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.     

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.     

In vivo study of fidelity of DNA double-strand break repair in bacteriophage T4

A method for in vivo studying the fidelity of DNA double-strand break (DSB) repair in bacteriophage T4 has been developed. The frequency of reversion of rII mutations to the wild phenotype was measured in i segC+ x i ets 1 segCDelta crosses, where ets 1 is an insertion in the initial part of the rII gene carrying a sequence recognized by SegC endonuclease; i designates a rIIB or rIIA mutation located at some distance from ets 1, and segCDelta is a deletion in the segC gene. In such cross, a DSB occurs in the site of ets 1. Their repair involves genetic recombination and DNA replication in the neighborhood of ets 1. In parallel, the frequency of reversion of the same i mutant in the absence of DSBs is measured in i x i self-crosses. Reversions of different types (base substitutions, deletions, insertions) can be studied with the use of structurally different i mutations located at varying distances from ets 1. The reversion frequencies were determined for three rIIB mutations and one rIIA mutation. The results obtained suggest that DSB repair in bacteriophage T4 is a process of high fidelity with the rate of errors that does not essentially exceed that in the case of usual phage multiplication.     

rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.     

Focused genetic recombination of bacteriophage t4 initiated by double-strand breaks

A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCDelta strain of bacteriophage T4 was developed. The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene. Under segC(+) conditions, the ets1 behaves as a recombination hotspot. Crosses of the ets1 against rII markers located to the left and to the right of ets1 gave similar results, thus demonstrating the equal and symmetrical initiation of recombination by either part of the broken chromosome. Frequency/distance relationships were studied in a series of two- and three-factor crosses with other rIIB and rIIA mutants (all segC(+)) separated from ets1 by 12-2100 bp. The observed relationships were readily interpretable in terms of the modified splice/patch coupling model. The advantages of this localized or focused recombination over that distributed along the chromosome, as a model for studying the recombination-replication pathway in T4 in vivo, are discussed.     

The Riia Gene of Bacteriophage T4. II. Regulation of Its Messenger RNA Synthesis

When the rII genes are first introduced into cells which had been previously infected by T4 phage deleted for these genes, the kinetics of synthesis of rIIA and rIIB RNA are rapid and identical. We show that this rapid synthesis depends on a functional motA gene for rIIB, but not for rIIA, RNA synthesis. By primer-extension mapping of T4 messenger RNA, we find three promoters close to the rIIA gene. One of them is an early promoter just before the rIIA.1 gene; it is used under all conditions tested. Another is in the coding portion of the rIIA.1 gene; it is weak, primarily because of a 19-bp spacing between the -10 and -35 elements, and its use is stimulated by T4 functions. The third is a motA-dependent (middle) promoter which has an unusual CCCGCTT box at -33. We present results which suggest that none of these promoters is likely to be the site at which the motB and motC gene products exercise their major influence on rIIA RNA synthesis.     

Mutations that detoxify an aberrant T4 membrane protein

We have investigated suppressors of the bacteriophage T4 rIIB toxic polypeptide encoded by the rIIB frameshift mutation FC238. We have found suppressors that eliminate the toxic polypeptide by creating new translational termination codons, that diminish the toxicity of the polypeptide by altering the amino acid sequence of the toxic protein, that alter the rIIA protein so as to influence toxicity, and that diminish the amount of toxic polypeptide by reducing the quantity of gene expression from the rIIB (FC238) gene. We propose that the toxicity of the FC238 polypeptide derives from its peculiar, bipartite structure and high membrane avidity. Suppressors that detoxify the FC238 polypeptide by missense probably disturb the bipartite structure and/or the affinity for the membrane. The distribution of transition mutations obtained with a variety of mutagens contributes to an appreciation of intrinsic mutability differences. Lastly, although suppressors of FC238 toxicity might emerge in phage genes other than rIIB and rIIA, none have been found.     

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 (12-2040 bp) from the ets1 site, and a6 is rIIA point mutation located at 2040 bp 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.     

Gene rIIB from bacteriophage T4 in genetic recombination

The effect of the rIIB gene on genetic recombination in bacteriophage T4 was studied. Relationships between recombination frequency and the physical distance were determined in three series of isomarker two-factor crosses between rII mutants. In the first series of intergenic crosses (rIIa x rIIb), the rII gene function was restored owing to complementation. In the second series of crosses, identical to the first one, the rIIB gene function was suppressed, because the rIIa parent carried an additional amberlike mutation in the rIIB gene. The recombinants were scored by plating lysates on the amber-suppressor Escherichia coli strain, on which an amberlike mutation was not expressed phenotypically. In the third series, all crosses were intragenic (rIIb x rIIb). In two series of crosses in the absence of the rIIB function, the relationships between recombination frequency and the physical distance were identical, whereas enhanced recombination frequencies were observed in the rIIB+ background. The magnitude of the rIIB-related effect depended on distance, reaching the maximum in the region located 100 to 200 bp from the beginning of the rIIB gene. The possible role of the rIIB protein in genetic recombination is discussed.     

Realignment of the Genetic Map of the Terminus of the rIIB Cistron of Bacteriophage T4

Duplication end-point mapping in the rIIB cistron indicates that the order of the BS-B10b segments is the inverse of that presented in Benzer's (1961) genetic maps. This findings is supported by two- and three-factor crosses and the phenotypes of rII deletions extending into the D region.     

Double-strand break repair in bacteriophage T4: recombination effects of 3'-5' exonuclease mutations

The role of 3'-5' exonucleases in double-strand break (DSB)-promoted recombination was studied in crosses of bacteriophage T4, 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 x ets1, where ets1 designates an insertion in the rIIB gene carrying the cleavage site for SegC and i's are rIIB or rIIA point mutations located at various distances (12-2040 bp) from the ets1 site. The frequency/distance relationship was obtained in crosses of the wild-type phage and dexA1 (deficiency in deoxyribonuclease A), D219A (deficiency in the proofreading exonuclease of DNA polymerase), and tsL42 (antimutator allele of DNA polymerase) mutants. In all the mutants, recombinant frequency in crosses with the i-markers located at 12 and 33 bp from ets1 was significantly enhanced, implying better preservation of 3'-terminal sequences at the ends of the broken DNA. The effects of dexA1 and D219A were additive, suggesting an independent action of the corresponding nucleases in the DSB repair pathway. The recombination enhancement in the dexA1 mutant was limited to short distances (<100 bp from ets1), whereas in the D219A mutant a significant enhancement was seen at all the tested distances. From the character of the frequency/distance relationship, it is inferred that the synthesis-dependent strand-annealing pathway may operate in the D219A mutant. The recombination-enhancing effect of the tsL42 mutation could be explained by the hypothesis that the antimutator 43Exo removes a shorter stretch of paired nucleotides than the wild-type enzyme does during hydrolysis of the unpaired terminus in the D-loop intermediate. The role of the proofreading exonuclease in the formation of a robust replicative fork is discussed.     

Point Mutants in the D2a Region of Bacteriophage T4 Fail to Induce T4 Endonuclease IV

We have studied the properties of presumptive point mutants in the D2a region of bacteriophage T4. Dominance tests showed that the D2a mutation was recessive to the wild-type allele. The mutations were shown to map in the D2a region by complementation against rII deletions. The D2a mutations were also located between gene 52 and rIIB by two- and three-factor crosses. The mutants are located at at least two distinct loci in the D2a region. The point mutants grow normally on all hosts tested and none of the mutants makes T4 endonuclease IV. We propose the name "denB" for the D2a locus.     

The influence of the reading context upon the suppression of nonsense codons

Summary We have examined the response of phage T4 nonsense mutations located at various sites within the same cistron to different suppression agents. A wide range of suppression efficiency is found for both ochre (UAA) and amber (UAG) mutations under conditions where suppression provides a measurement of the amount of chain propagation past the mutated site. We have established a relationship between our measurement-the size of the phage yield-and the amount of rIIB product present in the infection. Our data suggest that the 1000-fold range of variations in yields observed in the rIIB cistron corresponds to a 30-fold range of variation in the level of rIIB product, i.e. in the relative frequency of chain propagation past the various nonsense codons included in our test.From the parallelism of response of any particular mutant to very different suppression mechanisms we conclude that the efficiency of suppression is site specific, that is to say, that the main factor determining the frequency of chain propagation at a nonsense codon by any type of suppression mechanism is the nucleotide sequence adjacent to the nonsense codon (reading context).We propose that the recognition of a natural termination signal involves a sequence longer than a nonsense codon and that nonsense codons outside of their natural environment induce variable termination rates which are reflected in the suppression potential.     

The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase.

T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52-protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here.     

Endonuclease VII is a key component of the mismatch repair mechanism in bacteriophage T4

In previous papers we described an extra recombination mechanism in T4 phage, which contributed to general recombination only when particular mutations were used as geneticmarkers (high recombination or HR markers), whereas it was practically inactive towards other rIIB mutations (low recombination or LR markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombination heteroduplexes. We suggested that the first step in this pathway, recognition and incision of the mismatch, is performed by endonuclease VII (endo VII) encoded by the T4 gene 49. In the present paper, we tested this hypothesis in vivo. We used an experimental model system that combines site-specific double-strand breaks with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. We compared recombination of homoallelic HR and LR markers in the S17 and S17 E727 background (amber mutations in the uvsX and in the uvsX and 49 genes, respectively). In S17-crosses, the HR and LR markers retain their respective high-recombination and low-recombination behavior. In S17 E727-crosses, however, the HR and LR markers show no difference in the recombination frequency and both behave as LR markers. We conclude that endo VII is the enzyme that recognizes mismatches in recombinational heteroduplexes and performs their incision. This role for endo VII was suggested previously from biochemical studies, but this is its first in vivo demonstration.     

The Riia Gene of Bacteriophage T4. I. Its DNA Sequence and Discovery of a New Open Reading Frame between Genes 60 and Riia

We have determined the DNA sequence of the rIIA gene and have discovered a small open reading frame, rIIA.1, between genes 60 and rIIA. The predicted molecular weights of these proteins are 82,840 for rIIA and 8,124 for rIIA.1. The rIIA protein has a repeated motif which suggests that the gene has evolved by duplication. It also has a motif which suggests that it belongs to a group of ompR-like proteins that control regulation of gene expression in response to changes in the external environment. We have sequenced three different missense mutants whose mutations lie in the Ala segment of the rIIA genetic map. All three changes are found within the first 35 bp of the rIIA coding sequence. The region of control of protein synthesis is identical in the rIIA gene and in gene 44 of T4. We relate this finding to the high sensitivity of both RNAs to translational repression by the T4 regA gene product.