Biomedical subjects

M Salas

Publications and source records attributed to M Salas.

At least 73 records · Page 4Linked to original sources

PubMed1998

Binding of phage phi29 protein p4 to the early A2c promoter: recruitment of a repressor by the RNA polymerase.

Regulatory protein p4 from Bacillus subtilis phage Phi29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase alpha subunit. This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented. Here, the binding of protein p4 to the A2c promoter has been studied. In the absence of RNA polymerase, protein p4 was found to bind with low affinity to a site centered at position -39 relative to the transcription start site. When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position -71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase alpha-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high affinity. A mutational analysis revealed that the site at -71 is critical for a stable interaction between protein p4 and RNA polymerase, and for efficient repression, whereas mutation of the site at -39 had only a small effect on repression efficiency. Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance.

Bacillus subtilis↗

PubMed1998

Role of the first aspartate residue of the "YxDTDS" motif of phi29 DNA polymerase as a metal ligand during both TP-primed and DNA-primed DNA synthesis.

Almost all known nucleic acid polymerases require three acidic residues to bind the metal ion during catalysis of nucleotide incorporation. Nevertheless, recent crystallographic data on bacteriophage RB69 DNA polymerase indicate that the first aspartate residue belonging to the conserved motif "YxDTDS" could have a merely structural role. To address this question, a mutant protein at the homologous aspartate residue (Asp456) in phi29 DNA polymerase was made 3'-5' exonuclease deficient. This allowed us to analyse the functional importance of this residue in different metal-dependent reactions that can be performed using either terminal protein (TP) or DNA primers. When Mg2+ was used as the metal activator, the synthetic activities of the mutant phi29 DNA polymerase, TP-primed initiation and DNA-primed polymerisation, were about 50-fold less efficient than those of the wild-type enzyme. Interestingly, the use of Mn2+ as the metal activator partially restored the wild-type phenotype. When polymerisation required an efficient translocation along the template, mutation of Asp456 strongly affected the catalytic efficiency of phi29 DNA polymerase. The results presented here indicate that Asp456 has a catalytic role as a metal-activator ligand, but also contributes to enzyme translocation along the DNA, required during consecutive nucleotide incorporation cycles. Moreover, Asp456 appears to be critical to remodel the active site during transition from TP priming to DNA priming. The results are discussed in the light of structural information corresponding to distantly related polymerases.

Aspartic Acid↗

PubMed1998

phi29 DNA polymerase residue Ser122, a single-stranded DNA ligand for 3'-5' exonucleolysis, is required to interact with the terminal protein.

Three amino acid residues highly conserved in most proofreading DNA polymerases, a phenylalanine contained in the Exo II motif and a serine and a leucine belonging to the S/TLx2h motif, were recently shown to be critical for 3'-5' exonucleolysis by acting as single-stranded DNA ligands (de Vega, M., Lázaro, J.M., Salas, M. and Blanco, L. (1998) J. Mol. Biol. 279, 807-822). In this paper, site-directed mutants at these three residues were used to analyze their functional importance for the synthetic activities of phi29 DNA polymerase, an enzyme able to start linear phi29 DNA replication using a terminal protein (TP) as primer. Mutations introduced at Phe65, Ser122, and Leu123 residues of phi29 DNA polymerase severely affected the replication capacity of the enzyme. Three mutants, F65S, S122T, and S122N, were strongly affected in their capacity to interact with a DNA primer/template structure, suggesting a dual role during both polymerization and proofreading. Interestingly, mutant S122N was not able to maintain a stable interaction with the TP primer, thus impeding the firsts steps (initiation and transition) of phi29 DNA replication. The involvement of Ser122 in the consecutive binding of TP and DNA is compatible with the finding that the TP/DNA polymerase heterodimer was not able to use a DNA primer/template structure. Assuming a structural conservation among the eukaryotic-type DNA polymerases, a model for the interactions of phi29 DNA polymerase with both TP and DNA primers is presented.

Amino Acid Sequence↗

PubMed1998

Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets.

Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33. By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner. We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery.

Amino Acid Sequence↗

PubMed1998

The RGD sequence in phage phi29 terminal protein is required for interaction with phi29 DNA polymerase.

The RGD (Arg-Gly-Asp) motif functions as a recognition site for adhesive proteins responsible for a number of cell-cell interactions. Certain viruses use this sequence as a receptor-binding site by interaction with cellular integrins. To elucidate the role of the RGD sequence of the phi29 terminal protein (TP), seven modified TPs were generated by site-directed mutagenesis. Most of the TP mutants were not efficiently used as primers, leading to a reduction of the TP-dAMP complex formation in the presence of the phi29 TP-DNA template. Moreover, these mutant TPs were poorly deoxyadenylylated by phi29 DNA polymerase in the absence of template. Analysis of primer TP/DNA polymerase complex formation showed that the modified TPs were affected in the formation of the heterodimeric complex. These results indicate that the RGD sequence present in phi29 TP is primarily involved in interaction with the viral DNA polymerase.

Amino Acid Sequence↗

PubMed1998

Mutational analysis of phi29 DNA polymerase residues acting as ssDNA ligands for 3'-5' exonucleolysis.

Here, three highly conserved amino acid residues have been characterized to function as ssDNA binding ligands at the 3'-5' exonuclease active site of phi29 DNA polymerase. One of these residues, Phe65, belongs to motif Exo II, previously described to contain an invariant aspartate and an invariant asparagine involved in catalysis and ssDNA binding, respectively. The other two residues, Ser122 and Leu123, form a newly identified motif "(S/T)Lx2h", and are the homologous counterparts of Pol I residues Asp457 and Met458, and of T4 DNA polymerase residues Ser286 and Leu287, the latter three residues shown to contact ssDNA at their corresponding cocrystal 3D structures. Site-directed mutagenesis and biochemical analysis of eight phi29 DNA polymerase mutant proteins at residues Phe65, Ser122 and Leu123 indicated their functional importance for: (1) a stable interaction with ssDNA; (2) 3'-5' exonucleolysis of ssDNA substrates; (3) proofreading of DNA polymerization errors. Extrapolation to the crystal structures of Klenow and T4 DNA polymerases indicates that the invariant aromatic ring contiguous to the catalytic aspartate of the Exo II motif, corresponding to Tyr423 in Klenow, Phe218 in T4, and Phe65 in phi29 DNA polymerase, appears to be critical to orient the ssDNA substrate in a stable conformation to allow 3'-5' exonucleolytic catalysis. This is the first time that the functional importance of this invariant residue, belonging to the Exo II motif, has been demonstrated.

Amino Acid Sequence↗

PubMed1998

Phi 29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates.

A 44 kDa C-terminal fragment of phi 29 DNA polymerase has been separately expressed and purified from Escherichia coli cells. As expected, the truncated protein lacked the 3'-5' exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of phi 29 DNA polymerase. On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn2+ as metal activator, although the catalytic efficiency was greatly reduced with respect to that of the complete enzyme. Moreover, and in contrast to the high processivity exhibited by phi 29 DNA polymerase (> 70 kb), polymerization by its C-terminal domain was completely distributive. All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization. Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of phi 29 DNA replication. We conclude that the C-terminal portion of phi 29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis. These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more specific functions (TP-priming, high processivity and strand-displacement) inherent to phi 29 DNA polymerase.

Bacillus Phages↗

PubMed1998

Substitution of the C-terminal domain of the Escherichia coli RNA polymerase alpha subunit by that from Bacillus subtilis makes the enzyme responsive to a Bacillus subtilis transcriptional activator.

Regulatory protein p4 of Bacillus subtilis phage phi 29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase alpha subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical alpha subunit containing the N-terminal domain of the E. coli alpha subunit and the CTD of the B. subtilis alpha subunit. Reconstitution of RNA polymerases containing this chimerical alpha subunit, the E. coli beta and beta' subunits, and the vegetative sigma factor from either E. coli (sigma 70) or B. subtilis (sigma A), generated hybrid enzymes that were responsive to protein p4 and efficiently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis alpha-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.

Amino Acid Sequence↗

PubMed1998

Transcription activation and repression by interaction of a regulator with the alpha subunit of RNA polymerase: the model of phage phi 29 protein p4.

Regulatory protein p4, encoded by Bacillus subtilis phage phi 29, has proved to be a very useful model to analyze the molecular mechanisms of transcription regulation. Protein p4 modulates the transcription of phage phi 29 genome by activating the late A3 promoter (PA3) and simultaneously repressing the two main early promoters, A2b and A2c (or PA2b and PA2c). This review describes in detail the regulatory mechanism leading to activation or repression, and discusses them in the context of the recent findings on the role of the RNA polymerase alpha subunit in transcription regulation. Activation of PA3 implies the p4-mediated stabilization of RNA polymerase at the promoter as a closed complex. Repression of the early A2b promoter occurs by binding of protein p4 to a site that partially overlaps the -35 consensus region of the promoter, therefore preventing the binding of RNA polymerase to the promoter. Repression of the A2c promoter, located 96 bp downstream from PA2b, occurs by a different mechanism that implies the simultaneous binding of protein p4 and RNA polymerase to the promoter in such a way that promoter clearance is inhibited. Interestingly, activation of PA3 and repression of PA2c require an interaction between protein p4 and RNA polymerase, and in both cases this interaction occurs between the same surface of protein p4 and the C-terminal domain of the alpha subunit of RNA polymerase, which provides new insights into how a protein can activate or repress transcription by subtle variations in the protein-DNA complexes formed at promoters.

Bacillus Phages↗

PubMed1998

DNA polymerase template switching at specific sites on the phi29 genome causes the in vivo accumulation of subgenomic phi29 DNA molecules.

The accumulation of subgenomic phage phi29 DNA molecules with specific sizes was observed after prolonged infection times with delayed lysis phage mutants. Whereas the majority of the molecules had a size of 4 kb, additional DNA species were observed with sizes of 8.2, 6.5, 2.3, 2 and 1 kb. Most of the molecules were shown to originate from the right end of the linear Bacillus subtilis phage phi29 genome. The nature of the 4, 2.3, 2 and 1 kb molecules was studied. The 2 kb molecules were shown to be single-stranded self-complementary strands forming hairpin structures. The other molecules consisted of palindromic linear double-stranded DNA molecules. Most probably, the subgenomic DNA molecules were formed when the moving phage replication fork from the right origin encountered a block that induces the DNA polymerase to switch template. Once formed, the subgenomic molecules are then amplified in vivo. Determination of the centres of symmetry of the 4 and 1 kb molecules revealed that both contained the almost 16 bp perfect dyad symmetry element (DSE): 5'-TGTTtCAC-GTGg-AACA-3' being a likely candidate for a protein binding site. Database analysis showed that this sequence occurs four times in the phi29 genome. In addition, the almost identical sequence 5'-TgGTTTCAC-GTGGAAtCA-3' was found once. These five DSEs are all located in the right half of the phi29 genome, and the same sequences are also present in the linear DNA of related B. subtilis phages. Most interestingly, this sequence is also found in the spoOJ gene of the B. subtilis chromosome. Recently, it has been shown that the SpoOJ protein is associated in vivo with the same DSE. As the same subgenomic phi29 DNA molecules accumulate after infection of B. subtilis spoOJ deletion strains, it is likely that, in addition to and/or independently of SpoOJ, other protein(s) bind to DSE.

Bacillus Phages↗

PubMed1998

Transcriptional activation of the Bacillus subtilis spoIIG promoter by the response regulator Spo0A is independent of the C-terminal domain of the RNA polymerase alpha subunit.

In vitro transcription from the spoIIG promoter by Bacillus subtilis RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.

Bacillus subtilis↗

PubMed1998

Control mechanisms of bacteriophage phi 29 DNA expression.

The phage phi 29 regulatory protein p4 activates the late promoter A3 by stabilizing the binding of Bacillus subtilis RNA polymerase (RNAP) as a closed complex. Interaction between the two proteins occurs through amino acid Arg120 in protein p4 and the C-terminal domain of the RNAP alpha subunit (alpha-CTD). In addition to its role as activator of the late transcription, protein p4 represses early transcription from the A2b and A2c promoters, that are divergently transcribed. Binding of p4 to its recognition site at the A3 promoter displaces the RNAP from promoter A2b, both by steric hindrance and by the curvature induced upon p4 binding. At the A2c promoter, the RNAP cooperates with p4 binding in such a way that promoter clearance is prevented. Interestingly, amino acid Arg120 in p4 and the alpha-CTD in B. subtilis RNAP are involved in the interactions that lead to transcription repression at promoter A2c. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. In the absence of a -35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the idea that overstabilization of RNAP at the promoter over a threshold level leads to repression.

Bacillus Phages↗

PubMed1998

The hindlimb hyperextension associated to the urogenital response in newborn underfed rats.

BACKGROUND: The aim of the study was to investigate how neonatal undernutrition interacts with mother-infant relationships to interfere with the expression of the urogenital response in the newborn. METHODS: The hyperextensive reflex components associated to the urogenital response (HUR) were measured between postnatal days 1-21, in control and neonatally undernourished rats with or without fullness of the bladder. RESULTS: In Experiment 1, both male and female underfed rats with different degrees of bladder fullness exhibited an increment in HUR latency and vertical hindlimb displacement, reduced transversal separation and prolonged performance of hindlimb relaxation. Experiment 2 was performed in rats after urine elicitation provoked only reduced hindlimb transversal separation in males, and prolonged latency to HUR in females. DISCUSSION: These findings may be related to the vulnerability to neonatal undernutrition of the maturational processes which take place in the spinal cord, the condition of the bladder, and hindleg muscle maturation during this period of life. CONCLUSIONS: Data suggest that neonatal undernutrition may play a role in mother-infant interaction by interfering with HUR responses to maternal anogenital licking of the pups.

Animals↗

PubMed1998

Swimming and self-grooming development of pups nursed by neonatally underfed rats.

Swimming and self-grooming development were analyzed in experimental animals nursed by neonatally underfed dams by a method including the deprivation of both food and sensory cues which provoke long-term altered maternal behavior. Experimental animals obtained from these dams manifested a significant retardation in physical growth, as evidenced by a decrease in body weight and a 1-day delay in ear and eye-opening, as well as a significant delay in swimming ability, escaping from the water and an increase in self-grooming behavior. These alterations of experimental animals may be presumably related with the carelessness in maternal care and altered so-matosensory stimulation given by the neonatally underfed dams to her experimental pups. Data suggest that the poor somatosensory stimulation given by the mother to her pups, the reduced early social interaction or the motivational stage of pups may be important determinants for the development of brain mechanisms underlying reflex activity of the progeny.

Animals↗

PubMed1997

Phage phi29 protein p6 is in a monomer-dimer equilibrium that shifts to higher association states at the millimolar concentrations found in vivo.

Protein p6 from Bacillus subtilis phage phi29 (Mr = 11 800) binds in vitro to DNA forming a large nucleoprotein complex in which the DNA wraps a multimeric protein core. The high intracellular abundance of protein p6 together with its ability to bind the whole phi29 DNA in vitro strongly suggests that it plays a role in viral genome organization. We have determined by sedimentation equilibrium analysis that protein p6 (1-100 microM range), in the absence of DNA, is in a monomer-dimer equilibrium, with an association constant (K2) of approximately 2 x 10(5) M-1. The intracellular concentration of protein p6 (approximately 1 mM) was estimated measuring the number of copies per cell (7 x 10(5)) and the cell volume (1 x 10(-15) L). At concentrations around 1 mM, protein p6 associates into oligomers. This self-association behavior is compatible with a dimer-hexamer model (K2,6 = 3.2 x 10(8) M-2) or with an isodesmic association of the dimer (K = 950 M-1), because the apparent weight-average molecular mass (Mw,a) does not reach saturation at the highest protein concentrations. The sedimentation coefficients of protein p6 monomer and dimer were 1.4 and 2.0, respectively, compatible with translational frictional ratios (f/fo) of 1.15 and 1.30, which slightly deviate from the hydrodynamics of a rigid globular protein. Taking together these results and considering the structure of the nucleoprotein complex, we speculate that the observed oligomers of protein p6 could mimic a scaffold on which DNA folds to form the nucleoprotein complex in vivo.

Bacillus Phages↗

PubMed1997

Bacteriophage phi29 DNA replication arrest caused by codirectional collisions with the transcription machinery.

The consequences on replication of collisions between phi29 DNA polymerase, a monomeric replicase endowed with strand displacement capacity, and the transcription machinery have been studied in vitro. Codirectional collisions with stalled transcription ternary complexes at four different promoters in the phi29 genome were found to block replication fork progression. Upon collision, the DNA polymerase remained on the template and was able to resume elongation once the RNA polymerase was allowed to move. Collisions with RNA polymerase molecules moving in the same direction also interfered with replication, causing a decrease in the replication rate. These results lead to the proposal that in bacteriophage phi29 a transcription complex physically blocks the progression of a replication fork. We suggest that temporal regulation of transcription and the low probability that the replication and transcription processes colocalize in vivo contribute to achieving minimal interference between the two events.

Bacillus Phages↗

PubMed1997

An invariant lysine residue is involved in catalysis at the 3'-5' exonuclease active site of eukaryotic-type DNA polymerases.

A lysine residue, contained in the motif "Kx2h", has been invariantly found in the eukaryotic-type (family B) class of DNA-dependent DNA polymerases with a proofreading function. The importance of this lysine has been assessed by site-directed mutagenesis in the corresponding residue (Lys143) of phi29 DNA polymerase. Substitution of this residue either by arginine or isoleucine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, giving a characteristic distributive pattern that contrasts with the processive pattern displayed by the wild-type phi29 DNA polymerase. Exonuclease assays carried out in the presence of a DNA trap, together with direct analysis of enzyme/ssDNA interaction, allowed us to conclude that this altered pattern was due to a reduction in the catalytic rate of these mutants, but not to a weakened association with ssDNA. These phenotypes indicate that the lysine residue of motif Kx2h plays an auxiliary role in catalysis of the exonuclease reaction, in very good agreement with recent crystallographic data showing that the lysine homologue of T4 DNA polymerase is indirectly involved in metal binding at the 3'-5' exonuclease active site. In agreement with a critical role in proofreading, substitution of Lys143 of phi29 DNA polymerase by arginine or isoleucine produced mutator enzymes that displayed a high frequency of misincorporation. Mutants at Lys143 also showed a reduced DNA polymerization capacity, but only when DNA synthesis was coupled to strand-displacement, an intrinsic property of phi29 DNA polymerase that is specifically affected by mutations at residues directly or indirectly involved in metal binding at the 3'-5' exonuclease active site.

Amino Acid Sequence↗

PubMed1997

ø29 DNA polymerase residue Lys383, invariant at motif B of DNA-dependent polymerases, is involved in dNTP binding.

Bacteriophage ø29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. Among them, motif B, characterized by the consensus +x3Kx(6-7)YG (+ being a positively charged amino acid), appears to be specifically conserved in those polymerases that use DNA but not RNA as template. In particular, the lysine residue of this motif is invariant in all members of DNA-dependent polymerases. In this paper we report a mutational analysis of this invariant residue of motif B with the construction and characterization of two mutant proteins in the corresponding residue (Lys383) of ø29 DNA polymerase. Mutant proteins (K383R and K383P) were overexpressed, purified and analyzed under steady-state conditions. In agreement with the modular organization proposed for ø29 DNA polymerase, the exonuclease activity was not affected in either mutant protein. Conversely, mutant K383P showed no detectable capacity to incorporate dNTP substrates using either DNA or TP as primer, although its affinity for DNA was not affected. The conservative substitution of Lys383 by arginine (K383R) resulted in a considerable impairment to use dNTPs, in both processive and non-processive DNA synthesis; the Km for dNTPs being 200-fold higher than that of the wild-type enzyme. Mutant K383R recovered the wild-type polymerase/exonuclease ratio when Mn2+ was used instead of Mg2+ as metal activator, indicating a distorted binding of the [dNTP-metal] chelate at the mutant enzyme active site. The positive charge at residue Lys383 was also critical in the catalysis of deoxynucleotidylation of the terminal protein by ø29 DNA polymerase. The results obtained suggest a direct role for the lysine residue in motif B in forming an evolutionarily conserved DNA templated dNTP binding pocket. Additionally, K383R mutant protein was also affected in the progression from protein-primed initiation to DNA elongation, a switch between two modes of priming that characterizes ø29 DNA replication.

Amino Acid Sequence↗