The T4 phage UvsW protein contains both DNA unwinding and strand annealing activities

UvsW protein belongs to the SF2 helicase family and is one of three helicases found in T4 phage. UvsW governs the transition from origin-dependent to origin-independent replication through the dissociation of R-loops located at the T4 origins of replication. Additionally, in vivo evidence indicates that UvsW plays a role in recombination-dependent replication and/or DNA repair. Here, the biochemical properties of UvsW helicase are described. UvsW is a 3' to 5' helicase that unwinds a wide variety of substrates, including those resembling stalled replication forks and recombination intermediates. UvsW also contains a potent single-strand DNA annealing activity that is enhanced by ATP hydrolysis but does not require it. The annealing activity is inhibited by the non-hydrolysable ATP analog (adenosine 5'-O-(thiotriphosphate)), T4 single-stranded DNA-binding protein (gp32), or a small 8.8-kDa polypeptide (UvsW.1). Fluorescence resonance energy transfer experiments indicate that UvsW and UvsW.1 form a complex, suggesting that the UvsW helicase may exist as a heterodimer in vivo. Fusion of UvsW and UvsW.1 results in a 68-kDa protein having nearly identical properties as the UvsW-UvsW.1 complex, indicating that the binding locus of UvsW.1 is close to the C terminus of UvsW. The biochemical properties of UvsW are similar to the RecQ protein family and suggest that the annealing activity of these helicases may also be modulated by protein-protein interactions. The dual activities of UvsW are well suited for the DNA repair pathways described for leading strand lesion bypass and synthesis-dependent strand annealing.     

Crystallographic and NMR analyses of UvsW and UvsW.1 from bacteriophage T4

The uvsWXY system is implicated in the replication and repair of the bacteriophage T4 genome. Whereas the roles of the recombinase (UvsX) and the recombination mediator protein (UvsY) are known, the precise role of UvsW is unclear. Sequence analysis identifies UvsW as a member of the monomeric SF2 helicase superfamily that translocates nucleic acid substrates via the action of two RecA-like motor domains. Functional homologies to Escherichia coli RecG and biochemical analyses have shown that UvsW interacts with branched nucleic acid substrates, suggesting roles in recombination and the rescue of stalled replication forks. A sequencing error at the 3'-end of the uvsW gene has revealed a second, short open reading frame that encodes a protein of unknown function called UvsW.1. We have determined the crystal structure of UvsW to 2.7A and the NMR solution structure of UvsW.1. UvsW has a four-domain architecture with structural homology to the eukaryotic SF2 helicase, Rad54. A model of the UvsW-ssDNA complex identifies structural elements and conserved residues that may interact with nucleic acid substrates. The NMR solution structure of UvsW.1 reveals a dynamic four-helix bundle with homology to the structure-specific nucleic acid binding module of RecQ helicases.     

Mutator mutations in bacteriophage T4 gene 32 (DNA unwinding protein).

Bacteriophage T4 gene 32 encodes a DNA unwinding protein required for DNA replication, repair, and recombination. Gene 32 temperature-sensitive mutations enhance virtually all base pair substitution mutation rates.     

Bacteriophage T4 helicase loader protein gp59 functions as gatekeeper in origin-dependent replication in vivo

Bacteriophage T4 initiates origin-dependent replication via an R-loop mechanism in vivo. During in vitro reactions, the phage-encoded gp59 stimulates loading of the replicative helicase, gp41, onto branched intermediates, including origin R-loops. However, although gp59 is essential for recombination-dependent replication from D-loops, it does not appear to be required for origin-dependent replication in vivo. In this study, we have analyzed the origin-replicative intermediates formed during infections that are deficient in gp59 and other phage replication proteins. During infections lacking gp59, the initial replication forks from two different T4 origins actively replicated both leading- and lagging-strands. However, the retrograde replication forks from both origins were abnormal in the gp59-deficient infections. The lagging-strand from the initial fork was elongated as a new leading-strand in the retrograde direction without lagging-strand synthesis, whereas in the wild-type, leading- and lagging-strand synthesis appeared to be coupled. These results imply that gp59 inhibits the polymerase holoenzyme in vivo until the helicase-primase (gp41-gp61) complex is loaded, and we thereby refer to gp59 as a gatekeeper. We also found that all origin-replicative intermediates were absent in infections deficient in the helicase gp41 or the single-strand-binding protein gp32, regardless of whether gp59 was present or absent. These results argue that replication from the origin in vivo is dependent on both the helicase and single-strand-binding protein and demonstrate that the strong replication defect of gene 41 and 32 single mutants is not caused by gp59 inhibition of the polymerase.     

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).     

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.     

The phage T4 protein UvsW drives Holliday junction branch migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.     

Locations of bacteriophage T4 origins of replication.

Partially replicated bacteriophage T4 DNA containing cytosine was isolated from cells 6.5 and 7 min after infection and cleaved with restriction endonuclease BglII or BamHI. Positions of replication eyes relative to the cleavage sites were observed by electron microscopy. Four groups of eyes were found. They are consistent with replication from origins located at map positions 34, 60, 73, and 86 kilobases. In individual molecules that contained two or three eyes, the distribution of the eyes agreed with the initiation of replication at more than one of these four assigned origins and possibly at two additional origins located near 15 and 110 kilobases, which were reported by P. M. Macdonald, R. M. Seaby, W. Brown, and G. Mosig (p. 111-116, in D. Schlessinger, ed., Microbiology--1983, 1983) and M. E. Halpern, T. Mattson, and A. W. Kozinski (Proc. Natl. Acad. Sci. U.S.A. 76:6137-6141, 1979).     

Recombination-dependent replication of plasmids during bacteriophage T4 infection

The replication of plasmids containing fragments of the T4 genome, but no phage replication origins, was analyzed as a possible model for phage secondary (recombination-dependent) replication initiation. The replication of such plasmids after T4 infection was reduced or eliminated by mutations in several phage genes (uvsY, uvsX, 46, 59, 39, and 52) that have previously been shown to be involved in secondary initiation. A series of plasmids that collectively contain about 60 kilobase pairs of the T4 genome were tested for replication after T4 infection. With the exception of those known to contain tertiary origins, every plasmid replicated in a uvsY-dependent fashion. Thus, there is no apparent requirement for an extensive nucleotide sequence in the uvsY-dependent plasmid replication. However, homology with the phage genome is required since the plasmid vector alone did not replicate after phage infection. The products of plasmid replication included long concatemeric molecules with as many as 35 tandem copies of plasmid sequence. The production of concatemers indicates that plasmid replication is an active process and not simply the result of passive replication after the integration of plasmids into the phage genome. We conclude that plasmids with homology to the T4 genome utilize the secondary initiation mechanism of the phage. This simple model system should be useful in elucidating the molecular mechanism of recombination-dependent DNA synthesis in phage T4.     

Bacteriophage T4 UvsW protein is a helicase involved in recombination, repair and the regulation of DNA replication origins.

Bacteriophage T4 UvsW protein is involved in phage recombination, repair and the regulation of replication origins. Here, we provide evidence that UvsW functions as a helicase. First, expression of UvsW allows growth of an (otherwise inviable) Escherichia coli recG rnhA double mutant, consistent with UvsW being a functional analog of the RecG helicase. Second, UvsW contains helicase sequence motifs, and a substitution (K141R) in the Walker 'A' motif prevents growth of the E.coli recG rnhA double mutant. Third, UvsW, but not UvsW-K141R, inhibits replication from a T4 origin at which persistent RNA-DNA hybrids form and presumably trigger replication initiation. Fourth, mutations that inactivate UvsW and endonuclease VII (which cleaves DNA branches) synergistically block repair of double-strand breaks. These in vivo results are consistent with a model in which UvsW is a DNA helicase that catalyzes branch migration and dissociation of RNA-DNA hybrids. In support of this model, a partially purified GST/UvsW fusion protein, but not a GST/UvsW-K141R fusion, displays ssDNA-dependent ATPase activity and is able to unwind a branched DNA substrate.     

Crystallization of the gene 45 protein from the DNA replication fork of bacteriophage T4

The gene 45 protein from bacteriophage T4 has been purified and is crystallized. This protein is part of the T4 DNA replication complex. The crystallized protein is active in complementation assays. X-ray diffraction analysis is in progress; data are measured for the native and several heavy atom derivatives. The crystals diffract to about 3.5-A resolution.     

DNA replication and head assembly in bacteriophage T4.

Phage DNA was accumulated in cells of E. coli B, infected with the phage T4DtsLB3 (gene 42), without the synthesis of late proteins (in the presence of chloramphenicol). Then (stage II), chloramphenicol was removed and further replication of the phage DNA suppressed with hydroxyurea and by simultaneously raising the temperature to 40 degrees. The media M9 or M9 with 1% amino acid were used; the times of addition of chloramphenicol and the hydroxyurea concentration were also varied. It was also shown that in medium M9, at stage II, chiefly early proteins were synthesized. In the medium containing amino acids, at stage II the following was observed: 1) DNA synthesis was entirely suppressed and a degradation of DNA occurred; 2) both early and late proteins were synthesized, with a predominance of the latter; 3) an assembly of the elements of the phage tails and capsids occurred without the neck and flagellum, and a small number of phage particles were also found; 4) the capsids, isolated in a sucrose density gradient after lysis with chloroform, contained the proteins Palt, P20, P23, P24, several unidentified proteins, and did not contain Pwac, P23, and P22, 5) the yield of viable phage varied from 0.05 to 15% per cell. Thus, the entire morphogenesis of T4 phage can occur without accompanying replication of phage DNA.     

Properties of bacteriophage T4 proteins deficient in replication repair

An epistasis group of mutations engendering increased sensitivity to diverse DNA-damaging agents was described previously in bacteriophage T4. These mutations are alleles of genes 32 and 41, which, respectively, encode a single-stranded DNA-binding protein (gp32) and the replicative DNA helicase (gp41). The mechanism by which the lethality of DNA damage is mitigated is unknown but seems not to involve the direct reversal of damage, excision repair, conventional recombination repair, or translesion synthesis. Here we explore the hypothesis that the mechanism involves a switch in DNA primer extension from the cognate template to an alternative template, the just-synthesized daughter strand of the other parental strand. The activities of the mutant proteins are reduced about 2-fold (for gp32) or 4-fold (for gp41) in replication complexes catalyzing coordinated synthesis of leading and lagging strands, in binding single-stranded DNA, promoting DNA annealing, and promoting branch migration. In striking contrast, the mutant proteins are strongly impaired in promoting template switching, thus supporting the hypothesis of survival by template switching.