Advanced search    

Search: authors:"Margarita Salas"

46 papers found.
Use AND, OR, NOT, +word, -word, "long phrase", (parentheses) to fine-tune your search.

Self-replication of DNA by its encoded proteins in liposome-based synthetic cells

Replication of DNA-encoded information and its conversion into functional proteins are universal properties of life. In an effort toward the construction of a synthetic minimal cell, we implement here the DNA replication machinery of the Φ29 virus in a cell-free gene expression system. Amplification of a linear DNA template by self-encoded, de novo synthesized Φ29 proteins is...

Engineering Permissive Insertion Sites in the Bacteriophage Phi29 DNA-Linked Terminal Protein

Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP) is covalently linked to the 5’ ends of the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modules that can target the DNA to...

Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

Protein-primed replication constitutes a generalized mechanism to initiate DNA or RNA synthesis in a number of linear genomes of viruses, linear plasmids and mobile elements. By this mechanism, a so-called terminal protein (TP) primes replication and becomes covalently linked to the genome ends. Bam35 belongs to a group of temperate tectiviruses infecting Gram-positive bacteria...

Dissecting the role of the ϕ29 terminal protein DNA binding residues in viral DNA replication

Phage ϕ29 DNA replication takes place by a protein-priming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the ϕ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid...

Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events

Pablo Gella 0 Margarita Salas 0 Mario Menca 0 0 Centro de Biologa Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientficas - Universidad Aut onoma de Madrid), Universidad Aut onoma

Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage

The SOS response in Eubacteria is a global response to DNA damage and its activation is increasingly associated with the movement of mobile genetic elements. The temperate phage GIL01 is induced into lytic growth using the host's SOS response to genomic stress. LexA, the SOS transcription factor, represses bacteriophage transcription by binding to a set of SOS boxes in the...

New Insights into the RNA-Based Mechanism of Action of the Anticancer Drug 5′-Fluorouracil in Eukaryotic Cells

5-Fluorouracil (5FU) is a chemotherapeutic drug widely used in treating a range of advanced, solid tumours and, in particular, colorectal cancer. Here, we used high-density tiling DNA microarray technology to obtain the specific transcriptome-wide response induced by 5FU in the eukaryotic model Schizosaccharomyces pombe. This approach combined with real-time quantitative PCR...

The Essential Role of the 3′ Terminal Template Base in the First Steps of Protein-Primed DNA Replication

Bacteriophages φ29 and Nf from Bacillus subtilis start replication of their linear genomes at both ends using a protein-primed mechanism by means of which the DNA polymerase initiates replication by adding dAMP to the terminal protein, this insertion being directed by the second and third 3′ terminal thymine of the template strand, respectively. In this work, we have obtained...

DNA stabilization at the Bacillus subtilis PolX core—a binding model to coordinate polymerase, AP-endonuclease and 3′-5′ exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been...

Dual Role of φ29 DNA Polymerase Lys529 in Stabilisation of the DNA Priming-Terminus and the Terminal Protein-Priming Residue at the Polymerisation Site

Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3′ terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of φ29 DNA...

Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

. Ricardo Arias-Gonzalez 4 Jos e M. Valpuesta 3 Jos e L. Carrascosa 3 4 Margarita Salas 0 Borja Ibarra 4 0 Centro de Biolog a Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Aut o noma , Cantoblanco, 28049

Involvement of residues of the ϕ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase

Bacteriophage ϕ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group...

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ϕ29 DNA mimic protein p56

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B...

A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2′-deoxyguanosine

Miguel de Vega 0 Margarita Salas 0 0 Instituto de Biologa Molecular ''Eladio Vin uela'' (CSIC), Centro de Biologa Molecular ''Severo Ochoa'' (CSIC-UAM), Universidad Aut onoma , Canto Blanco, 28049

Differential Spo0A-mediated effects on transcription and replication of the related Bacillus subtilis phages Nf and ϕ29 explain their different behaviours in vivo

Castilla-Llorente 0 Wilfried J. J. Meijer 0 Margarita Salas 0 0 Instituto de Biologı ́a Molecular 'Eladio Vi n ̃uela' (CSIC), Centro de Biologı ́a Molecular 'Severo Ochoa' (CSIC-UAM), C/Nicola ́ s Cabrera

Novel dimeric structure of phage ϕ29-encoded protein p56: insights into uracil-DNA glycosylase inhibition

Protein p56 encoded by the Bacillus subtilis phage ϕ29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions. Each polypeptide chain contains...

Increased Na+/Ca2+ Exchanger Expression/Activity Constitutes a Point of Inflection in the Progression to Heart Failure of Hypertensive Rats

Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca2+-handling alterations are known to occur in SHR. However, the putative modifications of Ca2+-handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial...

Involvement of the TPR2 subdomain movement in the activities of ϕ29 DNA polymerase

The polymerization domain of ϕ29 DNA polymerase acquires a toroidal shape by means of an arch-like structure formed by the specific insertion TPR2 (Terminal Protein Region 2) and the thumb subdomain. TPR2 is connected to the fingers and palm subdomains through flexible regions, suggesting that it can undergo conformational changes. To examine whether such changes take place, we...

Editing of misaligned 3′-termini by an intrinsic 3′–5′ exonuclease activity residing in the PHP domain of a family X DNA polymerase

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolXBs), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolXBs possesses an intrinsic 3′–5′ exonuclease activity specialized in resecting unannealed 3′-termini in a gapped DNA substrate. Biochemical analysis of a...

DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts

The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4–DNA complex contains two identical protomers that make similar interactions with the target sequence 5′-AACTTTTT-15 bp-AAAATGTT-3′. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 binding to a series of mutated...