Double-strand break repair in bacteriophage T4: coordination of DNA ends and effects of mutations in recombinational genes

Coordination of DNA ends during double-strand break (DSB) repair was studied in crosses of bacteriophage T4 in which DSBs were induced site-specifically by SegC endonuclease in the DNA of only one of the parents. Coupling of the genetic exchanges to the left and to the right of the DSB was measured in the wild-type genetic background as well as in T4 strains bearing mutations in several recombination genes: 47, uvsX, uvsW, 59, 39 and 61. The observed quantitative correlation between the degree of coupling and position of the recombining markers in relation to the DSB point implies that the two variants of the splice/patch-coupling (SPC) pathway, the "sequential SPC" and the "SPC with fork collision", operate during DSB repair. In the 47 mutant with or without a das suppressor, coupling of the exchanges was greatly reduced, indicating a crucial role of the 47/46 complex in coupling of the genetic exchanges on the two sides of the DSB. From the observed dependence of the apparent coupling on the intracellular ratio of breakable and unbreakable chromosomes in different genetic backgrounds it is inferred that linking of the DNA ends by 47/46 protein is the mechanism that accounts for their concerted action during DSB repair. A mechanism of replicative resolution of D-loop intermediate (RR pathway) is suggested to explain the phenomenology of DSB repair in DNA arrest and uvsW mutants. A "left"-"right" bias in the recombinogenic action of two DNA ends of the broken chromosome was observed which was particularly prominent in the 59 (41-helicase loader) and 39 (topoisomerase) mutants. Phage topoisomerase II (gp39-52-60) is indispensable for growth in the DNA arrest mutants: the doubles 47(-)39(-), uvsX 39(-) and 59(-)39(-) are lethal.     

Double-strand break repair in tandem repeats during bacteriophage T4 infection

Recombinational repair of double-strand breaks in tandemly repeated sequences often results in the loss of one or more copies of the repeat. The single-strand annealing (SSA) model for repair has been proposed to account for this nonconservative recombination. In this study we present a plasmid-based physical assay that measures SSA during bacteriophage T4 infection and apply this assay to the genetic analysis of break repair. SSA occurs readily in broken plasmid DNA and is independent of the strand exchange protein UvsX and its accessory factor UvsY. We use the unique features of T4 DNA metabolism to examine the link between SSA repair and DNA replication and demonstrate directly that the DNA polymerase and the major replicative helicase of the phage are not required for SSA repair. We also show that the Escherichia coli RecBCD enzyme can mediate the degradation of broken DNA during early, but not late, times of infection. Finally, we consider the status of broken ends during the course of the infection and propose a model for SSA during T4 infections.     

Asymmetric repair of bacteriophage T7 heteroduplex DNA

Summary Heteroduplex DNA molecules were prepared in vitro using one strand of DNA carrying a point mutation and one strand of the corresponding wild-type DNA. The heteroduplex DNA was transfected into competent bacteria and the progeny genotypes in the resulting infective centers were determined. From the results were conclude that about 80% of all transfected DNA molecules are repaired before DNA replication starts. This fraction of repaired DNA is independent of the location of the mismatched nucleotide pair. However, mismatch correction occurs preferentially on the H strand of the heteroduplex DNA.The repair does not depend on a known phage coded function but requires the active bacterial genes mut U, mut H, mut S and probably mut L.     

Incorporation of large heterologies into heteroduplex DNA during double-strand-break repair in mouse cells

In this study, the formation and repair of large (>1 kb) insertion/deletion (I/D) heterologies during double-strand-break repair (DSBR) was investigated using a gene-targeting assay that permits efficient recovery of sequence insertion events at the haploid chromosomal immunoglobulin (Ig) mu-locus in mouse hybridoma cells. The results revealed that (i) large I/D heterologies were generated on one or both sides of the DSB and, in some cases, formed symmetrically in both homology regions; (ii) large I/D heterologies did not negatively affect the gene targeting frequency; and (iii) prior to DNA replication, the large I/D heterologies were rectified.     

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.     

DNA helicase requirements for DNA replication during bacteriophage T4 infection.

The lytic bacteriophage T4 uses multiple mechanisms to initiate the replication of its DNA. Initiation occurs predominantly at replication origins at early times of infection, but there is a switch to genetic recombination-dependent initiation at late times of infection. The T4 insertion-substitution system was used to create a deletion in the T4 dda gene, which encodes a 5'-3' DNA helicase that stimulates both DNA replication and recombination reactions in vitro. The deletion caused a delay in T4 DNA synthesis at early times of infection, suggesting that the Dda protein is involved in the initiation of origin-dependent DNA synthesis. However, DNA synthesis eventually reached nearly wild-type levels, and the final number of phages produced per bacterium was similar to that of the wild type. When the dda mutant phage also contained a mutation in T4 gene 59 (a gene normally required only for recombination-dependent DNA replication), essentially no DNA was synthesized. Recent in vitro studies have shown that the gene 59 protein loads a component of the primosome, the T4 gene 41 DNA helicase, onto DNA. A molecular model for replication initiation is presented that is based on our genetic data.     

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 ⁺ × i ets1 segCΔ crosses, where ets1 is an insertion in the initial part of the rIB gene carrying a sequence recognized by SegC endonuclease; i designates a rIIB or rIIA mutation located at some distance from ets1, and segCΔ is a deletion in the segC gene. In such cross, a DSB occurs in the site of ets1. Their repair involves genetic recombination and DNA replication in the neighborhood of ets1. In parallel, the frequency of reversion of the same i mutant in the absence of DSBs is measured in i × 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 ets1. 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.     

DNA injection during bacteriophage T4 infection of Escherichia coli.

The process of phage T4 DNA injection into the host cell was studied under a fluorescent microscope, using 4',6-diamidino-2-phenylindole as a DNA-specific fluorochrome. The phage DNA injection was observed when spheroplasts were infected with the artificially contracted phage particles having a protruding core. The DNA injection was mediated by the interaction of the core tip with the cytoplasmic membrane of the spheroplast. A membrane potential was not required for the process of DNA injection. On the other hand, DNA injection upon infection by intact noncontracted phage of the intact host cell was inhibited by an energy poison. Based on these observations, together with results from previous work, a model for the T4 infection process is presented, and the role of the membrane potential in the infection process is discussed.     

Involvement of bacteriophage T4 genes in radiation repair

One interpretation of Ebisuzaki's (1966) observation that the functional survival of certain early phage T4 genes is identical in v⁺ and v -infected cells is that the product of the early gene being studied is essential for the successful completion of excision repair (which is known to be mediated by the v gene). An experiment designed to test this hypothesis is described, with results which fully support the idea. Assuming then that this interpretation is valid, it became possible to determine the involvement in excision repair of a much wider range of early genes by establishing whether or not the v allele affects their functional survival. In addition a comparable series of experiments was performed with phages carrying the u.v.-sensitive y mutation which is known to mediate a quite different type of repair in T4-infected cells.The results indicate that genes 1, 30, 42, 43 and 56 are involved in excision repair, but not genes 32, 41, 43 or 44. All these genes are however involved in y-mediated repair. It appears therefore that this latter repair system (which bears some resemblance to that controlled by the rec genes in bacteria) depends on normal phage DNA synthesis for its completion. However the repair synthesis following the excision of pyrimidine dimers in u.v.-irradiated T4 DNA seems distinct from normal DNA synthesis in that it does not involve certain of the early phage genes, and in particular does not utilize the DNA polymerase coded by gene 43. It is suggested that the polymerase activity associated with this repair synthesis is provided by the bacterial Kornberg polymerase pol I.     

Activities involved in base excision repair of bacteriophage T4 and lambda DNA in vivo

Summary The in vivo excision repair functions of Escherichia coli exonuclease III and 3-methyladenine DNA glycosylase I, and bacteriophage T4 pyrimidine dimer-DNA glycosylase were investigated. Following exposure of bacteriophage T4 or lambda to methyl methanesulfonate or ultraviolet irradiation, survival was determined by plating on E. coli have various genetic backgrounds. Although exonuclease III was shown to participate in base excision repair initiated by 3-methyladenine DNA glcosylase I, it had no detectable role in base excision repair initiated by the T4 pyrimidine dimer-DNA glycosylase. Despite its 3 apurinic/apyrimidinic endonuclease activity in vitro, T4 pyrimidine dimer-DNA glycosylase, even in large quantities, did not complement mutants defective in exonuclease III in the repair of apurinic sites generated by 3-methyladenine DNA glycosylase I in vivo.     

Molecular arrangement of DNA in bacteriophage T4

The degree of preferred orientation and the coiling of the deoxyribonucleic acid within phage T₄ was studied by two independent techniques, namely, polarization of fluorescence and uv linear dichroism. A correlation between the two kinds of data was obtained, which indicated that a significant proportion (about 30%) of total phage DNA is aligned preferentially along the long axis of phage heads. Analyses of the data suggest that all of the phage DNA cannot be in a highly supercoiled helical configuration. A few models of the DNA arrangement in T₄ have been discussed in which linear sidewise packings of DNA would be predominant and may explain the observed longitudinal orientation of intraphage DNA.     

Partial exclusion of genes of bacteriophage T2 with T4-glucosylated DNA in crosses with bacteriophage T4

A recombinant strain (D41) between phage T2 and T4 was isolated which possessed the T2 region of the genome between genes 32 and 39 and both the T4 genesgt ⁺ andgt ⁺ for glucosyltransferase. D41 was crossed with T4amber mutants in the genes for early functions and in some genes for late funcitions. The progeny of the crosses was examined for the frequency of theam ⁺ markers from D41. Genes 32, 60 and 39 in the T2 region of the recombinant strain were as sensitive to exclusion as those in standard-type T2. The T4 glucosylation of the DNA of these T2 genes did not protect them against partial exclusion by T4. However, genes in the region from gene 56 to 55 in the recombinant were resistent to exclusion. In standard-type T2 this region of the genome is sensitive to partial exclusion by T4. There are at least four exclusion sensitive sites in T2: one near gene 32, one near gene 60, one linked to gene 56 and one between genes 42 and 55.This investigation was carried out partially within the frame of the Association between Euratom and the University of Leiden, contract nr. 052-64-1-BIAN.     

The bacteriophage T4 DNA injection machine

The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached. Upon binding of the fibers to their cell receptors, the baseplate undergoes a large conformational switch, which initiates sheath contraction and culminates in transfer of the phage DNA from the capsid into the host cell through the tail tube. The baseplate has a dome-shaped sixfold-symmetric structure, which is stabilized by a garland of six short tail fibers, running around the periphery of the dome. In the center of the dome, there is a membrane-puncturing device, containing three lysozyme domains, which disrupts the intermembrane peptidoglycan layer during infection.     

Participation of bacteriophage T4 gene 41 in replication repair

The location of the non-essential T4 mutant uvs79, with defective replication repair, is described. After crosses with double mutants dispersed over the early region of T4, a linkage was observed with the double mutant am41 : am42. For more accurate location, crosses were made with single mutants. Uvs79 proved to be located between mutants amC23 and amN81 in gene 41, as shown by 3-point crosses. No genetic complementation with respect to multiplicity reactivation was found between amN81 and uvs79 after a co-infection of an su^? host. Apparently, mutant amN81 is disturbed as to replication repair and, owing to its lack of DNA synthesis, also in replication-dependent recombination repair. Consequently, the product of gene 41 has a function additional to its RNA-primer induction during replication of undamaged DNA. Presumably, the product of gene 41 induces RNA primers opposite DNA regions containing lesions. This capability is believed to be specifically affected by the uvs79 mutation.     

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.