Top PDF Studies on the DNA of bacteriophage T7

the relation between molecular weight and cr Yeandle 6 rederived to include the electrical effects expected from the polyanionic character of the DNA, and confirmed 1 that even if the DN[r]

Gene 5 of bacteriophage T7 encodes a DNA polymer- ase essential for phage replication. A single point muta- tion in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature- sensitive phage at 42 °C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 °C or 42 °C. Upon infection at 30 °C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 °C or 42 °C approach levels observed using wild-type T7 phage. The amount of solu- ble gene 5 protein produced at 42 °C is comparable to that produced at 30 °C, indicating that the temperature- sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this ob- servation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-pro- duced at 30 °C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.

As the replication fork moves along a DNA duplex, DNA primase periodically deposits short RNA primers at specific priming sequences on the lagging strand, triggering the syn- thesis of Okazaki fragments that are subsequently processed to form a continuous DNA strand (7, 8). In most DNA replication systems, a separate primase protein transiently interacts with the DNA helicase to initiate primer synthesis on the lagging strand. In E. coli, the strength of this interaction affects the frequency of priming and thereby sets the average length of Okazaki fragments (9). The primase and helicase activities of bacteriophage T7 are fused in a single polypeptide that assem- bles into a ring-shaped hexamer (10 –12). The bifunctional pri- mase-helicase unwinds DNA ahead of the replication fork, and it primes the discontinuous synthesis of the lagging strand of the replication fork. The short tetranucleotides synthesized by the primase domain of the primase-helicase are not extended by T7 DNA polymerase alone (13–15); they are elongated by the polymerase only if the primase-helicase is also present during primer extension. It is not known how many subunits of the hexameric primase-helicase directly participate in the priming of DNA synthesis, nor is it known how the primase-helicase stimulates primer utilization by T7 DNA polymerase. The pri- mase-helicase protein consists of an N-terminal primase do- main and C-terminal helicase domain (12, 16, 17) that will separately catalyze tetraribonucleotide synthesis and DNA un- winding, respectively (16, 18, 19). However, the primase do- main alone does not support the extension of primers by T7 DNA polymerase (18). The dual requirement for the primase- helicase and the T7 DNA polymerase during RNA-primed syn- thesis of DNA suggests that these proteins associate in a com- plex that initiates the elongation of RNA primers synthesized by the primase (20).

Two lines of evidence suggest that at least a portion of the thioredoxin binding domain of gene 5 protein resides within the polymerase domain. T7 DNA polymerase purified in the ab- sence of thioredoxin is subject to proteolytic attack at three clustered sites that lie within the COOH-terminal region of the protein, and this proteolysis is inhibited by thioredoxin (9). Mapping of these sites showed the proteolytic cleavage to re- side at positions Ile-289, Lys-299, and Ala-323 of the T7 gene 5 protein (24). These results implicated the sequence Ile-289 to Ala-323 as a region that physically interacts with thioredoxin. This sequence, which does have a homologous counterpart in E. coli DNA polymerase I (30, 31), has an unusually high content of hydrophilic residues. In the structure of the large fragment of E. coli DNA polymerase I, this 76-amino acid sequence is located in a region referred to as the “thumb” that partially covers the crevice through which the DNA passes, a region that has been proposed to be involved in the interaction of the polymerase with duplex DNA (27). Further evidence suggest- ing that this unique amino acid sequence interacts with thiore- doxin comes from studies on suppressor mutations that allow T7 phage to use a genetically altered thioredoxin (22). One suppressor mutation (Glu-319 3 Lys) resides within this re- gion and restores the ability of T7 DNA polymerase to interact with this particular mutant thioredoxin (23).

The three-dimensional structure of bacteriophage T7 DNA poly- merase reveals the presence of a loop of 4 aa (residues 401– 404) within the DNA-binding groove; this loop is not present in other members of the DNA polymerase I family. A genetically altered T7 DNA polymerase, T7 pol⌬401– 404, lacking these residues, has been characterized biochemically. The polymerase activity of T7 pol ⌬ 401– 404 on primed M13 single-stranded DNA template is one-third of the wild-type enzyme and has a 3 ⴕ -to-5 ⴕ exonuclease activity indistinguishable from that of wild-type T7 DNA polymer- ase. T7 pol ⌬ 401– 404 polymerizes nucleotides processively on a primed M13 single-stranded DNA template. T7 DNA polymerase cannot initiate de novo DNA synthesis; it requires tetraribonucle- otides synthesized by the primase activity of the T7 gene 4 protein to serve as primers. T7 primase-dependent DNA synthesis on single-stranded DNA is 3- to 6-fold less with T7 pol ⌬ 401– 404 compared with the wild-type enzyme. Furthermore, the altered polymerase is defective (10-fold) in its ability to use preformed tetraribonucleotides to initiate DNA synthesis in the presence of gene 4 protein. The location of the loop places it in precisely the position to interact with the tetraribonucleotide primer and, pre- sumably, with the T7 gene 4 primase. Gene 4 protein also provides helicase activity for the replication of duplex DNA. T7 pol ⌬ 401– 404 and T7 gene 4 protein catalyze strand-displacement DNA synthesis at nearly the same rate as does wild-type polymerase and T7 gene 4 protein, suggesting that the coupling of helicase and polymerase activities is unaffected.

Compensatory evolution of T7 phage. The self-sufficient ge- nome organization of bacteriophage T7 and its robust lytic life cycle have made it an attractive model to investigate genetic adaptation under environmental pressure (13). One study has shown that T7 phage modifies expression of several genes in the course of its adaptation to compensate for loss of DNA ligase (40). Alterations in gene 3 were previously observed in T7 phage that suppresses the loss of the ligase gene, gene 1.3, when grown on a ligase-deficient host (41). These studies sug- gested that T7 phage modifies genes whose products are in- volved in DNA replication and/or recombination in order to adapt to alterations in an essential gene. However, since the enzymatic activity of some proteins is agonistic or antagonistic to other proteins, compensatory modification of genes must be precisely regulated in order to achieve effective overall func- tion. For example, we found that mutations in gene 3 that suppress the phenotype of gp5-4N arise when gene 3 is ex- pressed from phage. When both gene 3 and gene 5 are over- expressed from supplied plasmids, compensation of phage growth by mutated gene 3 is not observed (data not shown). This observation supports the suggestion that compensatory changes need to be balanced in a DNA metabolism network (3, 41). We have also observed mutations in gene 3 that can suppress defects in T7 phage that have mutations in other replication proteins such as gene 2.5 ssDNA binding protein (B. Marintcheva, unpublished results) or gene 4 helicase-pri- mase (S.-J. Lee, unpublished results).

The SPR sensorgrams in Fig. 2B show the binding between gp5/trx-primer/template and the different gp4 variants that were subsequently injected into the flow cell. Because the gp4 variants differ in mass from the WT gp4, we normalized the FIGURE 1. The replisome of bacteriophage T7 and variants of gp4. A, the replisome of bacteriophage T7 contains DNA polymerase (gp5) and its pro- cessivity factor, E. coli thioredoxin (trx), DNA primase-helicase (gp4), and ssDNA-binding protein (gp2.5). gp4 unwinds dsDNA and generates two ssDNA templates for the leading and lagging strand gp5/trx. gp2.5 coats the lagging strand ssDNA. The primase domain of gp4 catalyzes the synthesis of oligoribonucleotides (green) that function as primers for the initiation of each Okazaki fragment. B, gp4 consists of two major domains connected by a flex- ible linker: the C-terminal helicase and the N-terminal primase. The primase domain contains two subdomains: the N-terminal ZBD and the RPD that are also connected by a flexible linker. The C-terminal 17 amino acids of the heli- case domain are unstructured. The positions of residues important in the construction of the various forms of g4 are indicated. We constructed five truncated proteins: gp4 ⌬ helicase (residues 1–271) lacks the helicase domain but contains the flexible linker connecting the RPD with the helicase domain, gp4 ⌬ C (residues 1–549) lacks the C-terminal 17 amino acids, gp4 ⌬ ZBD (resi- dues 64 –566) lacks the N-terminal ZBD but includes a part of the flexible linker that connects the ZBD and RPD, gp4 ⌬ primase (residues 241–566) lacks both the N-terminal ZBD and RPD but contains the flexible linker that con- nects the RPD and the helicase domain, and gp4 ⌬ C ⌬ primase consists of the helicase domain and the flexible linker connecting helicase with the RPD but is lacking both the primase domain and the C-terminal acidic tail.

To map the deletion in T5stO on the T5 chromosome by electron microscopy, the intact strand of T5stO is hybridized with a mixture of two fragments as shown in Fig... Schematic representa[r]

The dialyzed membrane complex was further purified by centrifugation in a second cesium chloride gradient under identical conditions described above.. At the end of the centrifugation, f[r]

The 32S to 100S linear DNA also contained TJ restriction endonuclease fragments, predominantly the TJL-R fragment expected of a concatemer that consists of monomeric DNAs joined left end[r]

Our observation that the defects which occur in phage DNA synthesis during infection of tsnB cells closely resemble those which occur during infection of a wild-type cell with a T7 gene [r]

The positions of promoter and terminator sites for the T7-specific RNA polymerase were estimated from the molecular weights of transcripts from exonucleolytically shortened templates 7; [r]

Through the use of an agarose-formaldehyde gel system that permits the resolution of high-molecular-weight RNAs, transcription of template DNA that had been cut with various restriction [r]

The needle-like extension associated with the capsid indi- cated by arrow 5 of Figure 3 (magnified in the inset) is seen more clearly to penetrate the attached vesicle and has a length of 41 nm, but also appears to have an additional ~14 nm broken from the extension at the distal inner sur- face of the vesicle (black arrowhead in Figure 3 and inset). The high visibility within the vesicle is apparently caused by vesicle breakage/leakage that is indicated by the dis- continuity of the vesicle's boundary. The capsid of this complex is partially full of DNA. The outer diameter of the needle is 8–11 nm, significantly smaller than the diameter of the wider end of the tail. A central channel, 3–4 nm in diameter, is sometimes visible, as illustrated by the more magnified complexes in Figure 4a–d. Thirty-five needle- like extensions, all with diameter in this range, were observed. Some were on capsids that were not attached to vesicles. Some had lengths of 50–60 nm (Figure 4e). Most Electron microscopy: a single field of particles

When extracts were incubated at 30°C and the intracellular DNA was subsequently extracted and digested with BstNI plus BglI, a novel anomalous DNA fragment 49% of the length of mature DN[r]

More recently, the nonionic detergent Brij-58 was shown to produce a similarly fast-sedimenting structure, which in electron micrographs appeared to consist of up to 30 phage equivalents[r]

45[Suppl.]:51-74, 1955 as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin [r]

DNA from T7%8 phage was irradiated and incubated with extracts from T73- or T73,6-infected strain W3110 under conditions that would permit both replication and recombination.. Controls n[r]

The major portion of the newly synthesized T7 DNA cosedimented Control Pronase Trypsin DNase RNase Phospholipase A Percent of control exhibiting tast sedimentation rate Membranes DNA 10 [r]

It was observed that after infection with T7 wild type a major portion of the newly synthesized DNA sedimented in both alkaline and neutral sucrose gradients 1.3 to 1.6 times as fast as [r]