Studeis on the biochemical basis of mutation. IV. Effect of amino acid substitution on the enzymatic and biological properties of bacteriophage T4 DNA polymerase

The effects of substituting specific amino acids at specified loci in the bacterio-phage T4 DNA polymerase molecule have been studied. Gene 43 (DNA polymerase) amber mutants grown on suppressor strains which substitute serine, glutamine, or tyrosine at specific sites in the polymerase molecule, produce enzymes with substantially different physical, enzymatic and biological properties when compared to wild type. When amB22, a gene 43 mutant which makes a DNA polymerase fragment with only 3′-exonuclease activity, was grown in Escherichia coli B40(sup⁺1), -(sup⁺ 2) or -(sup⁺3), enzymes with different temperature sensitivities and nuclease to polymerase ratios were produced. Measurements of spontaneous mutation rates in these suppressed strains indicated that the two with higher than normal exonuclease activity were antimutators, and the one with a slightly lower exonuclease activity was a mutator. The substituted amino acids at the amB22 site perturbed the 3′-exonuclease activity creating either antimutator or mutator phenotypes. Thus, the B22 enzymes provide additional biochemical evidence to support the hypothesis that the exonuclease to polymerase ratio may influence the spontaneous mutation rate in phage T4.     

Suppression of Amber Mutations of Bacteriophage T4 Gene 43 (DNA Polymerase) by Translational Ambiguity

The growth properties of twelve different amber (am) mutants of bacteriophage T4 gene 43 (DNA polymerase) were examined by using nonpermissive (su(-)) as well as permissive (su(+)) Escherichia coli hosts. It was found that most of these mutants were measurably suppressed in su(-) hosts by translational ambiguity (misreading of codons during protein synthesis). The ability of these mutants to grow in response to this form of weak suppression probably means that the T4 gene 43 DNA polymerase can be effective in supporting productive DNA replication when it is supplied in small amounts. By similar criteria, studies with other phage mutants suggested that the products of T4 genes 62 (uncharacterized), 44 (uncharacterized), 42 (dCMP-hydroxymethylase), and 56 (dCTPase) are also effective in small amounts. Some T4 gene products, such as the product of gene 41 (uncharacterized), seem to be partially dispensable for phage growth since am mutants of such genes do propagate, although weakly, in streptomycin-resistant su(-) hosts which appear to have lost the capacity to suppress am mutations by ambiguity.     

Biochemistry of DNA-defective mutants of bacteriophage T4. VI. Biological functions of gene 42.

Bacteriophage T4 gene 1 and 42 amber mutants (defective in deoxynucleoside monophosphate kinase and deoxycytidylate hydroxymethylase, respectively) are able to synthesize DNA in cell-free lysates prepared as described by Barry and Alberts (1972), in contrast to their inabliity to do so in plasmolyzed and toluenized cell systems. Addition of extracts containing an active gene 1 or 42 product has no effect on synthesis in lysates defective in the respective gene. Thus, if these enzymes do play additional direct roles in replication, these roles are not manifest in the lysed-cell system. The gene 42 mutant am N122/m, a double mutant bearing an additional defect in DNA polymerase, is unable to synthesize DNA in these lysates. This inability is overcome by addition of extracts containing an active T4 DNA polymerase. m is a leaky amber mutation which reduces DNA polymerase activity to a very low level. However, this level is high enough to allow positive genetic complementation tests with gene 43 mutants. Two other gene 42 amber mutants contain additional defects: am 269 induces only half the normal level of DNA polymerase, and am C87 fails to induce a detectable level of thymidylate synthetase. These defects do not result from pleiotropic effects of the gene 42 mutations. In plasmolyzed cells, temperature-sensitive gene 42 mutants fail to synthesize DNA under conditions where replication forks and 5-hydroxymethyl-dCTP are present. This supports the idea that the gene 42 protein is directly involved in DNA synthesis.     

Genetic Mapping of the Amino-Terminal Domain of Bacteriophage T4 DNA Polymerase

The DNA polymerase of bacteriophage T4 is a multifunctional enzyme that harbors DNA-binding, DNA-synthesizing and exonucleolytic activities. We have cloned in bacterial plasmids about 99% of the structural gene for this enzyme (T4 gene 43). The gene was cloned in six contiguous 5'-terminal DNA fragments that defined seven intragenic mapping regions. Escherichia coli hosts harboring recombinant plasmids carrying the gene 43 subsegments were used in marker-rescue experiments that assigned a large number of ts and nonsense polymerase mutations to different physical domains of the structural gene. Conspicuously, only one missense mutation in a large collection of mutants mapped in the 5'-terminal 450 base-pair segment of the approximately 2700 base-pair gene. To test if this indicated a DNA polymerase domain that is relatively noncritical for biological activity, we mutagenized a recombinant plasmid carrying this 5'-terminal region and generated new conditional-lethal mutations that mapped therein. We identified five new ts sites, some having mutated at high frequency (nitrosoguanidine hot spots). New ts mutations were also isolated in phage genes 62 and 44, which map upstream of gene 43 on the T4 chromosome. A preliminary examination of physiological consequences of the ts gene 43 mutations showed that they exhibit effects similar to those of ts lesions that map in other gene 43 segments: some were mutators, some derepressed gene 43 protein synthesis and they varied in the severity of their effects on T4-induced DNA synthesis at nonpermissive temperatures. The availability of the gene 43 clones should make it possible to isolate a variety of lesions that affect different activities of the T4 DNA polymerase and help to define the different domains of this multifunctional protein.     

Morphogenesis of the tail fiber of bacteriophage T 4 . V. In vitro complementation between the gene 36 product and the genes 37-38 directed component

Summary An in vitro complementation was observed between the gene 36 product and the genes 37–38 directed component of the tail fiber of bacteriophage T₄. A possible role of the gene 36 product as well as the reconstitution process in the complementation were briefly discussed.Biozentrum, University of Basel, CH-4056 Basel, Swiss. 1 The following defectives in T₄ fiber genes were used: single defective mutants; amE1 (gene 36^–), amN52 (gene 37^–) and amB262 (gene 38^–); double defective mutants; amN52:B262 (genes 37^–38^–), amA455: N52 (genes 34^–37^–) and amB252:N52 (genes 35^–37^–); and triple defective mutants; am A455:B252:N52 (genes 34^–35^–37^–) and amA455:B252:B262 (genes 34^–35^–38^–).     

Studies on the biochemical basis of mutation VI. Selection and characterization of a new bacteriophage T4 mutator DNA polymerase

A new mutant of bacteriophage T4 has been isolated by a procedure which was designed to select for mutants with high spontaneous reversion rates. This mutant, M19, induces a defective DNA polymerase which has a degraded specificity and makes errors by inserting the incorrect nucleotide more frequently than the wild-type enzyme.In addition to M19, several other T4 polymerase amber and temperature-sensitive mutants have been located on a linear, fine-scale map. The mutants which most strongly affect mutation rates are found in two clusters at 25% and 80% of the gene. These two domains may represent the active site(s) of the polymerase and exonuclease activities.     

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.     

Host- and Phage-Mediated Repair of Radiation Damage in Bacteriophage T4

T4+ exhibits increased ultraviolet sensitivity on derivatives of Escherichia coli K12 or B lacking deoxyribonucleic acid (DNA) polymerase I. However, the sensitivity of T4v is not affected by the absence of host DNA polymerase. T4x and T4y also show increased sensitivity on DNA polymerase-deficient strains, but to a lesser extent than observed with wild-type T4. When T4x or T4y, but not T4+, are plated on a double mutant lacking both DNA polymerase and the uvrA gene product, a partial suppression of the polymerase effect is observed. Host ligase appears to be able to suppress to some extent the T4y phenotype but has no effect on wild-type T4 or other T4 mutants. T4xv incubated in E. coli B or B(s-1) in the presence of chloramphenicol (50 mug/ml) shows increased resistance over directly plated irradiated phage. Increased survival under the same conditions was not observed with T4+ or other T4 mutants. The repair of X-ray-damaged T4 was investigated by examining survival curves of T4+, T4x, T4y, T4ts43, and T4ts30. The repair processes were further defined by observing the effects of plating irradiated phage on various hosts including strains lacking DNA polymerase I or polynucleotide ligase. Two classes of effects were observed. Firstly, the x and y gene products seem to be involved in a repair system utilizing host ligase. Secondly, in the absence of host DNA polymerase, phage sensitivity is increased in an unknown manner which is enhanced by the presence of host uvrA gene product.     

Genetic control of capsid length in bacteriophage T4: clustering of ptg mutations in gene 23.

Fifty-two new bacteriophage T4 ptg mutations have been isolated by selecting for the giant-capsid phenotype they display. Genetic mapping placed all of them at eight sites, all located in gene 23. These sites were clustered in three locations, one near amber B17 (gene 23 nucleotide [NT] 268), another centrally placed between amE506 (NT 706) and amE1270 (NT 925), and the third between amC208 (NT 1297) and amE1236 (NT 1489). The lack of a selective system for identifying recombinant genotypes when dealing with the very close linkages found within these clusters opens the possibility that more than eight sites are represented in this set of mutations. Since one site was represented by only one mutation, it seems likely that further searching might uncover additional sites. It is suggested that the clustering of mutations observed here identifies regions of the gene 23 product that play a role in regulating the capsid length of T4.     

Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants.

Segments of DNA that contained the DNA polymerase gene of bacteriophage T5 were isolated. The physical locus of the gene was identified by transforming Escherichia coli with purified DNA fragments generated by restriction enzyme digestions, and the transformed cells were used to rescue amber mutants of T5 with mutations in the gene for DNA polymerase. The method is applicable to any other gene that has mutations with low reversion frequencies. We studied the following mutations of the T5 DNA polymerase gene, reading from left to right by the standard convention (D. J. McCorquodale, Crit. Rev. Microbiol. 4:101-159, 1975): D7, D8, aml, ts5E-ts53, am6, and D9. These loci were found to reside within three pieces of DNA with a total length of 3,600 base pairs. Because the structural gene for T5 DNA polymerase is estimated to be 2,600 base pairs long, the whole structural gene may reside in these segments. These are located 58.3 to 61.3% of the distance from the left end of the DNA. The left-end piece of the DNA (1,100 base pairs) containing the polymerase gene has loci D7 and D8, and the right-end piece (1,600 base pairs) has locus D9, according to the results of the transformation assay. These results are consistent with the genetic map.     

Primary structure of T4 DNA polymerase. Evolutionary relatedness to eucaryotic and other procaryotic DNA polymerases

Bacteriophage T4 gene 43 codes for the viral DNA polymerase. We report here the sequence of gene 43 and about 70 nucleotides of 5'- and 3'-flanking sequences, determined by both DNA and RNA sequencing. We have also purified T4 DNA polymerase from T4 infected Escherichia coli and from E. coli containing a gene 43 overexpression vector. A major portion of the deduced amino acid sequence has been verified by peptide mapping and sequencing of the purified DNA polymerase. All these results are consistent with T4 DNA polymerase having 898 amino acids with a calculated Mr = 103,572. Comparison of the primary structure of T4 DNA polymerase with the sequence of other procaryotic and eucaryotic DNA polymerases indicates that T4 DNA polymerase has regions of striking similarity with animal virus DNA polymerases and human DNA polymerase alpha. Surprisingly, T4 DNA polymerase shares only limited similarity with E. coli polymerase I and no detectable similarity with T7 DNA polymerase. Based on the location of specific mutations in T4 DNA polymerase and the conservation of particular sequences in T4 and eucaryotic DNA polymerases, we propose that the NH2-terminal half of T4 DNA polymerase forms a domain that carries out the 3'----5' exonuclease activity whereas the COOH-terminal half of the polypeptide contains the dNTP-binding site and is necessary for DNA synthesis.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Autogenous translational operator recognized by bacteriophage T4 DNA polymerase

The synthesis of the DNA polymerase of bacteriophage T4 is autogenously regulated. This protein (gp43), the product of gene 43, binds to a segment of its mRNA that overlaps its ribosome binding site, and thereby blocks translation. We have determined the Kd of the gp43-operator interaction to be 1.0 x 10(-9) M. The minimum operator sequence to which gp43 binds consists of 36 nucleotides that include a hairpin (containing a 5 base-pair helix and an 8 nucleotide loop) and a single-stranded segment that contains the Shine-Dalgarno sequence of the ribosome binding site. In the distantly related bacteriophage RB69 there is a remarkable conservation of this hairpin and loop sequence at the ribosome binding site of its DNA polymerase gene. We have constructed phage operator mutants that overproduce gp43 in vivo, yet are unchanged for in vivo replication rates and phage yield. We present data that show that the replicative and autoregulatory functions are mutually exclusive activities of this polymerase, and suggest a model for gp43 synthesis that links autoregulation to replicative demand.     

Effect of amino acid substitutions in the polypeptide chain of DNA-polymerase on manifestation of the mutator effect]

Thin map of gene 43, controlling the synthesis of T4 DNA polymerase, is obtained by mapping experiments performed with 39 amber mutants, and is used for analysis of the sites of DNA polymerase gene from the point of view of displaying the mutator effect. The mutant sites studied possessed different reaction on amino acid substitutions in the polypeptide chain of the enzyme. Most of sites of the DNA polymerase gene, with the exception of two "supersensitive", responsed only on the apparent type of the amino acid substitutions: the mutator effect of amber mutations, which are located at these sites, was exhibited only in the case of insertion of the definite amino acid in the respective point of polypeptide chain. The proposed system of amber mutations for studying the mutator effect, allowed the authors to obtain the data on the effect of concrete alterations in the polypeptide chain of the enzyme on the development of its mutator properties.     

Non-essential UV-sensitive bacteriophage T4 mutants affecting early DNA synthesis: a third pathway of DNA repair.

Non-essential bacteriophage T4 mutants uvs58 and uvs79 showed a lower UV sensitivity than either the excision-repair mutant v am5 or the replication-dependent recombination-repair mutant y10. The UV sensitivity of double and triple mutants carrying one of the mutations uvs58 or uvs79, and v am 5 or (and) y10 was higher than the sum of the sensitivities of the single mutants. The uvs58 mutation was mapped to the early gene region, close to amN81 (gene 41). The unirradiated mutants uvs58 and uvs79 accumulated newly synthesized DNA at a slower rate than wild-type T4. Double mutants uvs58:am59 and uvs79:am59 showed DNA synthesis in E. coli B su- to be arrested at a 3--5 times lower level than that in am59-infected cells. Chloramphenicol, added 9--12 min after infection, suppressed arrests of DNA synthesis, the double mutants showing a lag of 8 min as compared with am59. Results from analysis of sucrose gradients of parental uvs58 and uvs79 DNA were in agreement with the suggestion of a mutation in an early function. The mutants uvs58 and uvs79 are suggested to be defective in a component of the DNA replication apparatus with a function in the adaptation to irregularities in the DNA structure. The third pathway of UV repair is tentatively designated as non-catalytic replication repair.     

Influence of the growth rate on the macromolecular composition of Acinetobacter calcoaceticus in carbon-limited chemostat culture

Abstract Infectious phage particles can be formed in vitro when extracts of T1-infected cells are incubated with T1 DNA. The DNA packaging system is based on mixtures of complementing extracts from Escherichia coli sup⁰ cells infected with the amber mutants am 4 (gene 16) or am 10 (gene 13). Gene 16 mutants are defective in the formation of DNA-filled heads but make proheads; gene 13 mutants are defective in prohead formation. Three forms of DNA have been packaged: (1) endogenous concatemeric DNA present in mixtures of am 4 and am 10 mutant extracts; (2) concatemeric DNA; (3) virion DNA both when supplied exogenously to mixtures of am 4 · am 20 and am 10 · am 20 double mutant extracts ( am 20 inhibits T1 DNA synthesis). The reaction requires added ATP, Mg²⁺ and spermidine for optimum efficiency and produces about 1.5 × 10³ pfu/ μ g and about 1 × 10⁴ pfu/ μ g for exogenous concatemeric and virion DNA, respectively.