uvsX Gene Research Articles

Double-strand break repair and recombination-dependent replication of DNA in bacteriophage T4 in the absence of UvsX recombinase: Replicative resolution pathway

The effects of mutations in bacteriophage T4 genes uvsX and 49 on the double-strand break (DSB)-promoted recombination were studied in crosses, in which DSBs were induced site-specifically within the rIIB gene by SegC endonuclease in the DNA of only one of the parents. Frequency of rII+ recombinants was measured in two-factor crosses of the type i×ets1 and in three-factor crosses of the type i×ets1 a6, where ets1 is an insertion in the rIIB gene carrying the cleavage site for SegC; i's are rIIB or rIIA point mutations located at various distances (12–2040bp) from the ets1 site, and a6 is rIIA point mutation located at 2040bp from ets1. The frequency/distance relationships were obtained in crosses of the wild-type phage and of the amber mutant S17 (gene uvsX) and the double mutant S17 E727 (genes uvsX and 49). These data provide information about the frequency and distance distribution of the single-exchange (splices) and double-exchange (patches) events. The extended variant of the splice/patch coupling (SPC) model of recombination, which includes transition to the replication resolution (RR) alternative is substantiated and used for interpretation of the frequency/distance relationships. We conclude that the uvsX mutant executes recombination-dependent replication but does it by a qualitatively different way. In the absence of UvsX function, the DSB repair runs largely through the RR subpathway because of inability of the mutant to form a Holliday junction. In the two-factor crosses, the double uvsX 49− is recombinationally more proficient than the single uvsX mutant (partial suppression of the uvsX deficiency), while the patch-related double exchanges are virtually eliminated in this background.

On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage

The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase.

Complementation of bacteriophage induction and recombination defects in Escherichia coli RecA(-) mutants by expression of the cloned T4 bacteriophage uvsX gene.

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA(-) mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present.

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.

Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation

The recA gene of Escherichia coli is the prototype of therecA/RAD51/DMC1/uvsX gene family of strand transferases involved in genetic recombination. In order to find mutations in the recA gene important in catalytic turnover, a genetic screen was conducted for dominant lethal mutants. Eight single amino acid substitution mutants were found to prevent proper chromosome segregation and to kill cells in the presence or absence of an inducible SOS system. All mutants catalyzed some level of recombination and constitutively stimulated LexA cleavage. The mutations occur at the monomer-monomer interface of the RecA polymer or at residues important in ATP hydrolysis, implicating these residues in catalytic turnover. Based on an analysis of the E96D mutant, a model is presented in which slow RecA-DNA dissociation prevents chromosome segregation, engendering lexA -independent, lethal filamentation of cells.

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.

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).

Frameshift and double-amber mutations in the bacteriophage T4 uvsX gene: Analysis of mutant UvsX proteins from infected cells

The bacteriophage T4 uvsX gene encodes a 43 kDa, single-stranded DNA-dependent ATPase, double-stranded DNA-binding protein involved in DNA recombination, repair and mutagenesis. Mutants of uvsX have a DNA-arrest phenotype and reduced burst size. Western blot immunoassay of UvsX peptides made by a number of amber mutants revealed amber peptides ranging from 25-32 kDa. Wild-type UvsX protein was also detected in lysates of cells infected with uvsX amber mutants, suggesting that their mutations are suppressed by translational ambiguity. We investigated the effects of mutations near the 5' end of uvsX. A frameshift mutation was engineered at codon 33. Western immunoblots for UvsX protein demonstrated that the frameshift mutant expresses no detectable wild-type UvsX; instead, a 37 kDa reactive peptide was detected. In order to determine if this peptide represents truncated UvsX protein, the mutation was regenerated in the cloned uvsX gene and expressed in transformed Escherichia coli. Endopeptidase digestion of the 37 kDa protein from the cloned gene generated peptide fragments indistinguishable from those obtained from wild-type UvsX. A double-amber mutant of uvsX was also generated by oligonucleotide site-directed mutagenesis. No UvsX protein was detected in lysates of cells infected with the uvsXam64am67 double mutant. Plaque size and sensitivity to UV inactivation for both the double-amber and the frameshift mutants were indistinguishable from those of other uvsX mutants. Mutations in uvsY had no demonstrable effect on efficiency of plating or UV sensitivity of uvsX mutants. Thus, null mutants of uvsX are viable.

Transcript Analyses of the uvsX-40-41 Region of Bacteriophage T4: Changes in the RNA as Infection Proceeds

The bacteriophage T4 genes uvsX (recombination protein), 40 (stimulates head formation), and 41 (DNA replication protein, part of the primase-helicase) are located together on the T4 genome (5'----3' uvsX-40-41). Previous analyses have indicated that all three proteins are expressed within 5 min after infection and that the level of 41 protein is less than that of uvsX. The mapping of transcripts from this region (reported here) shows that this expression arises from polycistronic messages detected between 2-4 min after infection, a time when phage-encoded factors are beginning to alter the host transcriptional apparatus. Major RNA 5' ends, 900 and 200 bases upstream of uvsX, show homology with previously deduced T4 transcription sites dependent on the T4 transcription factor motA (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258). Analysis of the 3' end of uvsX RNAs shows that initially most transcripts extend through gene 40 and 41, although approximately equal to one-fourth end just past uvsX (within gene 40). Later, more of the uvsX messages are monocistronic, having 5' ends close to the gene (200 and 55 bases upstream) and having the 3' end within gene 40. Thus, during infection the level of 41 RNA is lowered relative to uvsX message. Mapping of RNA expressed from an uvsX-40-41 plasmid in an uninfected cell gives 5' ends 700, 450, and 55 bases upstream of uvsX, i.e. positions different from those during T4 infection. This indicates that infection significantly changes the 5' ends for uvsX RNA, either by altering transcription initiation or RNA processing sites. In contrast, the majority of the uvsX RNAs expressed by plasmid in the uninfected cell do end at the stop mapped during infection. Thus, the host alone can produce this 3' end.

Altered Expression of the Bacteriophage T4 Gene 41 (Primase-Helicase) in an Escherichia coli rho Mutant

Bacteriophage T4 gene 41 protein is an essential replication protein, part of the primase-helicase required for lagging strand DNA synthesis. In a T4+ infection, 41 RNA is first expressed as a polycistronic transcript attached to the upstream RNA of genes uvsX (recombination protein) and 40 (stimulates head formation (Hinton, D. M. (1989) J. Biol. Chem. 264, 14432-14439). As infection proceeds, less of the upstream RNA extends into gene 41 due to an RNA 3' end, approximately equal to 60 bases downstream of uvsX. DNA sequence analysis of this region positions this end within gene 40, immediately after a GC-rich hairpin. This end probably arises from host factor-dependent transcription termination or RNA processing since it is observed in RNA expressed by a uvsX-40-41 plasmid in vivo, but is not seen after in vitro transcription with purified Escherichia coli RNA polymerase. The E. coli transcription termination (rho) mutant rho026 has been characterized as a rho mutation whose terminating activity is not effectively overcome by phage lambda antitermination (Das, A., Gottesman, M. E., Wardwell, J., Trisler, P., and Gottesman, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 5530-5534). During a T4+ abortive infection of rho026, the levels of some phage proteins, including 41, are depressed; a T4 phage mutant in goF gives wild type protein patterns in rho026 (Stitt, B. L., and Mosig, G. (1989) J. Bacteriol., in press). The RNA analyses presented here demonstrate that the severalfold decrease in 41 protein in rho026 is accompanied by a similar decrease in 41 RNA. There is both a general reduction in polycistronic uvsX-40-41 RNA and a 2-2.5-fold increase in the proportion of uvsX RNA ending at the 3' end. Infection of rho026 by T4 goF1 returns the relative amount of RNA reading into 41 versus that stopped to near a wild type level. These results suggest that host rho and the T4 goF are involved in the expression of T4 41 RNA.

Tertiary initiation of replication in bacteriophage T4. Deletion of the overlapping uvsY promoter/replication origin from the phage genome.

Tertiary initiation of bacteriophage T4 DNA replication is resistant to the RNA polymerase inhibitor rifampicin and apparently involved in the activity of recombination hot spots in the T4 genome (Kreuzer, K. N., and Alberts, B. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3345-3349). One of the origins that function by the tertiary mechanism maps at the promoter for gene uvs Y. A deletion and a linker-insertion mutation in the uvsY promoter/origin region were generated by in vitro manipulations and then placed into the T4 genome using the insertion/substitution system (Selick, H. E., Kreuzer, K. N., and Alberts, B. M. (1988) J. Biol. Chem. 263, 11336-11347). Both resulting phage strains are uvsY- mutants, but they differ in that one has a deletion of the minimal tertiary origin and the other does not. The effects of the uvsY mutations on tertiary origin activity were assayed by infecting tertiary origin plasmid-bearing Escherichia coli with the two phage mutants. The tertiary origin plasmids replicated extensively after infection by either uvsY- phage mutant, demonstrating that the uvsY protein is not required for tertiary initiation. The extent of plasmid replication was increased dramatically as a result of either mutation, indicating that the uvsY protein plays some negative role in either the initiation or subsequent processing of plasmid replicative intermediates. The phage strain with an origin deletion induced the replication of a tertiary origin plasmid with which it shared no homology. Therefore, plasmid-phage recombination is not required for the replication of tertiary origin plasmids. The replication of a tertiary origin plasmid is also shown to be independent of the phage genes uvsX, 59, and 46, but markedly reduced by mutations in the T4-induced topoisomerase.

Function of cloned T4 recombination genes, uvsX and uvsY, in cells of Escherichia coli.

Genes uvsX and uvsY of bacteriophage T4 both control genetic recombination and repair of damaged DNA, and their mutant phenotypes bear a striking resemblance to each other. It has been shown recently that the uvsX gene product is analogous to the recA gene product of Escherichia coli (Yonesaki et al. 1985; Yonesaki and Minagawa 1985; Formosa and Alberts 1986), but the function of the uvsY gene is unknown. To obtain further insight into the function of these genes we introduced plasmidborne copies of the two genes separately or together into E. coli. The uvsX gene rendered recA- cells more resistant to UV and raised the recombination frequency of lambda phage and E. coli, but hampered induction of the lambda prophage and the SOS function of E. coli. The uvsY gene had no detectable function when introduced alone into E. coli but significantly enhanced the function of the uvsX gene when the two plasmid-borne genes were introduced together.

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein.

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.

Studies on the recombination genes of bacteriophage T4: suppression of uvsX and uvsY mutations by uvsW mutations.

Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42 degrees and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41 degrees the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30 degrees it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41 degrees was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.

Purification and characterization of the T4 bacteriophage uvsX protein.

Gene uvsX of bacteriophage T4 encodes a 40,000-dalton protein that plays a key role in the major pathway for genetic recombination in T4-infected cells. Mutations at the uvsX locus lead to increased sensitivity to various DNA-damaging agents, reduced phage bursts, decreased genetic recombination, and early arrest of DNA synthesis. Like the Escherichia coli recA protein, the purified uvsX protein is a DNA-dependent ATPase that catalyzes pairing between homologous single- and double-stranded DNA molecules in vitro (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H., (1985) Eur. J. Biochem. 148, 127-134). At physiological salt concentrations, the uvsX protein binds tightly and cooperatively to single-stranded DNA, covering about five nucleotides per protein monomer; at lower salt concentrations, a similar type of binding to double-stranded DNA is detected (Griffith, J., and Formosa, T., (1985) J. Biol. Chem. 260, 4484-4491). We show here that the ATPase activity of this protein is unusual in producing both ADP plus Pi and AMP plus PPi as products. Generating the fully active form of the ATPase is a cooperative process, apparently requiring that a protein monomer be bound to single-stranded DNA while surrounded by other ATP-bound monomers. The catalysis of homologous pairing by the uvsX protein is shown to be greatly stimulated by the presence of the T4 gene 32 protein, a helix-destablizing protein previously studied in this laboratory, and it requires continued ATP hydrolysis. We present a method that allows the purification of the uvsX protein to essential homogeneity. We also describe the complete purification of two proteins that bind to the uvsX protein: the T4 uvsY protein (16,000 daltons) and an E. coli host protein of 32,000 daltons whose gene is unknown. The host protein is likely to play a role in DNA metabolism, because it also binds to the T4 gene 32 protein and to DNA; the sequence of its amino-terminal 29 amino acids has been determined.

Cloning of the bacteriophage T4 uvsX gene and purification and characterization of the T4 uvsX recombination protein.

The bacteriophage T4 uvsX gene is a nonessential gene required for normal levels of DNA repair, recombination, and replication. We demonstrate that plasmids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologically active uvsX protein. This uvsX protein imparts increased survival to UV-irradiated T4 uvsX- phage and decreases the T4 uvsX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recombination nuclease. The uvsX protein purified from cells with a uvsX+ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix-destablizing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA. These results agree with the recent characterization of uvsX protein from T4-infected cells by Yonesaki et al. (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148, 127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B.M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 363-370). In addition, we find that under some reaction conditions strand exchange is catalyzed by uvsX protein in the absence of 32 protein. The level of the uvsX protein expressed by the uvsX+ plasmids is high and independent of the orientation of the T4 DNA within the vector. This suggests that transcription promoter(s) lie upstream of the uvsX gene on the cloned T4 DNA. In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the uvsX gene and extend through the gene.

T4 phage gene uvsX product catalyzes homologous DNA pairing.

Gene uvsX of phage T4 controls genetic recombination and the repair of DNA damage. We have recently purified the gene product, and here describe its properties. The protein has a single-stranded DNA-dependent ATPase activity. It binds efficiently to single- and double-stranded DNAs at 0 degrees C in a cooperative manner. At 30 degree C the double-stranded DNA-protein complex was stable, but the single-stranded DNA-protein complex dissociated rapidly. The instability of the latter complex was reduced by ATP. The protein renatured heat-denatured double-stranded DNA, and assimilated linear single-stranded DNA into homologous superhelical duplexes to produce D-loops. The reaction is stimulated by gene 32 protein when the uvsX protein is limiting. With linear double-stranded DNA and homologous, circular single-stranded DNA, the protein catalyzed single-strand displacement in the 5' to 3' direction with the cooperation of gene 32 protein. All reactions required Mg2+, and all except DNA binding required ATP. We conclude that the uvsX protein is directly involved in strand exchange and is analogous to the recA protein of Escherichia coli. The differences between the uvsX protein and the recA protein, and the role of gene 32 protein in single-strand assimilation and single-strand displacement are briefly discussed.

Genetic rearrangement of DNA induces knots with a unique topology: implications for the mechanism of synapsis and crossing-over.

We have determined the topological sign of the knots produced by a cycle of phage lambda integrative recombination. To insure that these knots reflect intrinsic features of the reaction mechanism, the substrate was constructed so that random interwrapping of segments of DNA played a minimal role in the topological outcome. The knotted DNA was coated with the bacteriophage T4 uvsX gene product and examined in the electron microscope to determine the nature of each crossing point or node. All of the knots were identical; they were trefoils with three nodes of positive sign. We interpret this result to mean that one recombination site, which previous work had indicated is organized into a nucleosome-like structure, is wrapped with a handedness identical to that found in nucleosomes. Therefore, this wrapping may explain the dependence of recombination on supercoiling of the substrate DNA. Moreover, we show that the topological result sharply limits acceptable mechanisms for the details of strand exchange.

Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvsX and uvsY.

The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination. Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Their relative molecular masses are 39 000 and 16 000, respectively. In the normal wild-type infection process they are produced early but not late in infection. Their synthesis continues for a longer period when DNA synthesis is blocked. We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye. The purification procedures suggest that these products may be membrane proteins. Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single-stranded fd DNA fragment into superhelical double-stranded fd DNA (RFI). The reaction requires ATP and Mg2+ besides substrate DNAs and uvsX protein. The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments.

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.