Biomedical subjects

Rafael Giraldo

Publications and source records attributed to Rafael Giraldo.

10 recordsLinked to original sources

PubMed2006

Early events in the binding of the pPS10 replication protein RepA to single iteron and operator DNA sequences.

RepA protein, encoded in the Pseudomonas pPS10 replicon, is a stable dimer in solution (dRepA), acting as a self-repressor of repA transcription through binding to an inverted repeat operator. However, RepA monomers (mRepA) are required to initiate plasmid replication upon binding to four directly repeated DNA sequences (iterons). RepA is composed of two winged-helix (WH) domains: C-terminal WH2 is the main DNA-binding domain (DBD) for both target sequences, whereas N-terminal WH1 acts as dimerization interface in dRepA, but becomes a second DBD in mRepA. On the basis of CD spectroscopy, hydrodynamics, X-ray crystallography and model building studies, we proposed previously that the activation of RepA initiator implies a large structural change in WH1, coupled to protein monomerization and interdomain compaction. Here, we report novel features in the process. Binding curves of RepA to an iteron, followed by fluorescence anisotropy in solution and by surface plasmon resonance on immobilized DNA, exhibit the profiles characteristic of transitions between three states. In contrast, RepA-R93C, a monomeric activated mutant, exhibits a single binding transition. This suggests the presence of an intermediate species in the iteron-induced dissociation and structural transformation of RepA. High concentrations of bovine serum albumin or ovalbumin (macromolecular crowding) enhance RepA affinity for an iteron in solution and, in gel mobility-shift assays, result in the visualization of novel protein-DNA complexes. RepA-induced DNA bending requires the binding of two WH domains: either both WH2 in dimers (operator) or WH1 plus WH2 in monomers (iteron).

Animals↗

PubMed2005

An intrinsic 5'-deoxyribose-5-phosphate lyase activity in DNA polymerase beta from Leishmania infantum supports a role in DNA repair.

Leishmania infantum is a parasitic protozoan which infects humans. This paper reports the expression in Escherichia coli and purification of the L. infantum gene product (AF182167), as well as its characterization as a DNA polymerase beta (Polbeta)-like, template-dependent DNA repair enzyme, with a metal preference for Mn2+ over Mg2+. As is the case with mammalian Polbeta and DNA polymerase lambda (Pollambda), L. infantum DNA polymerase beta (Li Polbeta) prefers gapped-DNA substrates having a 5'-phosphate end, in agreement with its role in DNA repair reactions. Purified Li Polbeta also displayed a 5'-deoxyribose-5-phosphate (dRP) lyase activity, consistent with a beta-elimination mechanism. The concerted action of dRP lyase and DNA polymerization activities of Li Polbeta on a uracil-containing DNA suggests its participation in "single-nucleotide" base excision repair (BER). Analysis of Li Polbeta DNA polymerization activity at different stages of the L. infantum infective cycle supports a role for Li Polbeta in nuclear DNA repair after the oxidative damage occurring inside the macrophage.

Amino Acid Sequence↗

PubMed2004

Twenty years of the pPS10 replicon: insights on the molecular mechanism for the activation of DNA replication in iteron-containing bacterial plasmids.

This review focuses on the contributions of the Pseudomonas replicon pPS10 to understanding the initiation of DNA replication in iteron-containing plasmids from Gram-negative bacteria. Dimers of the pPS10 initiator protein (RepA) repress repA transcription by binding to the two halves of an inverted repeat operator. RepA monomers are the active initiator species that bind to four directly repeated sequences (iterons). pPS10 initiator was the first Rep protein whose domains were defined (two "winged-helix," WH modules) and their binding sites were identified at each half of the iteron repeat. This was confirmed by the crystal structure of the monomer of a homologous initiator (RepE from F plasmid) bound to iteron DNA. The recently solved structure of the dimeric N-terminal domain (WH1) of pPS10 RepA, when compared to the RepE monomer, shows that upon dimer dissociation an alpha-helix at WH1 C-terminus becomes part of an interdomain beta-sheet. In solution, the iteron sequence, by itself, can induce the same kind of structural transformation in RepA. This seems to alter the package of both WH domains to adapt their DNA reading heads (HTH motifs) to the distinct spacing between half repeats in iterons and operator. Based on biochemical and spectroscopic work, structural and functional similarities were proposed between RepA and archaeal/eukaryal initiators. This was independently confirmed by the crystal structure of the archaeal initiator Cdc6. Characterization of mutants, either in pPS10 or in the Escherichia coli chromosome, has provided some evidence on a WH1-mediated interaction between RepA and the chromosomal initiator DnaA that results in a broadened-host range.

Amino Acid Sequence↗

PubMed2003

Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA.

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.

Amino Acid Motifs↗

PubMed2003

Non-cytotoxic variants of the Kid protein that retain their auto-regulatory activity.

Kid and Kis are, respectively, the toxin and antitoxin encoded by the parD operon of plasmid R1. The recently solved crystal structure of Kid has revealed that this protein closely resembles the CcdB toxin of plasmid F. In CcdB, the residues involved in toxicity are located at the carboxy-terminal end of the protein. However, an analogous information on the Kid toxin was not available. Here, we have characterized a collection of non-toxic mutants of the Kid protein and identified the residues that affected the toxicity but not the co-regulatory activity of Kid. These are located in two discrete regions of the protein, at the amino and carboxy-terminal ends. Particularly, residues E18 and R85, that are conserved in the Escherichia coli ChpAK and RelE toxins, are affected by amino-acid changes that alter neither the overall structure of the protein nor its state of association, as shown by CD and sedimentation equilibrium analyses. However, thermal denaturation and intrinsic tryptophan fluorescence emission data point to subtle local changes at the N-terminal end of the protein. The implications of these results in the current model on the structure and function of Kid-related bacterial toxins are discussed.

Amino Acid Sequence↗

PubMed2003

A conformational switch between transcriptional repression and replication initiation in the RepA dimerization domain.

Plasmids are natural vectors for gene transfer. In Gram-negative bacteria, plasmid DNA replication is triggered when monomers of an initiator protein (Rep) bind to direct repeats at the origin sequence. Rep dimers, which are inactive as initiators, bind to an inverse repeat operator, repressing transcription of the rep gene. Rep proteins are composed of N-terminal dimerization and C-terminal DNA-binding domains. Activation of Rep is coupled to dimer dissociation, converting the dimerization domain into a second origin-binding module. Although the structure of the monomeric F plasmid initiator (mRepE) has been determined, the molecular nature of Rep activation remains unknown. Here we report the crystal structure of the dimeric N-terminal domain of the pPS10 plasmid initiator (dRepA). dRepA has a winged-helix fold, as does its homologous domain in mRepE. However, dimerization transforms an interdomain loop and beta-strand (monomeric RepE) into an alpha-helix (dimeric RepA). dRepA resemble the C terminus of eukaryotic and archaeal Cdc6, giving clues to the phylogeny of DNA replication initiators.

Amino Acid Sequence↗

PubMed2003

Common domains in the initiators of DNA replication in Bacteria, Archaea and Eukarya: combined structural, functional and phylogenetic perspectives.

Although DNA replication is the universal process for the transmission of genetic information in all living organisms, until very recently evidence was lacking for a related structure and function in the proteins (initiators) that trigger replication in the three 'Life Domains' (Bacteria, Archaea and Eukarya). In this article new data concerning the presence of common features in the initiators of chromosomal replication in bacteria, archaea and eukaryotes are reviewed. Initiators are discussed in the light of: (i) The structure and function of their conserved ATPases Associated with various cellular Activities (AAA+) and winged-helix domains. (ii) The nature of the macromolecular assemblies that they constitute at the replication origins. (iii) Their possible phylogenetic relationship, attempting to sketch the essentials of a hypothetical DNA replication initiator in the micro-organism proposed to be the ancestor of all living cells.

Amino Acid Sequence↗

PubMed2002

Crystallization and preliminary X-ray crystallographic studies on the parD-encoded protein Kid from Escherichia coli plasmid R1.

DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase. The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E. coli is thought to target and block DnaB-dependent DNA replication. The toxicity of Kid is antagonized through interaction with the Kis antidote protein and the resultant complex can then act as a transcriptional regulator for the parD system. Crystals of selenomethionine-incorporated Kid have been obtained by the hanging-drop vapour-diffusion method using potassium phosphate as the precipitant. The crystals belong to the monoclinic system, space group P2(1), have unit-cell parameters a = 32.9, b = 45.0, c = 64.4 A, beta = 96.2 degrees and diffract to a d(min) of better than 1.8 A on a synchrotron-radiation source. The determination of the structure of Kid will permit a better understanding of its interactions with DnaB and Kis and allow the evolutionary relationships of Kid to other toxins of plasmid and chromosomal origin to be explored.

Bacterial Proteins↗

PubMed2002

Genetic identification of two functional regions in the antitoxin of the parD killer system of plasmid R1.

We report the identification and genetic analysis of mutants in the antitoxin of the parD (kis, kid) killer system of plasmid R1. Missense mutants placed at codons 10, 11, 12 and 18 maintained the antitoxin activity of Kis, but not the ability of this protein to co-regulate the parD system together with the Kid toxin. Deletion of the last 33 amino acids of Kis inactivated the antitoxin activity of the protein and reduced substantially, but not completely, its regulatory activity. These results define two functional regions in Kis: an amino-terminal region which is specifically involved in regulation, and a carboxy-terminal region of the protein, which is important both for its regulatory and antitoxin activities.

Amino Acid Sequence↗

PubMed2002

Structural and functional analysis of the kid toxin protein from E. coli plasmid R1.

We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.

Amino Acid Sequence↗