Wild-type bacteriophage T4 is restricted by the lambda rex genes.

The bacteriophage T4 rII genes and the lambda rex (r exclusion) genes interact; rII mutants are unable to productively infect rex+ lambda lysogens. The relationship between rex and rII has been found to be quantitative, and plasmid clones of rex have excluded not only rII mutants but T4 wild type and most other bacteriophages as well. Mutations in the T4 motA gene substantially reversed exclusion of T4 by rex.     

Properties of Bacteriophage T4 Mutants Defective in Gene 30 (Deoxyribonucleic Acid Ligase) and the rII Gene

In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.     

The isolation and characterization of TabR bacteria: Hosts that restrict bacteriophage T4 rII mutants

Summary We have isolated mutants of Escherichia coli B (called TabR) that restrict the growth of bacteriophage T4 rII mutants at high temperature. TabR strains lysed very rapidly after infection with rII mutants, and no progeny phage were produced. T4⁺-infected TabR cells also lysed quickly, but the cells remained intact long enough to give a small burst. We have selected pseudorevertants of rII deletion mutants that grow on TabR at high temperature; tk (thymidine kinase) is a component of one class of these pseudorevertants.T4 strains harboring mutations in genes 12, 16, 25, 34, 36, 45 and 63 were also specifically restricted on TabR strains at high temperature. Bacteriophages T2, T4, T5, T6, and T7 grew normally on TabR, while , 80, and P1 failed to grow at any temperature. The most restrictive TabR strains were auxotrophic for methionine at high temperature, and most spontaneous Met⁺ revertants had also lost the ability to restrict rII mutants, suggesting that the TabR phenotype and methionine auxotrophy result from the same mutation.Although the mechanism by which TabR strains exert their restriction has not been determined, one model is described. The potential uses of these and similar strains is discussed.     

Caffeine as a comutagen for ethylmethanesulfonate in strains of phage T4

Summary The mutation frequency of DNA polymerase mutants of phage T4 treated with ethyl methanesulfonate (EMS) then incubated in the presence and absence of caffeine was studied using an rII reversion system. The DNA polymerase mutation is shown to be antimutagenic for EMS induction of reversions which occur by a GC to AT transition. Caffeine acts as a comutagen for the induction by EMS of mutant phages and produces a significant increase in the frequency of reversions from rII to r⁺. Caffeine is slightly mutagenic for the phage strain carrying the wild type polymerase and inhibits the action of the 35 exonuclease function of T₄ DNA polymerase as measured in vitro. These findings suggest that caffeine acts by directly influencing nucleotide selection or the editing function of the DNA polymerase.     

Reversion of Frameshift Mutations Stimulated by Lesions in Early Function Genes of Bacteriophage T4

Temperature-sensitive (ts) mutants representative of a number of genes of phage T4 were crossed with rII mutants to allow isolation of ts, rII double-mutant recombinants. The rII mutations used were characterized as frameshift mutations primarily on the basis of their revertability by proflavine. For each ts, rII double mutant, the effect of the ts mutation on spontaneous reversion of the rII mutation was determined over a range of incubation temperatures. A strong enhancement in reversion of two different rII mutants was detected when they were combined with tsL56, a mutation in gene 43 [deoxyribonucleic acid (DNA) polymerase]. Three other mutants defective in gene 43 enhanced reversion about fourfold. Two mutations in gene 32, which specifies a protein necessary for DNA replication, enhanced reversion about 5-fold and 18-fold, respectively. Two additional mutations in gene 43 and two in gene 32 had no effect. Fivefold and threefold enhancements in reversion were also found with mutations in genes 44 (DNA synthesis) and 47 (deoxyribonuclease), respectively. No significant effect was found with mutations in seven additional genes. The results of other workers suggest that frameshift mutations arise from errors in strand alignment during repair synthesis occurring at chromosome tips. Our results show that such errors can be enhanced by mutations in the DNA polymerase, the gene 32 protein, and the enzymes specified by genes 44 and 47. This implies that these proteins are employed in the repair process occurring at chromosome tips and that mutational errors in these proteins can lead to loss of ability to recognize and reject strand misalignments.     

Point Mutants in the D2a Region of Bacteriophage T4 Fail to Induce T4 Endonuclease IV

We have studied the properties of presumptive point mutants in the D2a region of bacteriophage T4. Dominance tests showed that the D2a mutation was recessive to the wild-type allele. The mutations were shown to map in the D2a region by complementation against rII deletions. The D2a mutations were also located between gene 52 and rIIB by two- and three-factor crosses. The mutants are located at at least two distinct loci in the D2a region. The point mutants grow normally on all hosts tested and none of the mutants makes T4 endonuclease IV. We propose the name "denB" for the D2a locus.     

In vivo study of fidelity of DNA double-strand break repair in bacteriophage T4

A method for in vivo studying the fidelity of DNA double-strand break (DSB) repair in bacteriophage T4 has been developed. The frequency of reversion of rII mutations to the wild phenotype was measured in i segC+ x i ets 1 segCDelta crosses, where ets 1 is an insertion in the initial part of the rII gene carrying a sequence recognized by SegC endonuclease; i designates a rIIB or rIIA mutation located at some distance from ets 1, and segCDelta is a deletion in the segC gene. In such cross, a DSB occurs in the site of ets 1. Their repair involves genetic recombination and DNA replication in the neighborhood of ets 1. In parallel, the frequency of reversion of the same i mutant in the absence of DSBs is measured in i x i self-crosses. Reversions of different types (base substitutions, deletions, insertions) can be studied with the use of structurally different i mutations located at varying distances from ets 1. The reversion frequencies were determined for three rIIB mutations and one rIIA mutation. The results obtained suggest that DSB repair in bacteriophage T4 is a process of high fidelity with the rate of errors that does not essentially exceed that in the case of usual phage multiplication.     

Polynucleotide kinase mutant of bacteriophage T4

Summary We report the isolation and biological properties of two T4 phage mutants which are deficient in polynucleotide kinase. These two mutants are wild type with respect to replication, genetic recombination and DNA repair (as measured by sensitivity to UV irradiation, -irradiation and ³²P decay). These negative results suggest that this phage-induced enzyme is not a critical factor in DNA metabolism.     

Induction of mutation in phage T4 by extracellular treatment with methylene blue and visible light

Summary Photodynamic inactivation with methylene blue and light (MB+Li) of maximally sensitized phages T4 and T4rII nearly followed single-hit kinetics. Not so mutation induction, which in one phage (T4rII N₁₇) tested showed multi-hit kinetics. This difference and the fact that T4rII phages with a GC base pair at the site of the rII-mutation showed no enhanced backmutation when compared to rII phages with an AT base pair or to sign mutants suggests that there is no guanine specificity for MB+Li-induced mutation in phage T4.     

Functional Interactions between the DNA Ligase of ESCHERICHIA COLI and Components of the DNA Metabolic Apparatus of T4 Bacteriophage

T4 phage completely defective in both gene 30 (DNA ligase) and the rII gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rII (T4 30- rII- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rII- phage on ligase-defective hosts. One class mapped in T4 gene su30 (Krylov 1972) and improved T4 30- rII- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (helix-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the role of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.     

On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage

The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.     

Parent-to-Progeny Transfer and Recombination of T4rII Bacteriophage

Transfer of parental, light (not substituted with 5-bromodeoxyuridine) (32)P-deoxyribonucleic acid (DNA) from rII(-) mutants of T4 bacteriophage to heavy (5-bromodeoxyuridine-substituted) progeny in Escherichia coli B was less homogeneous than in wild phages. The net transfer was 5 to 20% of the value for wild T4 phage, and the parental contribution per progeny DNA molecule amounted to 7 to 100% of the genome. Three classes could be distinguished, based on the density distribution of parental label in CsCl analysis of the progeny phages. "Far recombined" phages contain parental material only in semiconservatively replicated subunits covalently attached to progeny DNA, amounting to 5 to 10% parental contribution per genome. "Intermediate recombinants" contain, aside from conventional recombinant DNA, parental DNA banding at the original, light density. This DNA may be unattached to heavy progeny DNA or attached by weak bonds which are very sensitive to shearing during the extraction procedure. The parental contribution is 10 to 50% per progeny DNA molecule in this class. "Conservative" phages band close to the parental, light density in CsCl; their DNA is purely light. When the parental phage is labeled with both (3)H-leucine (capsid) and (32)P (DNA), the specific activity of (3)H/(32)P in the "conservative progeny" is 10 to 40% of that in the parental, showing that at least some of the (32)P in this area belongs to phages with parental DNA as the sole DNA component inside an unlabeled capsid, i.e., parental DNA which has been injected into the host and matured in a new capsid without replication or recombination. This phenomenon occurs to about the same extent in both single and multiple infection.     

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.     

Bacteriophage T4 rnh (RNase H) Null Mutations: Effects on Spontaneous Mutation and Epistatic Interaction with rII Mutations

The bacteriophage T4 rnh gene encodes T4 RNase H, a relative of a family of flap endonucleases. T4 rnh null mutations reduce burst sizes, increase sensitivity to DNA damage, and increase the frequency of acriflavin resistance (Acr) mutations. Because mutations in the related Saccharomyces cerevisiae RAD27 gene display a remarkable duplication mutator phenotype, we further explored the impact of rnh mutations upon the mutation process. We observed that most Acr mutants in an rnh+ strain contain ac mutations, whereas only roughly half of the Acr mutants detected in an rnhDelta strain bear ac mutations. In contrast to the mutational specificity displayed by most mutators, the DNA alterations of ac mutations arising in rnhDelta and rnh+ backgrounds are indistinguishable. Thus, the increase in Acr mutants in an rnhDelta background is probably not due to a mutator effect. This conclusion is supported by the lack of increase in the frequency of rI mutations in an rnhDelta background. In a screen that detects mutations at both the rI locus and the much larger rII locus, the r frequency was severalfold lower in an rnhDelta background. This decrease was due to the phenotype of rnh rII double mutants, which display an r+ plaque morphology but retain the characteristic inability of rII mutants to grow on lambda lysogens. Finally, we summarize those aspects of T4 forward-mutation systems which are relevant to optimal choices for investigating quantitative and qualitative aspects of the mutation process.     

Nonreplicated DNA and DNA Fragments in T4 r? Bacteriophage Particles: Phenotypic Mixing of a Phage Protein

"Conservative phage" containing a genome derived from an infecting phage particle which has not undergone replication in the cell but nevertheless has become encapsulated and released in a normal phage particle, are found after infection of Escherichia coli with rII(-) or rI(-) mutants under conditions which result in rapid lysis. If such conservative phage are derived from a mixed infection with v(+) and v(1) phage, they display phenotypic mixing of the v gene product (an endonuclease carried in the phage particle). Populations of rI and rII mutant phage grown under conditions of rapid lysis include particles containing short DNA fragments. It is suggested that a "maturation defect", common to rI and rII mutants, but absent in rIII mutants, may account for the encapsulation of nonreplicated DNA as well as that of the DNA fragments.     

Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro

DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.     

Physiological and Genetic Aspects of Abortive Infection of a Shigella sonnei Strain by Coliphage T7

Phage T7 adsorbed to and lysed cells of Shigella sonnei D(2) 371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (ss(-) mutants) have been isolated which show normal growth in strain D(2) 371-48. Upon mixed infection of this strain with T7 wild type and an ss(-) mutant, infection was abortive; no complementation occurred. The DNA of the ss(-) mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D(2) 371-48 were isolated from ss(-) mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of ss(-) mutants did not produce ss(-) recombinants when they were crossed among themselves. When independently isolated ss(-) mutants were crossed with each other, wild-type recombinants were found; ss(-) mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [ss(+) function (for suicide in Shigella)] which leads to the breakdown of phage DNA in the Shigella host.