Recombination in amber-mutants of bacteriophage T4B. I. Recombination and complementation in phage T4 amber-mutants]

In studying intergenic and intragenic complementation in amber mutants in genes of phage T4 controlling the synthesis of phage tail fibres the data have been obtained indicating the dependency of the results of complementation tests on those of crosses of respective markers. The results obtained show that in complementation of amber mutants of phage T4 the phage yield varies widely and depends on the location of markers on the phage genetic map.     

Region-Specific Recombination in Phage T4. I. a Special Glucosyl-Dependent Recombination System

In this communication, we describe a recombination mechanism in bacteriophage T4D that acts only on glycosylated phage, acts in some regions of the genome, but not others, and is heat sensitive, showing decreasing activity with increasing temperature.     

Marker-Dependent Recombination in T4 Bacteriophage. IV. Recombinational Effects of Antimutator T4 DNA Polymerase

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3' -> 5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49) -> 3'->5' exonuclease (gp43) -> DNA polymerase (gp43) -> DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates.     

Region-Specific Recombination in Phage T4. II. Structure of the Recombinants

In this paper, we present results of crosses designed to elucidate the structure of recombinants in the tail-fiber region of bacteriophage T4, in which a glucosylation-dependent recombinations mechanism is operative, and the cause of the "special" recombination in glycosylated crosses is discussed. We present evidence that, when phage are nonglycosylated, recombination in the tail-fiber region proceeds via long heteroduplex overlaps. Mismatched bases within such regions (in nonglycosylated phage) are repaired efficiently (as contrasted to those of glucosylated phage), but asymmetrically; that is, there may be an equal probability of resolving the mismatch to mutant or wild type.     

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.     

Marker-Dependent Recombination in T4 Bacteriophage. III. Structural Prerequisites for Marker Discrimination

Distance- as well as marker-dependence of genetic recombination of bacteriophage T4 was studied in crosses between rIIB mutants with known base sequences. The notion of a "basic recombination," which is the recombination within distances shorter than hybrid DNA length in the absence of mismatch repair and any marker effects, was substantiated. The basic recombination frequency per base pair can serve as an objective parameter (natural constant) of general recombination reflecting its intensity. Comparative studies of the recombination properties of rIIB mutants with various sequence changes in the mutated sites showed that the main factor determining the probability of mismatch repair in recombination heteroduplexes is the length of a continuous heterologous region. A run of A:T pairs immediately adjoining the mismatch appears to stimulate its repair. In the case of mismatches with DNA strands of unequal length, formed by frameshift mutations, the repair is asymmetric, the longer strand (bulge) being preferentially removed. A pathway for mismatch repair including sequential action of endonuclease VII (gp49)----3'----5' exonuclease (gp43)----DNA polymerase (gp43)----DNA ligase (gp30) was proposed. A possible identity of the recombinational mismatch repair mechanism to that operating to produce mutations via sequence conversion is discussed.     

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid: III. Formation of Long Single Strands During Recombination

Evidence was presented to support the hypothesis that long single strands appearing at late times (15 min after infection) are produced as a result of recombination and not as a continuous elongation during the replication process. The production of long strands does not depend on the multiplicity of infection, and the first long strands appear at the time when 20 to 50 phage equivalent units of deoxyribonucleic (DNA) are synthesized, and not earlier. The addition of chloramphenicol at 5 min, which prevents molecular recombination but allows replication of DNA, prevents the formation of long, single strands. Chloramphenicol added between 8 and 10 min after infection, a time at which molecular recombination is fully expressed and covalent repair of recombinant molecules is allowed, does not prevent formation of long single strands. Cutting of single-strand DNA with a limited amount of endonuclease I allows confirmation that the fast-sedimenting characteristic of intracellular denatured DNA is caused primarily by the length of the strands, and not by the formation of aggregates. The computer simulation of two recombination models indicates the feasibility of random breakage and rejoining of molecules in generating long concatenates.     

Recombination in phage T4 gene-43 (DNA polymerase) mutants.

Summary The effect of phage T4 gene 43 (DNA polymerase) mutations on recombination between adjacent base pairs was measured in rII amber and opal mutants.The mutator allele tsL56 did not promote recombination frequencies at the two sites in which its effect was studied. The antimutator allele tsCB87 caused slight or no reduction in recombination frequencies at five sites.Abbreviations A, T, G and C are adenine, thymine, guanine and 5-hydroxymethylcytosine, respectively     

Replication and Recombination in Ligase-Deficient rII Bacteriophage T4D

Deoxyribonucleic acid replication and genetic recombination were investigated after infection of Escherichia coli with ligase-deficient rII bacteriophage T4D. The major observations are: (i) deoxyribonucleic acid synthesis is discontinuous, (ii) the discontinuities are more slowly repaired than in wild-type infection, (iii) host ligase is required for viability, and (iv) genetic recombination is increased.     

Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

Parent-to-Progeny Transfer and Recombination of T4rII Bacteriophage

Transfer of parental, light (not substituted with 5-bromodeoxyuridine) (32)P-deoxyribonucleic acid (DNA) from rII(-) mutants of T4 bacteriophage to heavy (5-bromodeoxyuridine-substituted) progeny in Escherichia coli B was less homogeneous than in wild phages. The net transfer was 5 to 20% of the value for wild T4 phage, and the parental contribution per progeny DNA molecule amounted to 7 to 100% of the genome. Three classes could be distinguished, based on the density distribution of parental label in CsCl analysis of the progeny phages. "Far recombined" phages contain parental material only in semiconservatively replicated subunits covalently attached to progeny DNA, amounting to 5 to 10% parental contribution per genome. "Intermediate recombinants" contain, aside from conventional recombinant DNA, parental DNA banding at the original, light density. This DNA may be unattached to heavy progeny DNA or attached by weak bonds which are very sensitive to shearing during the extraction procedure. The parental contribution is 10 to 50% per progeny DNA molecule in this class. "Conservative" phages band close to the parental, light density in CsCl; their DNA is purely light. When the parental phage is labeled with both (3)H-leucine (capsid) and (32)P (DNA), the specific activity of (3)H/(32)P in the "conservative progeny" is 10 to 40% of that in the parental, showing that at least some of the (32)P in this area belongs to phages with parental DNA as the sole DNA component inside an unlabeled capsid, i.e., parental DNA which has been injected into the host and matured in a new capsid without replication or recombination. This phenomenon occurs to about the same extent in both single and multiple infection.     

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid II. Single-Strand Breaks and Exposure of Uncomplemented Areas as a Prerequisite for Recombination

Deoxyribonucleic acid (DNA) from several "DNA-deficient" amber mutants was observed to be either nicked (amber 22, 82, 122, and wild type) or cut (amber 453) after injection into a nonpermissive host. This effect was inhibited by chloramphenicol (CM), indicating that it is due to phage-induced enzymes. Although most of the mutants tested for replication in a density-label system were in fact DNA-deficient (amber 22, 82, 122), one (amber 81) was found to replicate almost identically to the wild type, and another (amber 453) was found to assume a hybrid density only. The hybrid moiety was less than, or equal to, one phage equivalent length, and was more efficiently extracted from infected bacteria than was similarly replicated DNA from wild-type phage. Interparental recombination between heavy and light parental DNA was observed for amber 82, 122, and wild type, but was not observed for amber 453; it was inhibited by CM. In contrast to amber 82 and wild type, the amber 453 intracellular DNA does not have single-strand regions, Because amber 453, unlike amber 82 and wild type T4, does not recombine, nicking and exposure of single-strand regions is postulated to be a prerequisite for recombination.     

Recombination between bacteriophage T4 and plasmid pBR322 molecules containing cloned T4 DNA

Reciprocal recombination between T4 DNA cloned in plasmid pBR322 and homologous sequences in bacteriophage T4 genomes leads to integration of complete plasmid molecules into phage genomes. Indirect evidence of this integration comes from two kinds of experiments. Packaging of pBR322 DNA into mature phage particles can be detected by a DNA--DNA hybridization assay only when a T4 restriction fragment is cloned in the plasmid. The density of the pBR322 DNA synthesized after phage infection is also consistent with integration of plasmid vector DNA into vegetative phage genomes. Direct evidence of plasmid integration into phage genomes in the region of DNA homology comes from genetic and biochemical analysis of cytosine-containing DNA isolated from mature phage particles. Agarose gel electrophoresis of restriction endonuclease-digested DNA, followed by Southern blot analysis with nick-translated probes, shows that entire plasmid molecules become integrated into phage genomes in the region of T4 DNA homology. In addition, this analysis shows that genomes containing multiple copies of complete plasmid molecules are also formed. Among phage particles containing at least one integrated copy, the average number of integrated plasmid molecules is almost ten. A cloning experiment done with restricted DNA confirms these conclusions and illustrates a method for walking along the T4 genome.     

Marker-Dependent Recombination in T4 Bacteriophage. II. the Evaluation of Mismatch Repairabilities in Crosses within Indicator Distances

The contribution of mismatch repair to genetic recombination in T4 phage has been evaluated by three independent approaches: (1) testing for non-additivity of recombinant frequencies; (2) measurements of double exchange frequencies in three-factor crosses: (3) comparisons of recombination abilities of mutations occupying the same site. Quantitative agreement among the results of these approaches suggests that within distances much less than the mean length of hybrid regions, mismatch repair accounts perfectly for high negative interference as measured in three-factor crosses and as manifested by non-additivity in two-factor crosses. The mismatch repair mechanism readily recognizes only particular mismatches, the repair frequency being dependent on the base sequence in both strands of the mismatched region.     

Late Events in T4 Bacteriophage Production. II. Giant Bacteriophages Contain Concatemers Generated by Recombination

Analysis of "giant" phage, which package concatenated DNA into their capsids, shows that they are predominantly heterozygous. The results are compatible with the hypothesis that concatemers are generated by recombination.     

Variations in Genetic Recombination Due to Amber Mutations in T4D Bacteriophage

Recombination experiments were performed to assess the affect of amber mutations in 12 genes of T4D bacteriophage on genetic recombination. Crosses were performed in various suppressor-containing bacterial hosts to permit the production of progeny phage. Amber mutations in genes 32, 46, and 47 caused decreased recombination, amber mutations in genes 30, 41, 42, 43, 56, 61, and 62 caused increased recombination, whereas mutations in genes 63 and 37 showed no demonstrable effect on recombination.     

Marker-Dependent Recombination in T4 Bacteriophage. I. Outline of the Phenomenon and Evidence Suggesting a Mismatch Repair Mechanism

In standard crosses, some rIIB mutants of T4 phage were found to be susceptible to an extra recombination mechanism to which the other mutants were much less susceptible. The following observations were interpreted as evidence for the mismatch-repair nature of the phenomenon: (1) Marker-dependent recombination generates exclusively double exchanges at both sides of the marker. (2) Marker-dependent recombination is highly sensitive to DNA base sequence at the site of the marker and to that at the corresponding site on the chromosome of the other parent. (3) Within certain limits, the contribution of the marker-dependent mechanism to the total recombination frequency is distance-independent and thus constitutes a constant component.