Isolation of bacteriophage T4 DNA polymerase mutator mutants

More than 20 new bacteriophage T4 DNA polymerase mutants have been isolated by a procedure designed to select mutants with high spontaneous mutation rates. Some of the mutants produce the highest mutation frequencies that have been observed in T4 thus far. The design of the selection procedure allows for the isolation of mutator mutants that preferentially induce certain types of replication errors, and some of the mutator mutants have mutational specificities different from wild-type. The new mutants are clustered at just two sites in the DNA polymerase gene, and this result confirms an earlier observation.     

The isolation and characterization of bacterial strains (Tab32) that restrict bacteriophage T4 gene 32 mutants

Summary Gene 32 of bacteriophage T4 codes for a single-stranded DNA binding protein. We have isolated mutants of Escherichia coli (called Tab32) that specifically restrict the growth of gene 32 missense mutants and allow normal growth of T4⁺. During infections of Tab32 with 32^tsL171, large amounts of DNA are synthesized and late proteins are made, but very few progeny phage are produced. At least two bacterial mutations are necessary for the restrictive phenotype; these mutations have been mapped to about min 41 and min 64.     

Gene 32 protein of bacteriophage T4 moderates the activities of the T4 gene 46/47-controlled nuclease and of the Escherichia coli RecBC nuclease in vivo.

Genes 46 and 47 of phage T4 control a nuclease that is required for genetic recombination and may act similarly to the Escherichia coli RecBC nuclease. In vivo, the nucleolytic activities of both of these nucleases must be moderated so that recombining DNA intermediates are not destroyed. We conclude from our present experiments that the phage T4 gene 32 protein, specifically its C-terminal domain, participates in such moderation. We have investigated DNA degradation in different gene 32 and gene 32/46 mutants under conditions that are completely restrictive for progeny production in all the mutants. Under these conditions, DNA of those gene 32 mutants in which the C-terminal domain of the protein is not synthesized or is modified is degraded to acid-soluble material. T4 gene 46 or E. coli recB mutations reduce such degradation; together they abolish it completely. By contrast, single gene 32 mutants which produce an unaltered C-terminal domain show little or no degradation of their DNA. Residual protection against nucleases is unrelated to residual primary DNA replication or to overproduction of the mutant peptides in the different gene 32 mutants.     

Recognition of chemically damaged DNA by the gene 32 protein from bacteriophage T4

We have used fluorescence spectroscopy to investigate the binding of gene 32 protein from bacteriophage T4 to DNA which has been chemically modified with carcinogens or antitumor drugs. This protein exhibits a high specificity for single-stranded nucleic acids and binds more efficiently to DNA modified either with cis-diaminodichloroplatinum(II) or with aminofluorene derivatives than to native DNA. This increased affinity is related to the formation of locally unpaired regions which are strong binding sites for the single-strand binding protein. In contrast, gene 32 protein has the same affinity for native DNA, DNA containing methylated purines and DNA that has reacted with trans-diaminodichloroplatinum(II) or with chlorodiethylenetriaminoplatinum(II) chloride. These types of damage do not induce a sufficient structural change to allow gene 32 protein binding. Depurination of DNA does not create binding sites for the T4 gene 32 protein but nicked apurinic sites are strong ligands for the protein. This T4 single-strand binding protein does not exhibit a significantly increased affinity for nicked DNA as compared with native DNA. These results are discussed with respect to the recognition of DNA damage by proteins involved in DNA repair and to the possible role of single-strand binding proteins in DNA repair mechanisms.     

Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function

Previous studies on the selection of bacteriophage T4 mutator mutants have been extended and a method to regulate the mutator activity of DNA polymerase mutator strains has been developed. The nucleotide changes of 17 bacteriophage T4 DNA polymerase mutations that confer a mutator phenotype and the nucleotide substitutions of several other T4 DNA polymerase mutations have been determined. The most striking observation is that the distribution of DNA polymerase mutator mutations is not random; almost all mutator mutations are located in the N-terminal half of the DNA polymerase. It has been shown that the T4 DNA polymerase shares several regions of homology at the protein sequence level with DNA polymerases of herpes, adeno and pox viruses. From studies of bacteriophage T4 and herpes DNA polymerase mutants, and from analyses of similar protein sequences from several organisms, we conclude that DNA polymerase synthetic activities are located in the C-terminal half of the DNA polymerase and that exonucleolytic activity is located nearer the N terminus.     

Semiconservative DNA replication is initiated at a single site in recombination-deficient gene 32 mutants of bacteriophage T4.

We have investigated, by electron microscopy, replicative intermediate produced early after infection of Escherichia coli with two phage T4 gene 32 mutants (amA453 and tsG26) which replicate their parental DNA but are defective in secondary replications and in moderating the activities of recombination nucleases. Under conditions completely restrictive for progeny production, both of these mutant produced replicative intermediates, each containing a single internal loop. Both branches of these loops were double stranded; i.e., both leading and lagging strands were synthesized. The replicative intermediates of these mutants qualitatively and quantitatively resembled early replicating wild-type T4 chromosomes after solitary infection of E. coli. However, in contrast to intracellular wild-type T4 DNA isolated from multiple infection, the mutant DNAs showed neither multiple branches nor multiple tandem loops. These results demonstrate that a truncated gene 32 protein which consists of less than one-third of the wild-type T4 helix-destabilizing protein can facilitate the functions of T4 replication proteins, specifically those of T4 DNA polymerase and priming proteins. Our results also support the hypothesis that the generation of multiple tandem loops or branches in vegetative T4 DNA depends on recombination (Mosig et al., in B. Alberts, ed., Mechanistic Studies of DNA Replication and Genetic Recombination, p. 527-543, Academic Press, Inc., New York, 1980).     

Mutations of bacteriophage T4 59 helicase loader defective in binding fork DNA and in interactions with T4 32 single-stranded DNA-binding protein

Bacteriophage T4 gene 59 protein greatly stimulates the loading of the T4 gene 41 helicase in vitro and is required for recombination and recombination-dependent DNA replication in vivo. 59 protein binds preferentially to forked DNA and interacts directly with the T4 41 helicase and gene 32 single-stranded DNA-binding protein. The helicase loader is an almost completely alpha-helical, two-domain protein, whose N-terminal domain has strong structural similarity to the DNA-binding domains of high mobility group proteins. We have previously speculated that this high mobility group-like region may bind the duplex ahead of the fork, with the C-terminal domain providing separate binding sites for the fork arms and at least part of the docking area for the helicase and 32 protein. Here, we characterize several mutants of 59 protein in an initial effort to test this model. We find that the I87A mutation, at the position where the fork arms would separate in the model, is defective in binding fork DNA. As a consequence, it is defective in stimulating both unwinding by the helicase and replication by the T4 system. 59 protein with a deletion of the two C-terminal residues, Lys(216) and Tyr(217), binds fork DNA normally. In contrast to the wild type, the deletion protein fails to promote binding of 32 protein on short fork DNA. However, it binds 32 protein in the absence of DNA. The deletion is also somewhat defective in stimulating unwinding of fork DNA by the helicase and replication by the T4 system. We suggest that the absence of the two terminal residues may alter the configuration of the lagging strand fork arm on the surface of the C-terminal domain, so that it is a poorer docking site for the helicase and 32 protein.     

Mutations in a conserved motif inhibit single-stranded DNA binding and recombination mediator activities of bacteriophage T4 UvsY protein

The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4. UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange. UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsY-ssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors. A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis. Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised. These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY. Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNA-dependent ATP hydrolysis and DNA strand exchange. The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly. Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.     

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.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems

The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination. gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA polymerase or recombination enzymes. The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however. Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA interactions. Therefore, we examined the effects of six different gp32 B-domain mutants on T4 in vitro systems for DNA synthesis and homologous pairing. We find that the B-domain is essential for gp32's stimulation of these reactions. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e. wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B. However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.     

Amplification of snap-back DNA synthesis reactions by the uvsX recombinase of bacteriophage T4.

The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.     

den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA.

Endonuclease V of bacteriophage T4 has been described as an enzyme, coded for by the denV gene, that incises UV-irradiated DNA. It has recently been proposed that incision of irradiated DNA by this enzyme and the analogous "correndonucleases" I and II of Micrococcus luteus requires the sequential action of a pyrimidine dimer-specific DNA glycosylase and an apyrimidinic/apurinic endonuclease. In support of this two-step mechanism, we found that our preparations of T4 endonuclease V contained a DNA glycosylase activity that produced alkali-labile sites in irradiated DNA and an apyrimidinic/apurinic endonuclease activity that converted these sites to nicks. Both activities could be detected in the presence of 10 mM EDTA. In experiments designed to determine which of the activities is coded by the denV gene, we found that the glycosylase was more heat labile in extracts of Escherichia coli infected with either of two thermosensitive denV mutants than in extracts of cells infected with wild-type T4. In contrast, apyrimidinic/apurinic endonuclease activity was no more heat labile in extracts of the former than in extracts of the latter. Our results indicate that the denV gene codes for a DNA glycosylase specific for pyrimidine dimers.     

Effects of the bacteriophage T4 dda protein on DNA synthesis catalyzed by purified T4 replication proteins

The T4 bacteriophage dda protein is a DNA-dependent ATPase and DNA helicase that is the product of an apparently nonessential T4 gene. We have examined its effects on in vitro DNA synthesis catalyzed by a purified, multienzyme T4 DNA replication system. When DNA synthesis is catalyzed by the T4 DNA polymerase on a single-stranded DNA template, the addition of the dda protein is without effect whether or not other replication proteins are present. In contrast, on a double-stranded DNA template, where a mixture of the DNA polymerase, its accessory proteins, and the gene 32 protein is required, the dda protein greatly stimulates DNA synthesis. The dda protein exerts this effect by speeding up the rate of replication fork movement; in this respect, it acts identically with the other DNA helicase in the T4 replication system, the T4 gene 41 protein. However, whereas a 41 protein molecule remains bound to the same replication fork for a prolonged period, the dda protein seems to be continually dissociating from the replication fork and rebinding to it as the fork moves. Some gene 32 protein is required to observe DNA synthesis on a double-stranded DNA template, even in the presence of the dda protein. However, there is a direct competition between this helix-destabilizing protein and the dda protein for binding to single-stranded DNA, causing the rate of replication fork movement to decrease at a high ratio of gene 32 protein to dda protein. As shown elsewhere, the dda protein becomes absolutely required for in vitro DNA synthesis when E. coli RNA polymerase molecules are bound to the DNA template, because these molecules otherwise stop fork movement (Bedinger, P., Hochstrasser, M., Jongeneel, C.V., and Alberts, B. M. (1983) Cell 34, 115-123).     

Bacteriophage T4 baseplate components. III. Location and properties of the bacteriophage structural thymidylate synthetase.

Two T4D thymidylate synthetase (td) temperature-sensitive mutants have been isolated and characterized. Both mutants produce heat-labile phage particles. This observation supports the view that this viral-induced protein is a phage structural component. Further, antiserum to td has been shown to block a specific step in tail plate morphogenesis. The results indicated that the td protein is largely covered by the T4D tail plate gene 11 protein. Since the phageinduced dihydrofolate reductase (dfr) also is partially covered by the gene 11 protein, it appears that td was adjacent to the tail plate dfr. This location has been confirmed by constructing a T4D mutant which is dfrtstdts and showing that these two tail plate constituents interact and give altered physical properties to the phage particles produced. A structural relationship for the tail plate folate, dfr, and td has been reported.     

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.     

Structure-function studies of the bacteriophage T4 DNA polymerase. Isolation of a novel suppressor mutant

We describe here our first attempt in using suppressor mutations to study structure-function relationships of the bacteriophage T4 DNA polymerase. One intragenic suppressor mutation, J5(43) degrees, was isolated that suppresses the temperature sensitivity but not the mutator activity of tsM19, a DNA polymerase mutant. Thus, the substituted amino acid induced by the tsM19 lesion decreases DNA polymerase fidelity, even if the temperature sensitivity has been corrected by a second amino acid substitution in the DNA polymerase polypeptide. The isolation, mapping and characterization of the J5(43) degrees mutation as well as the purification and characterization of the tsM19-J5(43) degrees mutant DNA polymerase are presented. The suppressor isolation procedure has general applicability for the selection of suppressor mutations of other T4 DNA polymerase mutator mutants.     

Regulation of the synthesis of bacteriophage T4 gene 32 protein

The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment.Mutations in genes required for late gene expression (genes 33 and 53) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not dependent on DNA synthesis.These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by its own function. Thus, in the absence of P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently in the maturation of replicated DNA, the increased P32 production is caused by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41, 56, 58, 60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.     

Plasmid models for bacteriophage T4 DNA replication: requirements for fork proteins.

Bacteriophage T4 DNA replication initiates from origins at early times of infection and from recombinational intermediates as the infection progresses. Plasmids containing cloned T4 origins replicate during T4 infection, providing a model system for studying origin-dependent replication. In addition, recombination-dependent replication can be analyzed by using cloned nonorigin fragments of T4 DNA, which direct plasmid replication that requires phage-encoded recombination proteins. We have tested in vivo requirements for both plasmid replication model systems by infecting plasmid-containing cells with mutant phage. Replication of origin and nonorigin plasmids strictly required components of the T4 DNA polymerase holoenzyme complex. Recombination-dependent plasmid replication also strictly required the T4 single-stranded DNA-binding protein (gene product 32 [gp32]), and replication of origin-containing plasmids was greatly reduced by 32 amber mutations. gp32 is therefore important in both modes of replication. An amber mutation in gene 41, which encodes the replicative helicase of T4, reduced but did not eliminate both recombination- and origin-dependent plasmid replication. Therefore, gp41 may normally be utilized for replication of both plasmids but is apparently not required for either. An amber mutation in gene 61, which encodes the T4 RNA primase, did not eliminate either recombination- or origin-dependent plasmid replication. However, plasmid replication was severely delayed by the 61 amber mutation, suggesting that the protein may normally play an important, though nonessential, role in replication. We deleted gene 61 from the T4 genome to test whether the observed replication was due to residual gp61 in the amber mutant infection. The replication phenotype of the deletion mutant was identical to that of the amber mutant. Therefore, gp61 is not required for in vivo T4 replication. Furthermore, the deletion mutant is viable, demonstrating that the gp61 primase is not an essential T4 protein.     

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.