Rapid isolation of homogeneous cloned T7 gene 5 protein and T7 DNA polymerase by affinity chromatography on immobilized thioredoxin.

Phage T7 DNA polymerase consists of a strong 1:1 complex of T7 gene 5 protein (80 kDa) and the reduced form of Escherichia coli thioredoxin (12 kDa). Immobilization of E. coli thioredoxin on the agarose matrix Affi-Gel retained both its redox activity and its ability to bind T7 gene 5 protein. This was used to develop a simple and fast high-yield purification method. Cloned T7 gene 5 protein, expressed in a thioredoxin-negative host cell, was isolated in pure and highly active form after elution from Affi-Gel--thioredoxin with a pH gradient from 10 to 12. This purification step separated gene 5 protein from variable amounts of two sets of reconstituting large polypeptide fragments without catalytic activity. Proteolytic cleavage in vivo probably gave rise to the fragments, the generation of which was mimicked by trypsin cleavage of pure gene 5 protein. The gene 5 protein preparation had an inherent low DNA polymerase and double-stranded 3'-exonuclease activity, which was stimulated at least 30-fold by the presence of reduced thioredoxin. Highly active and pure T7 DNA polymerase was obtained by reconstitution of gene 5 protein with thioredoxin and was isolated by phosphocellulose or FPLC Mono Q chromatography. The gene 5 protein and T7 DNA polymerase preparations are suitable for further physicochemical characterization and as reagents in DNA sequencing.     

The basis for camptothecin enhancement of DNA breakage by eukaryotic topoisomerase I.

The eukaryotic topoisomerase I (topo I) is the target of the cytotoxic alkaloid camptothecin (CTT). In vitro, CTT enhances the breakage of DNA by topo I when the reaction is stopped with detergent. Although breakage at some sites is enhanced to a great extent while breakage at others is enhanced only minimally, CTT does not significantly change the breakage specificity of topo I in vitro. It has been suggested that CTT acts by slowing the reclosure step of the nicking-closing reaction. To test this hypothesis, we have measured the rate of reclosure for different break sites in the presence of CTT after adding 0.5 M NaCl to a standard low salt reaction. In support of the hypothesis, we find that topo I-mediated DNA breakage is enhanced the greatest at those sites where closure of the break is the slowest. These results suggest a mechanism for the toxicity of CTT in vivo.     

Role of the T5 gene D15 nuclease in the generation of nicked bacteriophage T5 DNA.

The processing of newly replicated concatameric T5 DNA into both single stranded DNA changed of unit length and single-stranded fragments of sizes comparable to those found in mature T5 virion DNA occurs in the absence of late T5 protein synthesis. The formation of unit-length, single-stranded DNA chains does not require the early T5 gene D15 nuclease: however, the subsequent formation of the single-stranded fragments does require that the D15 nuclease be functional. A reexamination of the properties of the purified D15 nuclease under a variety of conditions showed that, in addition to functioning as a 5' leads to 3' exonuclease, the enzyme can also introduce endonucleolytic scissions into mature T5 DNA in a reaction that requires duplex T5 DNA and preexisting, single-stranded interruptions.     

Second-step transfer of bacteriophage T5 DNA: purification and characterization of the T5 gene A2 protein.

Second-step transfer of bacteriophage T5 DNA requires the function of the T5 pre-early proteins A1 and A2. We have isolated and characterized the gene A2 protein as part of an effort to determine the mechanism of second-step transfer. The A2 protein was purified by DNA-cellulose column chromatography followed by gel filtration and ion-exchange column chromatography. The A2 protein's identity was confirmed by two-dimensional gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer gel filtration in 6 M guanidine hydrochloride demonstrated a molecular weight of 15,000 for the A2 polypeptide. Migration of the A2 protein through gel filtration columns under nondenaturing conditions, in combination with sedimentation behavior, indicated dimerization of the A2 polypeptide. The existence of the A2 dimer was confirmed by protein cross-linking with dimethyl suberimidate and analysis of the cross-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition, degree of polymerization, DNA-binding ability, and physical characteristics of the T5 gene A2 protein are consistent with a function of the A2 protein in DNA transfer.     

Cloning and DNA sequence of the 5'-exonuclease gene of bacteriophage T5

The nucleotide sequence of the BalI-PstI fragment of T5 DNA, 1347 bp in length, coding for 5'-exonuclease (D15 gene), has been determined. A coding region of the gene contains 873 bp and is preceded by a typical Shine-Dalgarno sequence. The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one. The sequence contains an open reading frame for 291 amino acid residues. The molecular mass of the 5'-exonuclease calculated from the predicted amino acid sequence is 33 400 Da.     

The halo-opsin gene. I. Identification and isolation

Halorhodopsin (HR), the light-driven chloride pump in halobacteria, was purified in the denatured as well as in the native state and chemically cleaved into peptide fragments. Isolation of peptide and liquid phase sequencing yielded approximately 20% of the halo-opsin (HO) structure in non-overlapping peptides. Chemically synthesized oligodeoxynucleotides corresponding to a peptide sequence obtained from both HR preparations were used to screen a cosmid gene bank of Halobacterium halobium strain L-33. A positive clone contained cosmid pAB H47 which by subcloning and nucleotide sequencing was shown to encode at least part of the HO gene.     

Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.     

Interaction of a DNA-binding protein, the product of gene D5 of bacteriophage T5, with double-stranded DNA: effects on T5 DNA polymerase functions in vitro.

The gene D5 product (gpD5) of bacteriophage T5 is a DNA-binding protein that binds preferentially to double-stranded DNA and is essential for T5 DNA replication, yet it inhibits DNA synthesis in vitro. Mechanisms of inhibition were studied by using nicked DNA and primed single-stranded DNA as a primer-template. Inhibition of T5 DNA polymerase activity by gpD5 occurred when double-stranded regions of DNA were saturated with gpD5. The 3' leads to 5' exonuclease associated with T5 DNA polymerase was not very active with nicked DNA, but inhibition of hydrolysis of substituents at 3'-hydroxyl termini by gpD5 could be observed. T5 DNA polymerase appears to be capable of binding to the 3' termini even when double-stranded regions are saturated with gpD5. The interaction of gpD5 with the polymerases at the primer terminus is apparently the primary cause of inhibition of polymerization.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the ADE1 gene.

The ADE1 gene of Saccharomyces cerevisiae was isolated by complementation in S. cerevisiae from a yeast genomic DNA library carried on plasmid YEp13. Electron microscopy of R-loop-containing DNA indicated the location of the ADE1 gene on the plasmid insert. Gene disruption and gene replacement were used to demonstrate that the ade1-complementing sequence was the actual ADE1 gene that maps on chromosome I. ade1 strains which normally form red colonies form white ones when transformed with the cloned ADE1 gene. This property should be very useful, since it enables detection of plasmids carrying this gene under nonselective conditions.     

Specific fragmentation of T7 phage DNA at low-melting sites.

A method has been developed for selective fragmentation of T7 DNA at AT-rich regions. The molecules have been subjected to complete digestion with single-strand-specific SI endonuclease after fixation of DNA AT-rich regions in the denatured state by glyoxal. The treatment resulted in three fragments having molecular weights of 13.6 +/- 0.4, 8.2 +/- 0.4 and 3.5 +/- 0.16 megadaltons as determined by electron microscopy. The position of these fragments along the T7 DNA molecule has been determined by means of analysis of the intermediates during SI-cleavage.     

Physical locus of the DNA polymerase gene and genetic maps of bacteriophage T5 mutants.

Segments of DNA that contained the DNA polymerase gene of bacteriophage T5 were isolated. The physical locus of the gene was identified by transforming Escherichia coli with purified DNA fragments generated by restriction enzyme digestions, and the transformed cells were used to rescue amber mutants of T5 with mutations in the gene for DNA polymerase. The method is applicable to any other gene that has mutations with low reversion frequencies. We studied the following mutations of the T5 DNA polymerase gene, reading from left to right by the standard convention (D. J. McCorquodale, Crit. Rev. Microbiol. 4:101-159, 1975): D7, D8, aml, ts5E-ts53, am6, and D9. These loci were found to reside within three pieces of DNA with a total length of 3,600 base pairs. Because the structural gene for T5 DNA polymerase is estimated to be 2,600 base pairs long, the whole structural gene may reside in these segments. These are located 58.3 to 61.3% of the distance from the left end of the DNA. The left-end piece of the DNA (1,100 base pairs) containing the polymerase gene has loci D7 and D8, and the right-end piece (1,600 base pairs) has locus D9, according to the results of the transformation assay. These results are consistent with the genetic map.     

Localization of single-chain interruptions in bacteriophage T5 DNA I. Electron microscopic studies.

Bacteriophage T5 DNA was examined in an electron microscope after limited digestion with exonuclease III from Escherichia coli. The effect of the exonuclease treatment was to convert each naturally occurring single-chain interruption in T5 DNA into a short segment of single-stranded DNA. The locations of these segments were determined for T5st(+) DNA, T5st(0) DNA, and fragments of T5st(0) DNA generated by EcoRI restriction endonuclease. The results indicate that single-chain interruptions occurr in a variable, but nonrandom, manner in T5 DNA. T5st(+) DNA has four principal interruptions located at sites approximately 7.9, 18.5, 32.6, and 64.8% from one end of the molecule. Interruptions occur at these sites in 80 to 90% of the population. A large number of additional sites, located primarily at the ends of the DNA, contain interruptions at lower frequencies. The average number of interruptions per genome, as determined by this method, is 8. A similar distribution of breaks occurs in T5st(0) DNA, except that the 32.6% site is missing. At least one of the principal interruptions is reproducibly located within an interval of 0.2% of the entire DNA.     

Mechanism of T5-induced DNA polymerase. I. Replication of short primer templates.

Bacteriophage T5-induced DNA polymerase shows an initial phase of rapid synthesis, followed by a slower steady rate for much longer periods, with short DNA primer-templates (400 to 600 nucleotides long), in vitro. On extrapolating the line of steady rate back to 0 min, an intercept is obtained on the ordinate. With large DNA primer-templates, such as denatured T5 DNA (average chain length approximately 50,000 bases), the rate of synthesis remains constant and is equal to the initial rate obtained with short primer-templates. The zero time intercept was proportional to the amount of enzyme used and independent of temperature. Polymer challenge experiments indicate that the initial phase of rapid synthesis can be attributed to the processive mode of synthesis by T5 DNA polymerase. After synthesizing a stretch of DNA processively for about 200 nucleotide residues, the enzyme apparently forms a "dead-end complex" with the primer-templates used and must dissociate from the primer-template in order to resume synthesis. The average size of the product made processively, during various phase of synthesis, remains invariant and is in good agreement with the size of the zero time intercept per enzyme molecule.     

Intron 5 alpha of the COXI gene of yeast mitochondrial DNA is a mobile group I intron.

We have found that intron 5 alpha of the COXI gene (al5 alpha) of yeast mtDNA is a mobile group I intron in crosses between strains having or lacking the intron. We have demonstrated the following hallmarks of that process: 1) co-conversion of flanking optional intron markers; 2) mutations that truncate the intron open reading frame block intron mobility; and 3) the intron open reading frame encodes an endonuclease activity that is required for intron movement. The endonuclease activity, termed I-Sce IV, cleaves the COXI allele lacking al5 alpha near the site of intron insertion, making a four-base staggered cut with 3' OH overhangs. Three cloned DNAs derived from different forms of the COXI gene, which differ in primary sequence at up to seven nucleotides around the cleavage site, are all good substrates for in vitro I-Sce IV cleavage activity. Two of the strains from which these substrates were derived were tested in crosses and are comparably efficient as al5 alpha recipients. When compared with omega mobility occurring simultaneously in one cross, al5 alpha is less efficient as a mobile element.