The phage T4 uvs Y recombination protein stabilizes presynaptic filaments

The bacteriophage T4 uvsY protein is required for efficient recombination in T4-infected Escherichia coli cells. Previous in vitro work has shown that the purified uvsY protein is an accessory protein; it stimulates homologous pairing catalyzed by the phage uvsX protein (a RecA-like recombinase) under certain conditions. We show here that this effect can be traced, at least in part, to a UvsY-dependent stabilization of uvsX protein-single-stranded DNA complexes. These presynaptic filaments are one of the early obligatory intermediates in the strand exchange reaction between homologous single- and double-stranded DNAs. The mechanism of filament stabilization seems to involve a slower loss of UvsX subunits. A model that accounts for the data is presented in which both recombination proteins are incorporated into the presynaptic filament.     

Topographical effects of ultraviolet irradiation upon recombination in phage T4

Summary The relative increment in UV stimulated recombination frequencies for 23 regions of the phage T4r II gene has been measured. Three kinds of region appear to exist and have been termed UV insensitive, average, and sensitive regions. Two UV sensitive regions exist, one in cistron A and one in cistron B. There appears to be no definite correlation of UV recombination sensitive regions with UV mutational hot spots.To explain recombination topographies, a model for phage heterozygosis together with some evidence has been presented and discussed.With 9 Figures in the TextThis work was supported by research grant G-14209 of the National Science Foundation.     

Allele specificity of genetic recombination in T4 phage using the indicator crossing study method]

The diagrams of relative correction ability of eighteen rII mutants of T4 phage were constructed on the basis of two-factor crosses, which were grouped into indicator series. In each series a pair of closely linked compared markers was crossed against indicator ones, the latter being distant enough so as to avoid simultaneous correction with the compared marker. The differences between the frequencies of wild type recombinants in crosses of two compared markers with indicator ones remained constant within the series and can be used as a measure of the differences between the compared markers in their correction ability. Mutants of base substitution type have small but statistically significant differences in correction ability. Simultaneous substitution of two bases in one codon yields a mutant which shows higher correction ability when compared to the mutant obtained as a result of substitution of only one base in the same codon. Frame shift mutants show much wider range of correctibility: some of them are corrected more rarely and others more frequently than base substitution mutants are.     

Contritution of correction to genetic recombination in T4 phage measured by the effect of map contraction

Marker-dependence of the fine structure map contraction in T4 phage is studied in two-factor crosses between rIIB mutants separated by indicator distances. The genetic intervals, which were short as compared with mean length of the heteroduplex region in hybrid DNA molecules but which exceeded the length of the DNA strand involved in a single correction event, were selected as indicator ones. On the basis of a deviation of measured frequencies from additivity (map contraction) the marker-specific frequencies of wild type recombinants arising as a result of correction to the wild type (kappa (- leads to +)) were calculated. For the most of the marker studied both of the base substitution and frame shift type the frequencies kappa (- leads to +) have the values below 2.10(-4). In the case of three most highly corrected frame shift markers with kappa (- leads to +) being 14.10(-4)--17.10(-4), about ten percent of all mismatched regions are corrected to the wild type.     

The structure of rII diploids of phage T4

Summary Crosses between the rII deletion 1589 and an overlapping deletion such as 638 which lies entirely within the rIIB cistron generate a few T4 phage particles, the so-called rII diploids, which contain two copies of the rII region, one derived from each parent in the cross. A specific model is proposed to account for the properties of these rII diploids. This model postulates: 1) the rII diploids contain a tandem duplication, 2) the duplicated region extends both to the left and right of the rII region itself, and 3) during phage multiplication recombination occurs between homologous regions of the duplication. These assumptions lead to precise predictions on the following points: 1) the frequency at which haploid 1589 and 638 phage particles are generated during multiplication, 2) the ratio of 1589 and 638 phage amongst the segregants, 3) the relative lengths of terminal redundancy to be found in the rII diploids, the 1589 and 638 segregants and wild-type T4, and 4) the formation and properties of homozygous diploids containing two copies either of the 1589 or 638 region.Experiments are reported which validate the model on these points and also indicate how the homozygous diploids can be utilized to generate new rII diploids with structures which would otherwise be unobtainable.     

T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms

Both T4 phage and DNA-free ghosts inhibit replication of RNA phage f2. Most but not all of the effects by T4 upon f2 growth can be blocked by the addition of rifampicin prior to T4 superinfection; by contrast, the inhibition of f2 synthesis by T4 ghosts cannot be blocked by rifampicin. This indicates that inhibition by intact T4 requires gene function, while inhibition by ghosts does not. There is a small, multiplicity-dependent inhibition by viable T4 on f2 growth in the presence of rifampicin which may be similar to the gene function-independent inhibition by T4 ghosts. With one viable T4 per cell, there appears to be no effect by viable T4 upon f2 growth which does not require T4 gene action. Moreover, increasing multiplicities of viable T4 appear to inhibit T4 replication as well.In the absence of rifampicin, pre-existing f2 single and double-stranded RNA are degraded after superinfection by viable T4, but remain stable after superinfection by ghosts. However, no new f2 RNA is synthesized after superinfection with either. In the presence of rifampicin, f2-specific protein synthesis is largely unaffected by viable T4, but is completely inhibited by ghosts. Both Escherichia coli, as well as f2-speciflc polysomes disappear in the presence of ghosts.We conclude that, at low multiplicities, T4 phage and T4 ghosts inhibit replication of f2 phage, and presumably host syntheses, by different mechanisms.     

Recombination apparatus of T4 phage

The substantial process of general DNA recombination consists of production of ssDNA, exchange of the ssDNA and its homologous strand in a duplex, and cleavage of branched DNA to maturate recombination intermediates. Ten genes of T4 phage are involved in general recombination and apparently encode all of the proteins required for its own recombination. Several proteins among them interact with each other in a highly specific manner based on a protein-protein affinity and constitute a multicomponent protein machine to create an ssDNA gap essential for production of recombinogenic ssDNA, a machine to supply recombinogenic ssDNA which has a free end, or a machine to transfer the recombinogenic single strand into a homologous duplex.     

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.     

Gene function of heterozygotes in phage T4

Summary Gene function of various T4-heterozygotes was tested. About half of the HETs containing wild type and anam-mutation disappeared under non-permissive conditions, if theam-defect concerned early functions. The same was found when phages, heterozygous forr ⁺ and anrII-point-mutation, were adsorbed to K12 (). A much more extensive loss of HETs in K could be observed if anrIIA- and anrIIB-point-mutation (block-mutations showed different results) occurred together in a non-recombinant heterozygote. The findings provide evidence that one class of T4-heterozygotes has a heteroduplex DNA-structure.With 3 Figures in the Text     

Folding kinetics of phage T4 thioredoxin

The folding mechanism for bacteriophage T4 thioredoxin is best described by a four-state box mechanism, N----Uc----Ut----It----N, where N indicates native, Uc the unfolded form with the cis proline isomer, Ut unfolded with the trans proline isomer, and It a compact form with a trans proline isomer. Both manual mixing fluorescence and size-exclusion chromatography indicate that there is a cis-trans proline isomerization that is important to the folding pathway. Furthermore, the data suggest that the cis-trans isomerization can also occur in a compact nativelike state which is referred to as It. The slow phase seen in fluorescence seems to be monitoring the cis-trans isomerization in the compact form, not the isomerization which occurs in the denatured state.