous indirect studies on T4 and agrees with determinations on bacteriophage T2. Many of the structural features of T4 observed in FDET preparations differ significantly from those observed by classical negative staining methods for TEM imaging. Most important among the differences are the conformation of the baseplate (a closed rosebud) and the positioning of the tail fibers (retracted). The retracted position of the tail fibers in the FDET preparations has been confirmed by negatively staining phage previously fixed suspended in solution with 2% glutaraldehyde. The FDET protocols appear to reveal important structural features not seen in negative stained preparations. These have implications for bacteriophage T4 conformation in solution, viral assembly and phage conformation states prior to tail contraction and DNA ejection.     

On the lack of host-cell reactivation of UV-irradiated phage T5. I. Interference of T5 infection with the host-cell reactivation of phage T1

UV-irradiated phage T5, in contrast to T1, T3 and T7, fail to display hostcell reactivation (HCR) when infecting excision-repair proficient Escherichia coli cells. Possible causes of this lack of HCR (which T5 shares with the T-even phages) have been investigated by studying HCR of T1 under conditions of superinfection by T5. Repair-proficient B/r cells were infected at low multiplicity with UV-irradiated phage T1 in the presence of 1.8 mg/ml caffeine and were superinfected after 15 min with heavily UV-irradiated T5 amber mutants at high multiplicity. The caffeine, which is later diluted out, prevents any T1 repair prior to T5 superinfection, and UV (254 nm) irradiation of T5 with 144 J/m² reduces the ability of this phage to exclude T1, thus permitting a reasonable fraction of the mixedly infected complexes to produce T1 progeny.Under these conditions, T5 superinfection causes loss of HCR in about 90% of the T1-producing complexes. Superinfection with unirradiated T5 likewise inhibits HCR of T1, but superinfection with irradiated T3 (a host-cell-reactivable phage) does not. This indicates that the observed HCR inhibition of T1 results from T5 infection rather than from competition of irradiated foreign DNA for the excision-repair enzymes of the bacterial host. Employment of apropriate T5 amber mutants has shown that “first-step transfer” (FST) of T5 DNA (involving only 8% of the T5 genome) is sufficient for HCR inhibition, but that transfer of the remainder DNA in addition inhibits a previously described minor T1 recovery process. HCR inhibition of T1, and thus presumably lack of HCR in T5 itself, is ascribed to a substance which is produced either post infection by a gene located in the FST segment of the T5 genome, or which is transferred from extracellular T5 together with the FST DNA.     

Inducible reactivation and mutagenesis of UV-irradiated bacteriophage P22 in Salmonella typhimurium LT2 containing the plasmid pKM101.

The inducible (Weigle) reactivation of UV-irradiated bacteriophage P22 has been examined on strains of Salmonella typhimurium with and without the mutagenesis-enhancing plasmid pKM101. A large inducible reactivation was observed in the plasmid-containing strain, but only a small response was observed in the strain lacking the plasmid. An increased frequency of clear-plaque mutants was detected among the survivors. The efficiencies of the plasmid-mediated and cellular repair processes have been determined. The kinetics of induction of the phage reactivation have been investigated. The relationship of the observed results to the inducible reactivation of UV-irradiated lambda in Escherichia coli and to error-prone repair is discussed.     

Processing of bacteriophage T4 tRNAs. The role of RNAase III

In order to assess the contribution of the processing enzyme RNAase III to the maturation of bacteriophage T4 transfer RNA, RNAase III⁺ and RNAase III^? strains were infected with T4 and the tRNAs produced were analyzed. Infection of the RNAase III⁺ strains of Escherichia coli with T4Δ27, a deletion strain missing seven of the ten genes in the T4 tRNA cluster, results in the appearance of a transient 10.1 S RNA molecule as well as the three stable RNAs encoded by T4Δ27, species 1, rRNA^Leu and tRNA^Gln. Infection of an RNAase III^? strain results in the appearance of a larger, transient RNA molecule, 10.5 S, and a severe reduction in the accumulation of tRNA^Gln. The 10.5 S RNA is similar to 10.1 S RNA but contains extra nucleotides (about 50) at the 5′ end. (10.1 S contains all the three final molecules plus about 70 extra nucleotides at the 3′ end.) Both 10.5 S and 10.1 S RNAs can be processed in vitro into the three final molecules. When 10.1 S is the substrate, the three final molecules are obtained whether extracts of RNAase III⁺ or RNAase III^? cells are used. However, when 10.5 S is the substrate RNAase III⁺ extracts bring out normal maturation, while using RNAase III^? extracts the level of tRNA^Gln is severely reduced. When 10.5 S is used with RNAase III⁺ extracts maturation proceeds via 10.1 S RNA, while when RNAase III^? extracts were used 10.1 S is not detected. The 10.5 S RNA can be converted to 10.1 S RNA by RNAase III in a reaction which produces only two fragments. The sequence at the 5′ end of the 10.5 S suggests a secondary structure in which the RNAase III cleavage site is in a stem. These experiments show that the endonucleolytic RNA processing enzyme RNAase III is required for processing at the 5′ end of the T4 tRNA cluster where it introduces a cleavage six nucleotides proximal to the first tRNA, tRNA^Gln, in the cluster.     

The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA.

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA $\text{in vitro}$ and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several $\text{E}$. $\text{coli}$ ribonucleases could serve as a messenger ribonuclease $\text{in vivo}$.     

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.