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Biomedical subjects

M de Vega

Publications and source records attributed to M de Vega.

12 recordsLinked to original sources
PubMed2003

[Treatment of T1N0 glottis carcinoma with radiotherapy. Results at our center and review of the literature].

OBJECTIVE: To evaluate the results of radiotherapy in the treatment of patients with T1N0 glottic squamous cell carcinoma. MATERIAL AND METHODS: Retrospective study of a cohort of 338 patients with T1N0 glottic carcinoma treated with radiotherapy at our institution between 1985-1997. A review of the literature published during the last ten years was carried out. RESULTS: Local control with radiotherapy in our patients was 82%, reaching 97% when salvage surgery was included. The local control with radiotherapy in most of the published series ranges between 81.90%. CONCLUSIONS: Treatment with radiotherapy achieves local control in early glottic carcinomas (T1N0) in 80-90% of cases. In our centre such treatment achieved local control in 82% of cases.

Carcinoma, Squamous Cell↗
PubMed2000

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein.

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.

Amino Acid Motifs↗
PubMed2000

Phage phi 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site.

Three highly conserved amino acid residues have been characterised here as ssDNA ligands at the 3'-5' exonuclease active site of o29 DNA polymerase. The functional role of Tyr59, His61 and Phe69 residues of o29 DNA polymerase (belonging to Exo II motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssDNA ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. These studies revealed that the mutations introduced severely affected their ssDNA binding capacity and, as a consequence, the 3'-5' exonuclease activity on ssDNA substrates was also severely impaired, producing drastic defects in the maintenance of replication fidelity. Crystal structures of Klenow fragment of Pol Ik and Thermococcus gorgonarius DNA polymerase complexed with ssDNA at their 3'-5' exonuclease active sites revealed that residues Gln419 of the former, and Tyr209 of the latter, the counterparts of His61 of o29 DNA polymerase, are making contacts with the penultimate phosphodiester bond of ssDNA substrate. Here, the functional role of this residue is described.

Amino Acid Motifs↗
PubMed1999

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

Bacillus Phages↗
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

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↗
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↗
PubMed1996

Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases.

By site-directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3'-5' exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3'-5' exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA-dependent DNA polymerases. Moreover, in contrast to the dual role in 3'-5' exonucleolysis and strand displacement previously observed for phi29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer-terminus at the 3'-5' exonuclease active site.

Amino Acid Sequence↗
PubMed1995

Backward updating of mental models during continuous reading of narratives.

Participants' ability to track the protagonist's position and surroundings, during continuous reading, was investigated. In Experiment 1, participants read passages involving either inside-outside or top-down topological relations. A typical story described the protagonist interacting with 1 object, which was either consistent or inconsistent with his or her location. The results show that it took longer to read the sentence in the inconsistent condition. Experiments 2-5 used recognition probe words to test the accessibility of both consistent and inconsistent objects. The results show that participants did not update the situation model when the last sentences did not mention again any target object (Experiments 2 and 3). However, the mention of an object by means of an ambiguous pronoun triggers the updating of the situation to resolve the antecedent. The updating starts immediately after reading the pronoun, and the target still remains activated at the end of the sentence (Experiments 4 and 5). The overall results establish boundary conditions for mental model updating.

Adult↗
PubMed1994

Terminal protein-primed DNA amplification.

By using appropriate amounts of four bacteriophage phi 29 DNA replication proteins--terminal protein, DNA polymerase, protein p6 (double-stranded DNA-binding protein), and protein p5 (single-stranded DNA-binding protein)--it has been possible to amplify limited amounts of the 19,285-bp-long phi 29 DNA molecule by three orders of magnitude after 1 hr of incubation at 30 degrees C. Moreover, the quality of the amplified material was demonstrated by transfection experiments, in which infectivity of the synthetic (amplified) phi 29 DNA, measured as the ability to produce phage particles, was identical to that of the natural phi 29 DNA obtained from virions. The results presented in this paper establish some of the requisites for the development of isothermal DNA amplification strategies based on the bacteriophage phi 29 DNA replication machinery that are suitable for the amplification of very large (> 70 kb) segments of DNA.

Bacillus Phages↗