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Margarita Salas

Publications and source records attributed to Margarita Salas.

At least 19 recordsLinked to original sources
PubMed2006

Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity.

This paper shows that the protein-primed DNA polymerases encoded by bacteriophages Nf and GA-1, unlike other DNA polymerases, do not require unwinding or processivity factors for efficient synthesis of full-length terminal protein (TP)-DNA. Analysis of their polymerization activity shows that both DNA polymerases base their replication efficiency on a high processivity and on the capacity to couple polymerization to strand displacement. Both enzymes are endowed with a proofreading activity that acts coordinately with the polymerization one to edit polymerization errors. Additionally, Nf double-stranded DNA binding protein (DBP) greatly stimulated the in vitro formation of the TP-dAMP initiation complex by decreasing the K(m) value for dATP of the Nf DNA polymerase by >20-fold. Whereas Nf DNA polymerase, as the phi29 enzyme, is able to use its homologous TP as well as DNA as primer, GA-1 DNA polymerase appears to have evolved to use its corresponding TP as the only primer of DNA synthesis. Such exceptional behaviour is discussed in the light of the recently solved structure of the DNA polymerase/TP complex of the related bacteriophage phi29.

Amino Acid Sequence↗
PubMed2006

Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication.

The transcription factor Spo0A is a master regulator for entry into sporulation in Bacillus subtilis and also regulates expression of the virulent B. subtilis phage phi29. Here, we describe a novel function for Spo0A, being an inhibitor of DNA replication of both, the phi29 genome and the B. subtilis chromosome. Binding of Spo0A near the phi29 DNA ends, constituting the two origins of replication of the linear phi29 genome, prevents formation of phi29 protein p6-nucleoprotein initiation complex resulting in inhibition of phi29 DNA replication. At the B. subtilis oriC, binding of Spo0A to specific sequences, which mostly coincide with DnaA-binding sites, prevents open complex formation. Thus, by binding to the origins of replication, Spo0A prevents the initiation step of DNA replication of either genome. The implications of this novel role of Spo0A for phage phi29 development and the bacterial chromosome replication during the onset of sporulation are discussed.

Bacillus Phages↗
PubMed2006

Involvement of phi29 DNA polymerase thumb subdomain in the proper coordination of synthesis and degradation during DNA replication.

Phi29 DNA polymerase achieves a functional coupling between its 3'-5' exonuclease and polymerization activities by means of important contacts with the DNA at both active sites. The placement and orientation of residues Lys538, Lys555, Lys557, Gln560, Thr571, Thr573 and Lys575 in a modelled phi29 DNA polymerase-DNA complex suggest a DNA-binding role. In addition, crystal structure of phi29 DNA polymerase-oligo (dT)5 complex showed Leu567, placed at the tip of the thumb subdomain, lying between the two 3'-terminal bases at the exonuclease site. Single replacement of these phi29 DNA polymerase residues by alanine was made, and mutant derivatives were overproduced and purified to homogeneity. The results obtained in the assay of their synthetic and degradative activities, as well as their coordination, allow us to propose: (1) a primer-terminus stabilization role at the polymerase active site for residues Lys538, Thr573 and Lys575, (2) a primer-terminus stabilization role at the exonuclease active site for residues Leu567 and Lys555 and (3) a primer-terminus binding role in both editing and polymerization modes for residue Gln560. The results presented here lead us to propose phi29 DNA polymerase thumb as the main subdomain responsible for the coordination of polymerization and exonuclease activities.

Bacillus Phages↗
PubMed2006

The structure of phage phi29 transcription regulator p4-DNA complex reveals an N-hook motif for DNA.

Protein p4 affects the transcriptional switch that divides bacteriophage phi29 infection in early and late phases. The synthesis of DNA replication proteins and p4 takes place in the early phase, while structural, morphogenesis, and lysis proteins are synthesized in the late phase. Transcriptional switch by p4 is achieved by activating the late promoter A3 and repressing the early promoters A2b and A2c. The crystal structure of p4 alone and in complex with a 41 bp DNA, including the A3 promoter binding site, helps us to understand how the phage cycle is controlled. Protein p4 has a unique alpha/beta fold that includes a DNA recognition motif consisting of two N-terminal beta turn substructures, or N-hooks, located at the tips of an elongated protein homodimer. The two N-hooks enter the major groove of the double helix, establishing base-specific contacts. A high DNA curvature allows p4 N-hooks to reach two major groove areas three helical turns apart, like a bow and its string.

Amino Acid Motifs↗
PubMed2006

Requirements for Bacillus subtilis bacteriophage phi29 DNA ejection.

Phage phi29 infects Bacillus subtilis and ejects its linear DNA with a right to left polarity in a two-step, "push-pull" mechanism. In the first step 65% of the DNA is pushed inside the cell, presumably by the pressure built inside the capsid. In the second step, the remaining DNA is pulled by a hypothetical motor that comprises at least viral protein p17, encoded by the right early operon, in an energy-dependent process. We have further studied phi29 DNA ejection by using energy poisons and DNA replication and transcription inhibitors. The first step is passive, as it does not require an external energy source. The second step is transcription-independent and is completely abolished by novobiocin, suggesting a requirement for negatively supercoiled DNA. Viral DNA pulling also requires an electrochemical proton gradient, as the process is highly impaired by specific energy poisons such as gramicidin and CCCP (carbonyl cyanide m-chlorophenylhydrazone). The fact that azide has no effect in the absence of p17 suggests that this protein is essential for energy transduction.

Bacillus Phages↗
PubMed2006

The phi29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition.

The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage phi29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 A resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.

Bacillus Phages↗
PubMed2006

A uracil-DNA glycosylase inhibitor encoded by a non-uracil containing viral DNA.

Uracil-DNA glycosylase (UDG) is an enzyme involved in the base excision repair pathway. It specifically removes uracil from both single-stranded and double-stranded DNA. The genome of the Bacillus subtilis phage 29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5'-end. Replication of 29 DNA starts by a protein-priming mechanism and generates intermediates that have long stretches of single-stranded DNA. By using in vivo chemical cross-linking and affinity chromatography techniques, we found that UDG is a cellular target for the early viral protein p56. Addition of purified protein p56 to B. subtilis extracts inhibited the endogenous UDG activity. Moreover, extracts from 29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by 29 to prevent the action of the base excision repair pathway if uracil residues arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a non-uracil-containing viral DNA.

Bacillus Phages↗
PubMed2005

Structural basis for membrane anchorage of viral phi29 DNA during replication.

Prokaryotic DNA replication is compartmentalized at the cellular membrane. Functional and biochemical studies showed that the Bacillus subtilis phage 29-encoded membrane protein p16.7 is directly involved in the organization of membrane-associated viral DNA replication. The structure of the functional domain of p16.7 in complex with DNA, presented here, reveals the multimerization mode of the protein and provides insights in the organization of the phage genome at the membrane of the infected cell.

Bacillus subtilis↗
PubMed2005

Molecular basis for the exploitation of spore formation as survival mechanism by virulent phage phi29.

Phage phi29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that phi29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host-encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected phi29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses phi29 development by repressing the main early phi29 promoters via different and novel mechanisms and also by preventing activation of the single late phi29 promoter.

Bacillus Phages↗
PubMed2005

A new plasmid vector for regulated gene expression in Bacillus subtilis.

We have developed a novel regulated expression vector for Bacillus subtilis based on the Staphylococcus aureus plasmid pUB110. This vector, named pPR54, carries the P(R) promoter and the cI857 gene (encoding a temperature-sensitive transcriptional repressor) from the Escherichia coli phage lambda. Using the gfp gene from the jellyfish Aequorea victoria as a reporter, we show that pPR54 is a useful vector for controllable production of heterologous proteins in B. subtilis.

Bacillus subtilis↗
PubMed2005

A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity.

Recent crystallographic studies of phi29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a phi29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of phi29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.

Amino Acid Sequence↗
PubMed2005

Involvement of the "linker" region between the exonuclease and polymerization domains of phi29 DNA polymerase in DNA and TP binding.

For several DNA-dependent DNA polymerases it has been shown that their synthetic and degradative activities are organized in two separated modules. The functional coordination required between them to accomplish successfully the replication process is provided by important contacts with the substrate contributed by residues coming from both modules. These domains are connected by a central "linker" region adjacent to the "YxGG/A" motif, the putative limit of the polymerization domain. We describe here the mutational analysis of phi29 DNA polymerase in several residues of this region, connecting the N- and C-terminal domains and conserved in DNA polymerases able to start replication by protein-priming. The mutant polymerases with the less conservative changes showed reduced DNA binding activity. Additionally, their TP binding capacity was reduced, affecting the TP-deoxynucleotidylation in the absence of template. Interestingly, the role of the residues studied here in DNA binding seems to be especially important to start replication, when the polymerase enters from the closed binary into the ternary complex. These results allow us to propose that this interdomain region of phi29 DNA polymerase is playing an important role for substrate binding including both DNA and TP.

Amino Acid Sequence↗
PubMed2005

Structure of the functional domain of phi29 replication organizer: insights into oligomerization and dna binding.

The Bacillus subtilis phage phi29-encoded membrane protein p16.7 is one of the few proteins involved in prokaryotic membrane-associated DNA replication that has been characterized at a functional and biochemical level. In this work we have determined both the solution and crystal structures of its dimeric functional domain, p16.7C. Although the secondary structure of p16.7C is remarkably similar to that of the DNA binding homeodomain, present in proteins belonging to a large family of eukaryotic transcription factors, the tertiary structures of p16.7C and homeodomains are fundamentally different. In fact, p16.7C defines a novel dimeric six-helical fold. We also show that p16.7C can form multimers in solution and that this feature is a key factor for efficient DNA binding. Moreover, a combination of NMR and x-ray approaches, combined with functional analyses of mutants, revealed that multimerization of p16.7C dimers is mediated by a large protein surface that is characterized by a striking self-complementarity. Finally, the structural analyses of the p16.7C dimer and oligomers provide important clues about how protein multimerization and DNA binding are coupled.

Binding Sites↗
PubMed2005

A precise DNA bend angle is essential for the function of the phage phi29 transcriptional regulator.

Bacteriophage phi29 protein p4 is essential for the regulation of the switch from early to late phage transcription. The protein binds to two regions of the phage genome located between the regulated promoters. Each region contains two inverted repeats separated by 1 bp. We used circular permutation assays to study the topology of the DNA upon binding of the protein and found that p4 induced the same extent of bending independent of the topology of the binding region. In addition, the results revealed that the p4-induced bending is not dependent on the affinity to the binding site but is intrinsic to p4 binding. Independent binding sites were identified through the characterization of the minimal sequence required for p4 binding. The protein has different affinity for each of its binding sites, with those overlapping the A2c and A2b promoter cores (sites 1 and 3), having the highest affinity. The functionality of the p4 binding sites and the contribution of p4-mediated promoter restructuring in transcription regulation is discussed.

Bacillus Phages↗
PubMed2005

Compartmentalization of prokaryotic DNA replication.

It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented.

Bacillus subtilis↗
PubMed2005

Homologies and divergences in the transcription regulatory system of two related Bacillus subtilis phages.

Transcription regulation relies on the molecular interplay between the RNA polymerase and regulatory factors. Phages of the phi29-like genus encode two regulatory proteins, p4 and p6. In phi29, the switch from early to late transcription is based on the synergistic binding of proteins p4 and p6 to the promoter sequence, resulting in a nucleosome-like structure able to synergize or antagonize the binding of RNAP. We show that a nucleosome-like structure of p4 and p6 is also formed in the related phage Nf and that this structure is responsible for the coordinated control of the early and late promoters. However, in spite of their homologies, the transcriptional regulators are not interchangeable, and only when all of the components of the Nf regulatory system are present is fully active transcriptional regulation of the Nf promoters achieved.

Bacillus Phages↗
PubMed2004

Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29.

The DNA polymerase from phage phi29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein. The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities. Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site. This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands. Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase. The high processivity of phi29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA.

Amino Acid Sequence↗