Recombination-dependent DNA replication in phage T4.

Studies in the 1960s implied that bacteriophage T4 tightly couples DNA replication to genetic recombination. This contradicted the prevailing wisdom of the time, which staunchly supported recombination as a simple cut-and-paste process. More-recent investigations have shown how recombination triggers DNA synthesis and why the coupling of these two processes is important. Results from T4 were instrumental in our understanding of many important replication and recombination proteins, including the newly recognized replication/recombination mediator proteins. Recombination-dependent DNA replication is crucial to the T4 life cycle as it is the major mode of DNA replication and is also central to the repair of DNA breaks and other damage.

An antitumor drug-induced topoisomerase cleavage complex blocks a bacteriophage T4 replication fork in vivo.

Many antitumor and antibacterial drugs inhibit DNA topoisomerases by trapping covalent enzyme-DNA cleavage complexes. Formation of cleavage complexes is important for cytotoxicity, but evidence suggests that cleavage complexes themselves are not sufficient to cause cell death. Rather, active cellular processes such as transcription and/or replication are probably necessary to transform cleavage complexes into cytotoxic lesions. Using defined plasmid substrates and two-dimensional agarose gel analysis, we examined the collision of an active replication fork with an antitumor drug-trapped cleavage complex. Discrete DNA molecules accumulated on the simple Y arc, with branch points very close to the topoisomerase cleavage site. Accumulation of the Y-form DNA required the presence of a topoisomerase cleavage site, the antitumor drug, the type II topoisomerase, and a T4 replication origin on the plasmid. Furthermore, all three arms of the Y-form DNA were replicated, arguing strongly that these are trapped replication intermediates. The Y-form DNA appeared even in the absence of two important phage recombination proteins, implying that Y-form DNA is the result of replication rather than recombination. This is the first direct evidence that a drug-induced topoisomerase cleavage complex blocks the replication fork in vivo. Surprisingly, these blocked replication forks do not contain DNA breaks at the topoisomerase cleavage site, implying that the replication complex was inactivated (at least temporarily) and that topoisomerase resealed the drug-induced DNA breaks. The replication fork may behave similarly at other types of DNA lesions, and thus cleavage complexes could represent a useful (site-specific) model for chemical- and radiation-induced DNA damage.

UvsW protein regulates bacteriophage T4 origin-dependent replication by unwinding R-loops.

The UvsW protein of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including repair, recombination, and recombination-dependent replication. UvsW has also been implicated in the repression of origin-dependent replication at late times of infection, when UvsW is normally synthesized. Two well-characterized T4 origins, ori(uvsY) and ori(34), are believed to initiate replication through an R-loop mechanism. Here we provide both in vivo and in vitro evidence that UvsW is an RNA-DNA helicase that catalyzes the dissociation of RNA from origin R-loops. Two-dimensional gel analyses show that the replicative intermediates formed at ori(uvsY) persist longer in a uvsW mutant infection than in a wild-type infection. In addition, the inappropriate early expression of UvsW protein results in the loss of these replicative intermediates. Using a synthetic origin R-loop, we also demonstrate that purified UvsW functions as a helicase that efficiently dissociates RNA from R-loops. These and previous results from a number of studies provide strong evidence that UvsW is a molecular switch that allows T4 replication to progress from a mode that initiates from R-loops at origins to a mode that initiates from D-loops formed by recombination proteins.

Mutations of the bacteriophage T4 type II DNA topoisomerase that alter sensitivity to antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide and an antibacterial quinolone.

Various antitumor and antibacterial agents target type II DNA topoisomerases, stabilizing a cleaved DNA reaction intermediate and thereby converting topoisomerase into a cellular poison. Two 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant bacteriophage T4 topoisomerases have previously been characterized biochemically, and we have now determined the sequence of the causative mutations. In one case, a mutation (E457K) in a conserved domain of gp39 (ATPase subunit) causes resistance to antitumor agent m-AMSA but hypersensitivity to the quinolone oxolinic acid. In the second case, a combination of two amino acid substitutions (S79F and G269V) in gp52 (DNA-cleaving subunit) causes resistance to both m-AMSA and oxolinic acid. The S79F mutation is responsible for drug resistance, whereas the G269V mutation suppresses a topoisomerase deficiency caused by S79F. Surprisingly, the G269V mutation by itself causes a dramatic hypersensitivity to both inhibitors, defining a new class of topoisomerase mutants. Because S79 and the adjacent N78 are homologous to two key residues of DNA gyrase that affect quinolone sensitivity, we generated additional amino acid substitutions at these two positions. The substitutions alter sensitivity to m-AMSA and to oxolinic acid, sometimes in opposite directions. Furthermore, the quinolone sensitivities of the various mutants paralleled those of corresponding gyrase mutants. These results support models in which both quinolones and antitumor agents bind to a conserved site that overlaps the active site of the enzyme.

Bacteriophage T4, a model system for understanding the mechanism of type II topoisomerase inhibitors.

Bacteriophage T4 provides a simple model system for analyzing the mechanism of action of antitumor agents that inhibit DNA topoisomerases. The phage-encoded type II topoisomerase is sensitive to many of the same antitumor agents that inhibit mammalian type II topoisomerase, including m-AMSA, ellipticines, mitoxantrone and epipodophyllotoxins. Results from the T4 model system provided a convincing demonstration that topoisomerase is the physiological drug target and strong evidence that the drug-induced cleavage complex is important for cytotoxicity. The detailed molecular steps involved in cytotoxicity, and the mechanism of recombinational repair of inhibitor-induced DNA damage, are currently being analyzed using this model system. Studies with the T4 topoisomerase have also provided compelling evidence that topoisomerase inhibitors interact with DNA at the active site of the enzyme, with each class of inhibitor favoring a different subset of cleavage sites based on DNA sequence. Finally, analysis of drug-resistance mutations in the T4 topoisomerase have implicated certain regions of the protein in drug interaction and provided a strong link between the mechanism of action of the antibacterial quinolones, which inhibit DNA gyrase, and the various antitumor agents, which inhibit mammalian type II topoisomerase.

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins.

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin.

Expression and function of the uvsW gene of bacteriophage T4.

The uvsW gene of bacteriophage T4 is involved in many aspects of phage DNA metabolism, including replication, recombination and repair. To approach the function of uvsW, the structure and expression of the uvsW gene were first explored. Molecular analyses defined the promoter region, the transcriptional start site, and the probable initiation codon. The required promoter region contains a sequence resembling the consensus for T4 late promoters. Furthermore, transcriptional analyses indicated that uvsW is expressed as a late gene, providing a time frame for uvsW action. Several novel observations restrict possible models for uvsW function. A uvsW-deletion mutation reduced overall phage-phage recombination 1.7-fold, but reduced plasmid integration tenfold relative to the wild-type. Thus, the UsvW protein plays a critical role in a specific recombination pathway involving simple reciprocal exchange. One of the most intriguing phenotypes associated with uvsW mutations is the restoration of arrested DNA synthesis caused by mutations that block secondary initiation, the major mode by which replication initiates at late times in wild-type infections. Experiments with plasmid model systems indicate that a uvsW mutation does not restore the arrested DNA synthesis by rescuing secondary initiation directly. Rather, a uvsW mutation appears to allow some alternative mode of late replication, implying that the UvsW protein normally represses this alternative pathway. The rifampicin resistance of uvsW-repressed replication suggests that it involves either tertiary initiation or some novel mode of initiation. Finally, the inappropriate early expression of uvsW from a heterologous promoter blocks most early phage DNA synthesis in a uvsY-mutant infection, suggesting that the UvsW protein is normally the key regulatory factor in the switch from early to late DNA replication. According to this suggestion, the restored late replication in a uvsW mutant is an abnormal continuation of an early mode(s) of replication.

Role of MotA transcription factor in bacteriophage T4 DNA replication.

At least two bacteriophage T4 replication origins, ori(uvsY) and ori(34), contain a T4 middle-mode promoter that is necessary for origin function. We wanted to analyze the requirement of these two replication origins for the MotA protein, which is the phage-encoded activator of middle-mode promoters. To ensure the complete absence of MotA protein, we deleted the motA gene from the T4 genome. Unexpectedly, the deletion mutant was not viable unless the MotA protein was provided from a recombinant plasmid. Therefore, MotA is an essential protein for T4 growth. The motA delta mutation reduced the synthesis of several proteins that are encoded by genes with middle-mode promoters, delayed and reduced the synthesis of late proteins, and substantially reduced phage genomic replication. The motA delta mutation also reduced the replication of an ori(uvsY)-containing plasmid and virtually abolished replication of an ori(34)-containing plasmid. The replication defects of the two origins correlated with transcriptional defects: the motA delta mutation modestly reduced transcription from the plasmid-borne ori(uvsY) promoter and strongly reduced transcription from the ori(34) promoter. These results provide strong evidence that MotA protein is normally involved in origin-dependent replication. However, MotA is not required for origin-directed replication as long as transcription can occur from the origin promoter.

RNA-DNA hybrid formation at a bacteriophage T4 replication origin.

The bacteriophage T4 replication origins ori(uvsY) and ori(34) each contain two distinct components: a T4 middle-mode promoter that is strictly required for replication and a downstream region of about 50 bp that is required for maximal levels of replication. Here, we present evidence that structure of the downstream region is important for replication initiation. Based on sensitivity to a single-stranded DNA-specific nuclease in vitro the downstream region behaves as a DNA unwinding element. The propensity to unwind is probably important for origin activity in vivo, because replication activity is maintained when the native downstream region is replaced with a heterologous DNA unwinding element from pBR322 in either orientation. We analyzed the origin DNA for possible unwinding in vivo by using potassium permanganate, a chemical that reacts with unpaired pyrimidine bases. The non-template strand, but not the template strand, became hypersensitive to permanganate after T4 infection regardless of whether replication could occur. Strand-specific permanganate hypersensitivity was also observed in artificial origins containing the pBR322 DNA unwinding element in either orientation. Hypersensitivity was only detected when the origin contained a promoter that would be active during T4 infection. Furthermore, the origin transcript itself appears to be necessary for hypersensitivity since insertion of a transcriptional terminator abolishes hypersensitivity downstream of the termination site. Our results strongly suggest that the downstream region functions as a DNA unwinding element during replication initiation, leading to the formation of a persistent RNA-DNA hybrid at the origin.

The MotA protein from bacteriophage T4 contains two domains. Preliminary structural analysis by X-ray diffraction and nuclear magnetic resonance.

Controlled protease cleavage experiments and N-terminal sequence analyses were used to show that the transcriptional activator MotA from bacteriophage T4 has a two-domain structure. The N and C-terminal domains have M(r) values of 10,300 and 11,800, respectively, and were separately cloned and overexpressed in Escherichia coli. One and two-dimensional NMR spectroscopy indicate that both domains have stably folded structures and contain extensive secondary structure. The N-terminal domain is substantially alpha-helical, whereas the C-terminal domain has a high content of beta-strand. The N-terminal domain has been crystallized under three different conditions, all with the space group P3(1(2))21 and similar unit cell dimensions. The best crystals are grown from ammonium sulfate, have cell dimensions a = b = 46.7 A, c = 139.6 A, and diffract to beyond 2.4 A. The high quality of the NMR and diffraction data will allow a complete structural analysis of MotA by a combination of these techniques.

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.

Integration of plasmids into the bacteriophage T4 genome.

We have analyzed the integration of plasmids into the bacteriophage T4 genome via homologous recombination. As judged by genetic selection for a plasmid-borne marker, a mutation in phage gene uvsX or uvsY essentially blocked the integration of a plasmid with homology to the T4 genome but no phage replication origin (non-origin plasmid). The strict requirement for these two proteins suggests that plasmid integration can proceed via a strand-invasion reaction similar to that catalyzed in vitro by the T4-encoded strand-exchange protein (UvsX) in concert with UvsY and gp32. In contrast to the results with the non-origin plasmid, a mutation in uvsX or uvsY reduced the integration of a T4 replication origin-containing plasmid by only 3-10-fold. These results suggest that the origin-containing plasmid integrates by both the UvsXY-dependent pathway used by the non-origin plasmid and by a UvsXY-independent pathway. The origin-containing plasmid integrated into the phage genome during a uvsX- or uvsY-mutant infection of a recA-mutant host, and therefore origin-dependent integration can occur in the absence of both phage- and host-encoded strand-exchange proteins (UvsX and RecA, respectively).

Repair of double-strand breaks in bacteriophage T4 by a mechanism that involves extensive DNA replication.

We investigated double-strand break (dsb) repair in bacteriophage T4 using a physical assay that involves a plasmid substrate with two inverted DNA segments. A dsb introduced into one repeat during a T4 infection induces efficient dsb repair using the second repeat as a template. This reaction is characterized by the following interesting features. First, the dsb induces a repair reaction that is directly coupled to extensive plasmid replication; the repaired/replicated product is in the form of long plasmid concatemers. Second, repair of the dsb site is frequently associated with exchange of flanking DNA. Third, the repair reaction is absolutely dependent on the products of genes uvsX, uvsY, 32, 46, and 59, which are also required for phage genomic recombination-dependent DNA replication. Fourth, the coupled repair/replication reaction is only partly dependent on endonuclease VII (gp49), suggesting that either another Holliday-junction-cleaving activity or an alternate resolution pathway is active during T4 infections. Because this repair reaction is directly coupled to extensive replication, it cannot be explained by the SZOSTAK et al. model. We present and discuss a model for the coupled repair/replication reaction, called the extensive chromosome replication model for dsb repair.

Bacteriophage T4 mutants hypersensitive to an antitumor agent that induces topoisomerase-DNA cleavage complexes.

Many antitumor agents and antibiotics affect cells by interacting with type II topoisomerases, stabilizing a covalent enzyme-DNA complex. A pathway of recombination can apparently repair this DNA damage. In this study, transposon mutagenesis was used to identify possible components of the repair pathway in bacteriophage T4. Substantial increases in sensitivity to the antitumor agent m-AMSA [4'-(9-acridinylamino)methanesulfon-m-anisidide] were found with transposon insertion mutations that inactivate any of six T4-encoded proteins: UvsY (DNA synaptase accessory protein), UvsW (unknown function), Rnh (RNase H and 5' to 3' DNA exonuclease), alpha-gt (alpha-glucosyl transferase), gp47.1 (uncharacterized), and NrdB (beta subunit of ribonucleotide reductase). The role of the rnh gene in drug sensitivity was further characterized. First, an in-frame rnh deletion mutation was constructed and analyzed, providing evidence that the absence of Rnh protein causes hypersensitivity to m-AMSA. Second, the m-AMSA sensitivity of the rnh-deletion mutant was shown to require a drug-sensitive T4 topoisomerase. Third, analysis of double mutants suggested that uvsW and rnh mutations impair a common step in the recombinational repair pathway for m-AMSA-induced damage. Finally, the rnh-deletion mutant was found to be hypersensitive to UV, implicating Rnh in recombinational repair of UV-induced damage.

Repair of topoisomerase-mediated DNA damage in bacteriophage T4.

Type II topoisomerase inhibitors are used to treat both tumors and bacterial infections. These inhibitors stabilize covalent DNA-topoisomerase cleavage complexes that ultimately cause lethal DNA damage. A functional recombinational repair apparatus decreases sensitivity to these drugs, suggesting that topoisomerase-mediated DNA damage is amenable to such repair. Using a bacteriophage T4 model system, we have developed a novel in vivo plasmid-based assay that allows physical analysis of the repair products from one particular topoisomerase cleavage site. We show that the antitumor agent 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) stabilizes the T4 type II topoisomerase at the strong topoisomerase cleavage site on the plasmid, thereby stimulating recombinational repair. The resulting m-AMSA-dependent repair products do not form in the absence of functional topoisomerase and appear at lower drug concentrations with a drug-hypersensitive topoisomerase mutant. The appearance of repair products requires that the plasmid contain a T4 origin of replication. Finally, genetic analyses demonstrate that repair product formation is absolutely dependent on genes 32 and 46, largely dependent on genes uvsX and uvsY, and only partly dependent on gene 49. Very similar genetic requirements are observed for repair of endonuclease-generated double-strand breaks, suggesting mechanistic similarity between the two repair pathways.

Double-strand break repair in tandem repeats during bacteriophage T4 infection.

Recombinational repair of double-strand breaks in tandemly repeated sequences often results in the loss of one or more copies of the repeat. The single-strand annealing (SSA) model for repair has been proposed to account for this nonconservative recombination. In this study we present a plasmid-based physical assay that measures SSA during bacteriophage T4 infection and apply this assay to the genetic analysis of break repair. SSA occurs readily in broken plasmid DNA and is independent of the strand exchange protein UvsX and its accessory factor UvsY. We use the unique features of T4 DNA metabolism to examine the link between SSA repair and DNA replication and demonstrate directly that the DNA polymerase and the major replicative helicase of the phage are not required for SSA repair. We also show that the Escherichia coli RecBCD enzyme can mediate the degradation of broken DNA during early, but not late, times of infection. Finally, we consider the status of broken ends during the course of the infection and propose a model for SSA during T4 infections.

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.

Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not.

Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.

Deletion of the essential gene 24 from the bacteriophage T4 genome.

We deleted the essential gene 24 from the genome of bacteriophage T4. The delta 24 phage is a conditional lethal mutant that can grow only when the host strain supplies the product of gene 24 in trans, or when the phage acquires a functional gene 24 by some type of recombination event. Thus, gene 24 can be used as a selectable marker, for example permitting transposition into the T4 genome and analyses of plasmid-phage recombination [Woodworth and Kreuzer, Mol. Microbiol. 6 (1992) 1289-1296; H.W.E. and K.N.K., manuscript submitted]. We also found that the promoter region of gene 24 allows a low level of autonomous plasmid replication in T4-infected cells, raising the possibility of a previously unrecognized mode of T4 replication initiation.

Differentially labeled mutant oligonucleotides for analysis of protein-DNA interactions.

We have developed a method to produce a set of four duplex oligonucleotides, each with a different labeled base at a given position, from one template-primer combination. The template oligonucleotide is synthesized with a mixture of all four bases at the position of interest, and the primer oligonucleotide hybridizes to the template at all bases 3' from the position of interest. Specifically labeled substrates are then produced by differential incorporation of each of the four labeled nucleotides in four separate reactions. This method is more cost-effective than synthesizing four separate duplex oligonucleotides with different base pairs at the position of interest. We have successfully used this method to test nucleotide substitutions at several positions of a DNA recognition site for the phage T4 type II DNA topoisomerase.