Genetic Expression in Heterozygous Replicative Form Molecules of phiX174

Heterozygous replicative form molecules of bacteriophage X174 deoxyribonucleic acid (DNA) have been constructed in vitro. These are composed of viral strands extracted from purified preparations of phage bearing ts mutations and complementary strands of either half length or full length synthesized with purified DNA polymerase, in vitro, on DNA from am3 phage. In infections with such heterozygous DNA, involving mutations in each of four different cistrons, phage with the genotype of the complementary strand comprised 1 to 20% of the total phage produced by a spheroplast population. From single-burst analysis of the progeny from DNA heterozygous in one cistron (B), it appears that those phage with the genotype of the complementary strand arise as major components in a small proportion of the infected cells rather than comprising a minor component in most cells. The implications of such a pattern of expression are discussed with respect to mechanisms of phage DNA synthesis.     

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.     

Construction of viable and lethal mutations in the origin of bacteriophage 'phi' X174 using synthetic oligodeoxyribonucleotides

Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX² DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung^? (uracil N-glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al., 1980a).Using (5′-³²P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA.Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro. Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas &; Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA.Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.     

The mechanism of replication of phi X174 single-stranded DNA. II. The role of viral proteins

We have studied the replication of φX174 DNA in Escherichia coli infected with various amber mutants (cistrons I to VII) of φX. Previous research showing that some of these mutants are able to form replicative form (RF) DNA but are unable to produce net amounts of viral progeny single-stranded DNA has been confirmed and extended. Evidence is presented that a defect in any one of four viral cistrons prevents the asymmetric replication of the RF to produce progeny viral DNA. At least four virus-coded proteins, three of which are part of the mature virion, must be present before single-stranded DNA synthesis can even be initiated; the possibility that single-stranded DNA is made and then degraded or converted to RF is eliminated. Mutants in one cistron (II) do permit the asymmetric replication of RF at late times, but the displaced viral strand is incorporated into a defective particle and subsequently may be partially degraded. Both RFI (superhelix) and RFII are present in roughly comparable amounts throughout the normal latent period in infections with wild-type phage or any of the phage mutants.     

In vivo methylation of replicating bacteriophage phi chi174 DNA

The pattern of DNA methylation during the infection of Escherichia coli C cells with bacteriophage φX174, has been studied. In vivo methylated DNA was isolated and analyzed using the following techniques: velocity sedimentation through neutral and alkaline sucrose gradients, isopycnic analysis, chromatography on benzoylated DBAE-cellulose columns and specific enzymatic digestion. All these analytical methods indicated that the DNA molecules that are methylated during the process of phage φX DNA replication are the replicating intermediates composed of a circular complementary strand and a viral strand larger than one genome length. It is concluded that methylation occurs on the nascent DNA strand of the replicating intermediates involved in the synthesis of progeny single-stranded DNA.     

Mechanism of replication of phichi174 single-stranded DNA. V. Dispersive and conservative transfer of parental DNA into progeny DNA

We have studied parent-to-progeny transfer of bacteriophage φX174 DNA during infection of Escherichia coli with isotopically-labeled, lysis-defective phage. After 60 minutes of infection at low multiplicities, 25 to 30% of the input viral DNA is transferred from the double-stranded replicative form into progeny phage; another 10 to 20% is transferred into the progeny single-stranded DNA pool. Thus, at times beyond the normal time of lysis, about 35 to 50% of the parental deoxyribonucleotides are found in progeny single-stranded DNA. Three quarters of the parental label found in the progeny phage is transferred by a dispersive process and one-quarter is transferred by a conservative, or non-dispersive, process such that the parental strand remains intact. At high multiplicities of infection the fraction of parental label transferred decreases.     

Infectivity of Lambda Heteroduplex Deoxyribonucleic Acid Molecules

Heteroduplex deoxyribonucleic acid (DNA) molecules were formed in vitro by denaturing and renaturing a mixture of DNAs from a variety of lambda strains. The infectivity of these heteroduplexes was studied by using host-controlled modification and restriction to prevent infection by contaminating parental homoduplexes. Either strand was able to protect against degradation by restriction nucleases in vivo. A large proportion of progeny phage, which had been produced after infection with heteroduplexes containing noncomplementary base pairs at multiple loci, retained the genotype of one of the parental homoduplexes. The results indicate that conversion of heteroduplexes to homoduplexes in vivo by a DNA repair mechanism does not occur frequently. Molecules heterozygous for the c17 or vir operator mutations were not infectious; it is suggested that these mutations involve multiple base pairs.     

Mike, a chimeric filamentous phage designed for the separate production of either DNA strand of pKUN vector plasmids by F+ cells

To enable the separate production of either DNA strand of recombinant pKUN plasmids [Peeters et al., Gene 41 (1986) 39-46] by conjugation-deficient F+ cells a chimeric Ff/IKe filamentous phage, Mike, has been constructed. Its genome contains the functions required for asymmetric DNA replication from the N-plasmid specific filamentous phage IKe, and the functions required for host cell penetration, single-stranded DNA accumulation, phage assembly, and secretion from the F-plasmid specific filamentous phage Ff (i.e. M13, fl, or fd).     

Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

The gapped duplex DNA approach to oligonucleotide-directed mutation construction.

A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (-) strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (-) strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (-) strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.     

Physical analyses of E. coli heteroduplex recombination products in vivo: on the prevalence of 5' and 3' patches

Background

Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3′-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands.

Methodology/Principle Findings

This paper re-examines heteroduplex-patch-strand polarity using phage λ and the λdv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for λ recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5′ or 3′ DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation.

Conclusions/Significance

These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism.     

Initiation of DNA synthesis on the isolated strands of bacteriophage f1 replicative-form DNA.

Viral and complementary strand circular DNA molecules were isolated from intracellular bacteriophage f1 replicative-form DNA. Soluble protein extracts of Escherichia coli were used to examine the initiation of DNA synthesis on these DNA templates. The initiation of DNA synthesis on f1 viral strand DNA was catalyzed by E. coli DNA-dependent RNA polymerase, as was initiation of f1 viral strand DNA isolated from mature phage particles. The site of initiation was the same as that used in vivo. In contrast, no de novo initiation of DNA synthesis was detected on f1 complementary strand DNA. Control experiments demonstrated that the E. coli dnaB, dnaC, and dnaG initiation proteins were active under the conditions employed. The results suggest that the viral strand of the f1 replicative-form DNA molecule carries the same DNA synthesis initiation site as the viral strand packaged in mature phage, whereas the complementary strand of the replicative-form DNA molecule carries no site for de novo primer synthesis. These in vitro observations are consistent with the simple rolling circle model for f1 DNA replication in vivo proposed by Horiuchi and Zinder.     

Partial replication of UV-irradiated T4 bacteriophage DNA results in amplification of specific genetic areas.

Upon infection of Escherichia coli with bromodeoxyuridine-labeled t4 phage that had received 10 lethal hits of UV irradiation, a sizable amount of phage DNA was synthesized (approximately 36 phage equivalent units of DNA per infected bacterium), although very little multiplicity reactivation occurs. This progeny DNA was isolated and analyzed. This DNA was biased in its genetic representation, as shown by hybridization to cloned segments of the T4 genome immobilized on nitrocellulose filters. Preferentially amplified areas corresponded to regions containing origins of T4 DNA replication. The size of the progeny DNA increased with time after infection, possibly due to recombination between partial replicas and nonreplicated subunits or due to the gradual overcoming of the UV damage. As the size of the progeny DNA increased, all of the genes were more equally represented, resulting in a decrease in the genetic bias. Amplification of specific genetic areas was also observed upon infection with UV-irradiated, nonbromodeoxyuridine-substituted (light) phage. However, the genetic bias observed in this case was not as great as that observed with bromodeoxyuridine-substituted phage. This is most likely due to the higher efficiency of multiplicity reactivation of the light phage.     

The essential role of bacteriophage T7 endonuclease (gene 3) in molecular recombination

Density transfer and shearing experiments show that the bacteriophage T7 endonuclease (gene 3) is necessary for the dispersion of parental DNA in the newly replicated DNA. These experiments on parental to progeny recombination support previous genetic data (Powling & Knippers, 1974; Kerr & Sadowski, 1975) that the gene 3 protein is essential for T7 recombination. Concatemers containing the newly replicated DNA have been sheared to the size of mature phage DNA and also to quarter molecules. In the presence of gene 3 protein, parental DNA and newly replicated DNA are interspersed. In the absence of gene 3 protein, the parental strand of each sheared DNA molecule is usually found intact.     

Initiation of heteroduplex-loop repair by T4-encoded endonuclease VII in vitro.

Heteroduplex DNAs with single-stranded loops of 51 nt or 8 nt were constructed in vitro and used in reactions with purified endonuclease VII (endo VII) from phage T4. The enzyme makes double-strand breaks by introducing pairs of staggered nicks flanking the loops. Regardless of loop-size the nicking sites map exclusively at the 3' side of the loop in the looping strand and at the 3' side of the base of the loop in the non-looping strand. The number of potential cleavage sites is small (less than 5) and their distribution depends on DNA sequence. The two closest staggered nicks are 4 bp apart, 2 bp on either side of the loop. Nicking always occurs in the double-stranded part of the molecules; the single-stranded loops are not attacked by endo VII. The nicks are introduced in a stepwise fashion and selection of the strand for the first nick depends on the sequence of 31 base pairs flanking the loops.     

The effect of ultraviolet light on the production of bacterial virus protein

The amount of phage-specific protein in T2-infected bacteria growing in a medium containing radiosulfur, S³⁵, has been studied by measuring the radioactivity in specific antiphage serum precipitates of lysates. In the course of normal infection, non-infective phage antigen has been found to make its first intracellular appearance shortly before the end of the eclipse period, in agreement with the findings of Maaløe and Symonds with phage T4. No such phage antigen is produced either in bacteria infected with UV-inactivated T2 or in T2-infected bacteria whose survival as an infective center has been destroyed by UV irradiation during the early stages of the eclipse period. If the infected bacteria are UV-irradiated only at later stages of the eclipse period however, then phage antigenic protein continues to be synthesized in those infected cells in which DNA synthesis and, a fortiori, production of infective progeny have been almost completely suppressed. It is concluded from these results that once the mechanism for formation of phage-specific protein has been established within the infected cell under the influence of the parental DNA, synthesis of phage-specific protein can continue independently of the synthesis of phage DNA. The possibility that the phage DNA controls the specificity of the phage protein indirectly through substances other than DNA is discussed.     

Replication of bacteriophage M13. 8. Differential effects of rifampicin and nalidixic acid on the synthesis of the two strands of M13 duplex DNA

Replication of bacteriophage M13 replicative forms is inhibited by rifampicin, an antibiotic that specifically inhibits the Escherichia coli RNA polymerase, and by nalidixic acid, an inhibitor of phage and bacterial DNA replication. Synthesis of the M13 complementary strand during RF³ replication was at least tenfold more sensitive to inhibition by rifampicin and by nalidixic acid than was that of the viral strand. Since M13 complementary strand synthesis is relatively insensitive to chloramphenicol, an inhibitor of protein synthesis, its inhibition by rifampicin suggests that complementary strands are initiated during RF replication by an RNA priming mechanism similar to that involved in parental RF formation. The nalidixic acid-sensitivity of complementary strand synthesis during RF replication clearly distinguishes this process from the nalidixic acid-resistant formation of the parental complementary strand in the conversion of the infecting single strand to RF.Production of progeny viral strands is indirectly affected by rifampiein in two ways. It prevents the conversion of supercoiled RF (RFI) to the open form (RFII), an essential step both in RF replication and in single-strand synthesis. In addition, rifampiein interferes with the expression of gene 5, an M13 gene function required for the accumulation of progeny viral strands.     

On the mechanism of production of tandem genetic duplications in phage lambda

We have asked whether the mechanism by which tandem genetic duplications arise in the chromosome of phage lambda is inter- or intramolecular. Two parental phages carrying genetic markers at opposite ends of the phage chromosome have been grown in mixed infection, and progeny phages carrying newly-arising tandem duplications have been analysed to determine whether they carry the markers in parental or recombinant configuration. Ordinary genetic recombination of the markers has been prevented by mutations in the phage and host. Phages carrying tandem duplications are isolated by use of CsCl density gradients and an Escherichia coli strain that does not plate deletion phages. Of the duplication mutants isolated under these conditions, 13% carry the input markers in recombinant configuration. This suggests that tandem duplications can be produced via an intermolecular route which joins sequences originally present on different DNA molecules.     

Fate of mat1 DNA strands during mating-type switching in fission yeast

The mating-type switching of the fission yeast, Schizosaccharomyces pombe, is highly regulated. Two consecutive asymmetric divisions are required to produce one mating-type switched cell among the four progeny. Using DNA density-gradient centrifugation we demonstrate that one-fourth of the mat1 DNA is not replicated by the conventional semi-conservative mode, but instead both DNA strands are synthesized de novo. Our data are consistent with a gene conversion event, initiated by a site- and strand-specific DNA break (SSB). We further demonstrate that the virgin switched mat1-containing chromatid no longer contained the nick, while it is reintroduced during the lagging strand synthesis of the mat1 locus on the sister chromatid. This finding establishes at the molecular level a firm experimental link between the phenotype and genotype in the process of asymmetric mating-type switching during mitotic divisions.