Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation.
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The outcome of collisions between Bacillus subtilis phage Phi29 DNA polymerase and oppositely oriented transcription complexes has been studied in vitro. We found that the replication fork was unable to go past a transcription ternary complex stalled head-on. However, head-on collisions did not lead to a deadlock. Both DNA and RNA polymerase remained bound to the template and, when the halted transcription complex was allowed to move, the replication machinery resumed normal elongation. These results suggested that a replication fork that encounters an RNA polymerase head-on whose movement is not impeded would bypass the transcription machinery. Our results for head-on collisions between concurrently moving replication and transcription complexes are indeed consistent with the existence of a resolving mechanism. The ability of Phi29 DNA polymerase to resolve head-on collisions with itself during symmetrical replication of Phi29 DNA in vivo is likely to be related to its ability to pass a head-on oriented RNA polymerase. (+info)
Mapping the inter-RNA interaction of bacterial virus phi29 packaging RNA by site-specific photoaffinity cross-linking.
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During replication, the lengthy genome of double-stranded DNA viruses is translocated with remarkable velocity into a limited space within the procapsid. The question of how this fascinating task is accomplished has long been a puzzle. Our recent investigation suggests that phi29 DNA packaging is accomplished by a mechanism similar to the driving of a bolt with a hex nut and that six packaging RNAs (pRNAs) form a hexagonal complex to gear the DNA-translocating machine (Chen, C., and Guo, P. (1997) J. Virol. 71, 3864-3871; Zhang, F., Lemieux, S., Wu, X., St.-Arnaud, S., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell 2, 141-147; Guo, P., Zhang, C., Chen, C., Garver, K., and Trottier, M., (1998) Mol. Cell 2, 149-155). In the current study, circularly permuted pRNAs were used to position an azidophenacyl photoreactive cross-linking agent specifically at a strategic site that was predicted to be involved in pRNA-pRNA interaction. Cross-linked pRNA dimers were isolated, and the sites of cross-link were mapped by primer extension. The cross-linked pRNA dimer retained full activity in phi29 procapsid binding and genomic DNA translocation, indicating that the cross-link distance constraints identified in dimer formation reflect the native pRNA complex. Both cross-linked dimers either containing or not containing the interlocking loops for programmed hexamer formation bound procapsid equally well; however, only the one containing the interlocking loops programmed for hexamer formation was active in phi29 DNA packaging. The cross-linked pRNA dimers were also identified as the minimum binding unit necessary for procapsid binding. Primer extension of the purified cross-linked pRNA dimers revealed that base G(82) was cross-linked to bases G(39), G(40), A(41), C(49), G(62), C(63), and C(64), which contribute to the formation of the three-way junction, suggesting that these bases are proximate in the formation of pRNA tertiary structure. Interestingly, the photoaffinity agent in the left interacting loop did not cross-link directly to the right loop as expected but cross-linked to bases adjacent to the right loop. These data provide a background for future modeling of pRNA tertiary structure. (+info)
Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins.
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DNA replication of phi29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage phi29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of phi29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of phi29 DNA polymerase to ssDNA and their stimulatory effect on replication by phi29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during phi29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by phi29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the phi29 and Nf SSBs do not show any effect. On the other hand, the phi29 SSB is the only one of the three SSBs able to increase the replication rate of phi29 DNA polymerase in primed M13 ssDNA replication. From the fact that the phi29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of phi29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB-ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on phi29-like DNA replication has been proposed. (+info)
Template specificity of transcription during sporulation of Bacillus subtilis.
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Partially purified extracts from sporulating Bacillussubtilis cultures transcribed different natural DNAs with different efficiencies. This template specificity results in an increased or a decreased synthetic activity with respect to extracts from vegetative cells, depending on the template used. With SPP1 DNA a decrease in activity occurs, whereas with T7 DNA an increased activity was observed, which is due to a higher efficiency of initiation. This is not an intrinsic property of RNA polymerase, but is due to some fraction(s) which can be separated from the enzyme. Together with invivo experiments on transcription and SPP1 phage production during sporulation, these results suggest a possible role of promoter recognition in sporulation. (+info)
Rifampicin-resistant bacteriophage PBS2 infection and RNA polymerase in Bacillus subtilis.
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Bacteriophage PBS2 replication is unaffected by rifampicin and other rifamycin derivatives, which are potent inhibitors of Bacillus subtilis RNA synthesis. Extracts of gently-lysed infected cells contain a DNA-dependent RNA polymerase activity which is specific for uracil-containing PBS2 DNA. The PBS2-induced RNA polymerase is insensitive to rifamycin derivatives which inhibit the host's RNA polymerase. (+info)
Identification of residues within two regions involved in self-association of viral histone-like protein p6 from phage theta29.
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Protein p6 of Bacillus subtilis phage theta29 is involved in the initiation of viral DNA replication and transcription by forming a multimeric nucleoprotein complex with the phage DNA. Based on this, together with its abundance and its capacity to bind to the whole viral genome, it has been proposed to be a viral histone-like protein. Protein p6 is in a monomer-dimer-oligomer equilibrium association. We have identified protein p6 mutants deficient in self-association by testing random mutants obtained by degenerated polymerase chain reaction in an in vivo assay for dimer formation. The mutations were mainly clustered in two regions located at the N terminus, and the central part of the protein. Site-directed single mutants, corresponding to those found in vivo, have been constructed and purified. Mutant p6A44V, located at the central part of the protein, showed an impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of O29 DNA replication. Mutant p6I8T has at least 10-fold reduced self-association capacity, does not bind DNA nor activate O29 DNA initiation of replication. C-terminal deletion mutants showed an enhanced dimer formation capacity. The highly acidic tail, removed in these mutants, is proposed to modulate the protein p6 self-association. (+info)
Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7.
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We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7. (+info)
Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification.
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All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging. (+info)