Mutator Mutations in Bacteriophage T4 Gene 42 (Dhmc Hydroxymethylase)

Temperature-sensitive mutations of bacteriophage T4 gene 42 produce diverse effects upon spontaneous mutation rates. G:C → A:T transition rates are increased, often strongly; frameshift mutation rates are weakly increased; A:T → G:C transition rates (and perhaps also A:T → Py:Pu transversion rates) are decreased; and one G:C → Py:Pu transversion rate is also decreased. These results, together with certain interactions between gene-42 mutator effects and both base analogue mutagenesis and the viral error-prone repair system, suggest that the dHMC hydroxymethylase coded by gene 42 affects mutation rates in a more complex manner than by the simple regulation of the concentration of the DNA precursor dHMCTP.     

Ultraviolet mutagenesis in bacteriophage T-4. I. Irradiation of extracellular phage particles

Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. I. Irradiation of extracellular phage particles. J. Bacteriol. 91:1775-1780. 1966.-Ultraviolet (UV) irradiation of extracellular T4 phage particles induces about 2 x 10(-4)r mutations per lethal hit. The mutants largely escape detection unless the irradiated phages are plated with very soft overlay agar. Multiplicity reactivation is not a prerequisite for mutagenesis. A much higher frequency of base pair substitution-type mutants is induced than is found in the spontaneous background, but sign mutants are also induced. Nearly half of the mutants map into previously identified UV hot spots. The rII mutants induced extracellularly are very similar to those induced intracellularly. The mutants also appear to result from direct radiation effects upon the bacteriophage deoxyribonucleic acid.     

Photodynamic inactivation and mutagenesis by angelicin (isopsoralen) or thiopyronin (methylene red) in wild-type and repair-deficient strains of bacteriophage T4.

Photodynamic inactivation of bacteriophage T4 particles, mediated by either angelicin or thiopyronin, is enhanced by defects in the T4 uvsW-uvsX-uvsY postreplication repair system but not by a defect in the denV pyrimidine-dimer-excision system. There was no evidence for functional interactions between the two repair systems. As observed previously with 8-methoxypsoralen, photodynamic mutagenesis with angelicin is abolished by defects in the uvsW-uvsX-uvsY system.     

The Specificity of Topoisomerase-Mediated DNA Cleavage Defines Acridine-Induced Frameshift Specificity within a Hotspot in Bacteriophage T4

Acridine-induced frameshift mutations in bacteriophage T4 occur at the precise location in the DNA at which acridines stimulate DNA cleavage by the T4-encoded type II topoisomerase in vitro. The mutations are duplications or deletions that begin precisely at the broken phosphodiester bond. In vivo, acridine-induced frameshift mutagenesis is reduced nearly to background levels when the topoisomerase is genetically inactivated. These observations are consistent with a model in which cleaved DNA, induced by the topoisomerase and acridine, serves as the substrate for the production of frameshift mutations at the same site. Our model predicts that the specificity and frequency of cleavage direct the specificity and frequency of mutagenesis. This prediction was tested by examining the influence of DNA sequence changes on topoisomerase-mediated cleavage and on mutagenesis in the T4 rIIB gene. The model successfully predicted the results. When DNA sequence changes altered the position of acridine-induced, topoisomerase-mediated DNA cleavage in vitro, frameshift mutations were found at the new positions. DNA sequence changes that strongly decreased in vitro cleavage also reduced mutagenesis at that site. These results demonstrate that acridine-induced frameshift mutation specificity is directed by the characteristics of the acridine-topoisomerase reaction and do not suggest that slipped pairing in repeated sequences plays a major role in acridine-induced frameshifts in bacteriophage T4.     

Ligase-Defective Bacteriophage T4 I. Effects on Mutation Rates

Temperature-sensitive mutations in bacteriophage T4 gene 30 (polynucleotide ligase) were examined for their effects on spontaneous and proflavine-induced frameshift mutagenesis in the rII and ac (acridine resistance) cistrons. Only small (fourfold or less) effects on mutation rates were observed, even when selection artifacts involving suppression of gene 30 mutations by rII mutations were taken into account. The deoxyribonucleic acid ligase gene of T4 therefore appears to be only a minor determinant of frameshift mutation rates. This result is consistent with the particular nature of frameshift mutagenesis in bacteriophage T4.     

Molecular modification of T4 bacteriophage proteins and its potential application — <Emphasis Type="Italic">Review</Emphasis>

Bacteriophage T4 is a virus with well-known genetics, structure, and biology. Such techniques as X-ray crystallography, cryo-EM, and three-dimensional (3D) image reconstruction allowed describing its structure very precisely. The genome of this bacteriophage was completely sequenced, which opens the way for the use of many molecular techniques, such as site-specific mutagenesis, which was widely applied, e.g., in investigating the functions of some essential T4 proteins. The phage-display method, which is commonly applied in bacteriophage modifications, was successfully used to display antigens (PorA protein, VP2 protein of vvIBDV, and antigens of anthrax and HIV) on T4’s capsid platform. As first studies showed, the phage-display system as well as site-specific mutagenesis may also be used to modify interactions between phage particles and mammalian cells or to obtain phages infecting species other than the host bacteria. These may be used, among others, in the constantly developing bacteriophage therapy. All manipulations of this popular bacteriophage may enable the development of vaccine technology, phage therapy, and other branches of biological and medical science.     

Hotspot sites for acridine-induced frameshift mutations in bacteriophage T4 correspond to sites of action of the T4 type II topoisomerase

The type II topoisomerase of bacteriophage T4 is a central determinant of the frequency and specificity of acridine-induced frameshift mutations. Acridine-induced frameshift mutagenesis is specifically reduced in a mutant defective in topoisomerase activity. The ability of an acridine to promote topoisomerase-dependent cleavage at specific DNA sites in vitro is correlated to its ability to produce frameshift mutations at those sites in vivo. The specific phosphodiester bonds cleaved in vitro are precisely those at which frameshifts are most strongly promoted by acridines in vivo. The cospecificity of in vitro cleavage and in vivo mutation implicate acridine-induced, topoisomerase-mediated DNA cleavages as intermediates of acridine-induced mutagenesis in T4.     

Amino acid similarities to other proteins offer insights into roles of UmuD and UmuC in mutagenesis

The products of the umuD and umuC genes are required for most uv and chemical mutagenesis in Escherichia coli. The genes are organized in an operon that is repressed by LexA and regulated as part of the SOS response. The umuD protein shares homology with the carboxyl-terminal domain of LexA. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. Similarities of UmuD to gene 45 protein of bacteriophage T4 and of UmuC to gene 44 protein and gene 62 protein suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.     

Bacteriophage T4 DNA polymerase determines the amount and specificity of ultraviolet mutagenesis

Summary Ultraviolet mutagenesis in bacteriophage T4 proceeds via error-prone repair (EPR) and requires the functional integrity of the uvsWXY system which mediates genetic recombination, recombinational repair, and mutability by diverse DNA damaging agents. Current opinion holds that mutagens acting through EPR generate DNA damage which blocks the progress of the replication complex and that EPR consists of the facilitated bypass of such inaccurate, damaged templates. This notion predicts that the T4 DNA polymerase (encoded by gene 43) mediates EPR in UV irradiated phage T4. This prediction is verified by the discovery that gene 43 mutations often enhance or reduce UV mutagenesis (which is scored by the induction of r mutants) and sometimes change its specificity.     

Purification, crystallization and preliminary X-ray diffraction analysis of the phage T4 vertex protein gp24 and its mutant forms

The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6A resolution compared to wild-type gp24 at 3.80A resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.     

Deletion and duplication sequences induced in CHO cells by teniposide (VM-26), a topoisomerase II targeting drug,can be explained by the processing of DNA nicks produced by the drug-topoisomerase interaction

Frameshift mutations induced by acridines in bacteriophage T4 have been shown to be due to the ability of these mutagens to cause DNA cleavage by the type II topoisomerase of T4 and the subsequent processing of the 3′ ends at DNA nicks by DNA polymerase or its associated 3′ exonuclease followed by ligation of the processed end to the original 5′ end. An analysis of the ability of nick-processing models is presented here to test the ability of nick processing to account for the DNA sequences of duplications and deletions induced in the aprt gene of CHO cells by teniposide (VM-26) [Han et al. (1993) J. Mol. Biol., 229, 52]. Although teniposide is not an acridine, it induces topoisomerase II-mediated DNA cutting in aprt sequences in vitro and mutagenesis in vivo. Although the previous study noted a correlation between mutation sites and nearby DNA discontinuities induced by the enzyme in vitro, neither the nick-processing model responsible for T4 mutations, nor double-strand break models alone were able to account for most of the mutant sequences. Thus, no single model explained the correlation between teniposide-induced DNA cleavage and mutagenic specificity. This report describes an expanded analysis of the ways that nick-processing models might be related to mutagenesis and demonstrates that a modified nick-processing model provides a biochemical rationale for the mutant speficities. The successful nick-processing model proposes that either 3′ ends at nicks are elongated by DNA polymerase and/or that 5′ ends of nicks are subject to nuclease activity; 3′-nuclease activity is not implicated. The mutagenesis model for nick-processing of teniposide-induced nicks in CHO cells when compared to the mechanism of nick-processing in bacteriophage T4 at acridine-induced nicks provides a framework for considering whether the differences may be due to cell-specific modes of DNA processing and/or due to the precise characteristics of topoisomerase-DNA intermediates created by teniposide or acridine that lead to mutagenesis.     

Methyl Methanesulfonate Mutagenesis in Bacteriophage T4

MMS induces diverse rII mutations from a wild-type background in bacteriophage T4. About 56% are base pair substitutions, about 30% are frameshift mutations, and the remainder is a miscellaneous set of rapidly reverting or leaky mutants of unknown composition; but deletions were not detected. MMS-induced forward mutation is sharply reduced by the mutations px and y, which also reduce ultraviolet, photodynamic and γ-ray mutagenesis and increase killing by all of these agents. Thus, many of the mutations arise via the T4 WXY system. The induction of G:C → A:T transitions was detected even in a px or y background using sensitive reversion tests, and the few forward rII mutations that were induced from this background also behaved like transition mutations. Thus, some MMS-induced mutations arise independently of the WXY system, perhaps as a result of the (rather weak) ability of MMS to alkylate the O⁶ position of guanine.     

The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.     

Frameshift mutagenesis in bacteriophage T7.

We have undertaken an initial characterization of frameshift mutagenesis in bacteriophage T7 and have identified a subset of very low reversion frameshift mutations in the T7 ligase gene (gene 1.3). We used this information to construct bacteriophage T7 strains that contain one extra or one less base pair in gene 1.3 such that a frameshift event restores the reading frame of that gene. These events can be quantified and the frameshift mutation isolated within a localized region of the ligase gene. We have also identified a portion of the T7 ligase protein that will accept tracts of nonsense amino acids yet still give a ligase positive phenotype. This allows flexibility in the design of the target DNA sequence with which to study frameshift mutagenesis. These assays for frameshift mutagenesis performed in E. coli cells infected with the appropriate T7 strain, were used to measure the frequency of both plus and minus frameshifts in vivo.     

Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.     

Random mntagenesis of the gene for bacteriophage T7 RNA polymerase

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.     

On the role of the single-stranded DNA binding protein of bacteriophage T4 in DNA metabolism. I. Isolation and genetic characterization of new mutations in gene 32 of bacteriophage T4

Summary The product of gene 32 of bacteriophage T4 is a single-stranded DNA binding protein involved in T4 DNA replication, recombination and repair. Functionally differentiated regions of the gene 32 protein have been described by protein chemistry. As a preliminary step in a genetic dissection of these functional domains, we have isolated a large number of missense mutants of gene 32. Mutant isolation was facilitated by directed mutagenesis and a mutant bacterial host which is unusually restrictive for missense mutations in gene 32. We have isolated over 100 mutants and identified 22 mutational sites. A physical map of these sites has been constructed and has shown that mutations are clustered within gene 32. The possible functional significance of this clustering is considered.     

Presence of inducible DNA repair in Bacillus thuringiensis

Weigle reactivation of ultraviolet-irradiated luminal diameter 8 bacteriophage was observed after ultraviolet treatment of Bacillus thuringiensis cells. A slight increased frequency of clear plaque mutants was detected among the survivors. The kinetics of induction of the phage reactivation and phage mutagenesis have been determined. The presence of chloramphenicol before and after irradiation abolished the induction of repair and mutagenesis. These experiments suggest that, in spite of the relatively small mutagenic response in bacteriophage progeny, B. thuringiensis has an inducible repair system responsible to the significant Weigle reactivation of irradiated phage.     

Ribonucleoside and deoxyribonucleoside triphosphate pools during 2-aminopurine mutagenesis in T4 mutator-, wild type-, and antimutator-infected Escherichia coli

Ribonucleoside and deoxyribonucleoside triphosphate pools have been measured in Escherichia coli infected with bacteriophage T4 DNA polymerase mutator, wild type, and antimutator alleles during mutagenesis by the base analogue 2-aminopurine. ATP and GTP pools expand significantly during mutagenesis, while CTP and UTP pools contract slightly. The DNA polymerase (gene 43) alleles and an rII lesion perturb normal dNTP pools more than does the presence of 2-aminopurine. We find no evidence that 2-aminopurine induces mutations indirectly by causing an imbalance in normal dNTP pools. Rather, it seems likely that, by forming base mispairs with thymine and with cytosine, 2-aminopurine is involved directly in causing bidirectional A.T in equilibrium G.C transitions. The ratios for 2-aminopurine deoxyribonucleoside triphosphate/dATP pools are 5-8% for tsL56 mutator and 1-5% for tsL141 antimutator and 43+ alleles. We conclude that the significant differences observed in the frequencies of induced transition mutations in the three alleles can be attributed primarily to the properties of the DNA polymerases with their associated 3'-exonuclease activities in controlling the frequency of 2-aminopurine.cystosine base mispairs.