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Biomedical subjects

A Bernad

Publications and source records attributed to A Bernad.

At least 37 records · Page 2Linked to original sources
PubMed1994

Ex vivo expansion and selection of retrovirally transduced bone marrow: an efficient methodology for gene-transfer to murine lympho-haemopoietic stem cells.

An efficient procedure for the insertion of genetic markers into a large proportion of the mouse haemopoietic system was developed, based on the in vitro expansion of retrovirally infected bone marrow and selection of the transduced cells. Bone marrow cells harvested 4 d after 5-FU treatment were incubated under IL-3/SCF stimulation and their growth dynamic, susceptibility to retroviral infection and reconstitution capacity evaluated throughout the incubation period. On the third day of culture a maximum expansion in the CFU-GM and CFU-S12 progenitor pools was observed (130- and 15-fold, respectively), with no apparent impairment in long-term repopulating precursors. This expansion was, however, accompanied by a net decrease in the CFU-GM susceptibility to the infection by supernatants containing a Moloney-derived ecotropic retroviral vector carrying the neor gene. The designed protocol thus involved the infection of freshly harvested 5-FU-treated bone marrow, followed by expansion under IL-3/SCF stimulation and selection for resistance to G418. This procedure allowed us to harvest up to 780 CFU-GM and 50 CFU-S12 per 10(5) bone marrow cells, free from non-genetically marked progenitors. Most of the animals reconstituted with the transduced marrow bore, for at least 5 months, a very high proportion of bone marrow, spleen and thymus cells tagged with the reporter gene. These results, together with the high percentage of haemopoietic precursors bearing the neor gene and expressing resistance to G418 5 months after the transplantation indicates that long-term lympho-haemopoietic repopulating cells were efficiently transduced and selected in vitro under conditions that preserve their self-renewal and differentiation properties. This gene-transfer methodology may improve the development of gene therapy protocols where the purging of non-transduced precursors would guarantee a lasting and uniform expression of exogenous genes.

Animals↗
PubMed1994

Accelerated and long-term hematopoietic engraftment in mice transplanted with ex vivo expanded bone marrow.

Using a murine experimental model, we investigated whether ex vivo expansion of BM grafts under IL-3/IL-6 stimulation accelerates the early hematopoietic recovery of recipients of BM transplants. To facilitate the ex vivo expansion of hematopoietic progenitors, BM was first enriched in proliferatively active hematopoietic stem cells by a single treatment with 5-fluorouracil (5FU) 4 days prior to the BM harvest. The results showed that the number of CFU-GM and CFU-S12 progenitors in the graft was significantly increased (56-fold and 14-fold, respectively), as a result of a 3 day incubation in the presence of IL-3 and IL-6. Daily analysis of animals transplanted with 5 x 10(4) BM cells, either freshly harvested or expanded for 3 days, showed that the expanded grafts consistently allowed a faster hematopoietic recovery of recipients. Differences between both groups of transplanted animals were most evident when the number of either femoral or splenic CFU-GMs were compared, with increases close to 70-fold at the fifth day of engraftment being observed. Similarly, mice transplanted with expanded grafts showed a hastened recovery in the cellularity of both organs that was most significant during the second week following transplantation, with maximal increases of 15 and 40-fold in the BM and spleen, respectively. Differences in peripheral leukocyte numbers between both groups of recipients were much less remarkable than those observed in the hematopoietic organs, although from the nadir period to the 11th day post-transplantation differences ranging from twofold to sixfold were apparent, consistent with a higher rate of mouse survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.

Bacillus Phages↗
PubMed1992

Phi 29 DNA polymerase active site. Mutants in conserved residues Tyr254 and Tyr390 are affected in dNTP binding.

Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs "D-NSLYP" and "K--NS(L/V)YG," regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.

Amino Acid Sequence↗
PubMed1992

Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases.

The complete amino acid (aa) alignment of the N-terminal domain of 33 DNA-dependent DNA polymerases encompassing the putative segments Exo I, Exo II and Exo III, proposed by Bernad et al. [Cell 59 (1989) 219-228] to form a conserved 3'-5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases, allowed us to identify and/or correct some of the most conserved segments (Exo I, II and III) in certain DNA polymerases. In particular, the aa region of T4 DNA polymerase and other eukaryotic (viral and cellular) DNA polymerases previously proposed as Exo I segment 1, did not align with the Exo I segment of Escherichia coli DNA polymerase I (PolI)-like and protein-primed DNA polymerases; instead, a new conserved region of aa similarity was identified in T4 DNA polymerase and eukaryotic (viral and cellular) DNA polymerases as their corresponding Exo I segment. Therefore, according to our alignment, the recently reported T4 DNA polymerase site-directed mutants, D189A and E191A [Reha-Krantz et al., Proc. Natl. Acad. Sci. USA 88 (1991) 2417-2421], do not correspond to what we now consider the critical Exo I motif of PolI. As discussed in this communication, the functional importance of conserved segments Exo I, Exo II and Exo III is supported by site-directed mutagenesis in PolI, and in phi 29, T7 and delta(Sc) DNA polymerases. Furthermore, genetically selected T4 DNA polymerase mutator mutants form two main clusters, centered in the conserved segment Exo III and in the newly identified Exo I segment.

Amino Acid Sequence↗
PubMed1992

The bacteriophage phi 29 DNA polymerase, a proofreading enzyme.

The bacteriophage phi 29 DNA polymerase, involved both in the protein-primed initiation and elongation steps of the viral DNA replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded DNA. This exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus. These characteristics enable the phi 29 DNA polymerase to act as a proofreading enzyme. A comparative analysis of the wild-type phi 29 DNA polymerase and a mutant lacking 3',5'-exonuclease activity indicated that a productive coupling between the exonuclease and polymerase activities is necessary to prevent fixation of polymerization errors. Based on these data, the phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication, appears to share the same mechanism for the editing function as that first proposed for T4 DNA polymerase and Escherichia coli DNA polymerase I on the basis of functional and structural studies.

Bacteriophages↗
PubMed1992

Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication.

Analysis of metal activation on the synthetic and degradative activities of phi 29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3'----5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'----5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi 29 DNA polymerase. Furthermore, DNA competition experiments using phi 29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases. Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.

Binding Sites↗
PubMed1992

DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase.

In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation.

Bacteriophages↗
PubMed1992

Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities.

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.

Amino Acid Sequence↗
PubMed1991

Characterization and mapping of the pyrophosphorolytic activity of the phage phi 29 DNA polymerase. Involvement of amino acid motifs highly conserved in alpha-like DNA polymerases.

The phi 29 DNA polymerase, an alpha-like DNA polymerase, shows an inorganic pyrophosphate-dependent degradative activity with similar requirements to the corresponding one of Escherichia coli DNA polymerase I: (a) it requires a high concentration of inorganic pyrophosphate and is reversed by polymerization; (b) like DNA polymerization, it needs a duplex DNA with protruding 5' single-strand; (c) it acts in the 3' to 5' direction releasing free dNTPs, thus, it can be considered as the reversal of polymerization; (d) although a correctly base-paired 3' primer terminus is the preferred substrate, the pyrophosphorolytic activity is able to remove mismatched 3' ends. In agreement with the structural and functional model previously proposed for the phi 29 DNA polymerase, the analysis of point mutations has revealed that the pyrophosphorolytic activity, like the polymerization activity, is located at the C-terminal portion of the molecule, involving the amino acid motif YCDTD, highly conserved in alpha-like DNA polymerases. Furthermore, the analysis of phi 29 DNA polymerase mutants indicates that pyrophosphorolysis, like DNA polymerization, also requires an efficient translocation of the enzyme along the template.

Bacteriophages↗
PubMed1991

A general structure for DNA-dependent DNA polymerases.

In addition to the general 3'-5' exonuclease domain described by Bernad et al. [Cell 59 (1989) 219-228] significant amino acid (aa) sequence similarity has been found in the C-terminal portion of 27 DNA-dependent DNA polymerases belonging to the two main superfamilies: (i) Escherichia coli DNA polymerase I (PolI)-like prokaryotic DNA polymerases, and (ii) DNA polymerase alpha-like prokaryotic and eukaryotic (viral and cellular) DNA polymerases. The six most conserved C-terminal regions, spanning approx. 340 aa, are located in the same linear arrangement and contain highly conserved motifs and critical residues involved in the polymerization function. According to the three-dimensional model of PolIk (Klenow fragment), these six conserved regions are located in the proposed polymerization domain, forming the metal and dNTP binding sites and the cleft for holding the DNA template. Site-directed mutagenesis in the phi 29 DNA polymerase supports some of these structural predictions. Therefore, it is likely that a 'Klenow-like core', containing the DNA polymerase and 3'-5' exonuclease activities, has evolved from a common ancestor, giving rise to the present-day prokaryotic and eukaryotic DNA polymerases.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of the YCDTDS amino acid motif of the phi 29 DNA polymerase.

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains amino acid consensus sequences common to other alpha-like DNA polymerases. Using site-directed mutagenesis we have studied the functional significance of the most conserved C-terminal segment mainly represented by the YCDTDS motif. A series of single point mutants has been constructed and the corresponding proteins have been overproduced and characterized. Measurements, on crude fractions, of the activity of the mutant proteins in the formation of the protein p3-dAMP initiation complex and in an in situ DNA polymerase assay, indicate that the YCDTDS domain is involved both in initiation and in elongation reactions.

Amino Acid Sequence↗
PubMed1990

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported. The results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. Interestingly, three of these mutations consist in the single amino acid change Ala to Val at position 492 of the protein. The ts2(24) and ts2(98) mutant phi 29 DNA polymerases were expressed, purified and their thermosensitivity was studied at two different steps of DNA replication: 1) protein-primed initiation and 2) elongation of the DNA chain. Whereas the ts2(24) mutation gave rise to a temperature-sensitive phenotype in both reactions, the ts2(98) mutant protein was rather insensitive to the temperature increase. In addition, the ts2(98) mutant protein showed clear differences in the activation by divalent cations. The relationship of these results with structural and functional domains in the phi 29 DNA polymerase are discussed.

Amino Acid Sequence↗
PubMed1990

The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site.

Amino Acid Sequence↗
PubMed1989

A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases.

The 3'----5' exonuclease active site of E. coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology. Three amino acid regions containing the critical residues in the E. coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases. Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme. These results reflect a high evolutionary conservation of this catalytic domain. Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.

Amino Acid Sequence↗
PubMed1989

Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication.

The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.

Bacillus subtilis↗
PubMed1988

Protein-primed replication of bacteriophage phi 29 DNA.

The replication of phi 29 DNA-protein p3 represents a simple model system to study the protein-priming mechanism of initiation of replication. The phi 29 DNA polymerase involved both in the initiation and elongation steps of phi 29 DNA-protein p3 replication, is a very processive enzyme and it is able to produce strand-displacement in the absence of other proteins. To correlate functional and structural domains in the phi 29 DNA polymerase point mutants in the most carboxyl region of amino-acid homology with other DNA polymerases have been constructed. Most of the mutations had a decreased initiation and elongation activity, but normal 3'----5' exonuclease activity, suggesting that this region contributes to the active domain for initiation and elongation. Point and deletion mutants in the terminal protein have allowed the mapping of one DNA-binding region and two DNA-polymerase-binding regions. The viral protein p6, which stimulates the initiation of replication, binds to a set of specific signals present at both phi 29 DNA ends. A good correlation of binding and stimulation of replication has been obtained by using fragments containing phi 29 DNA-terminal sequences and deletion mutants of protein p6. The viral protein p5 has been shown to bind to single-stranded DNA, to protect the latter against nuclease digetion, and to stimulate phi 29 DNA-protein p3 replication in vitro.

Bacteriophages↗