Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage

The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damagedSpanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015 to J.A.T.]; Fundación Ramón Areces (Institutional Grant to the Centro de Biologıa Molecular Severo Ochoa); Spanish Ministry of Economy and Competitiveness (predoctoral fellowships to M.V.V and M.A.O-B.); Universidad Autónoma de Madrid (predoctoral fellowship to M.G-F.); Consejo Superior de Investigaciones Cientıficas (JAE-Doc contract to M.S.). Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2010-16989 and Consolider Ingenio CSD2007-00015]Peer Reviewe

Procedimiento para la producción en levaduras de cápsidas virales vacías compuestas por proteínas derivadas de pVP2 del virus causante de la enfermedad de la bursitis infecciosa (IBDV)

Referencia OEPM: P200401044.-- Fecha de solicitud: 30/04/2004.-- Titulares: Consejo Superior de Investigaciones Científicas (CSIC), Bionostra, S.L.Procedimiento para la producción en levaduras de cápsidas virales vacías compuestas por proteínas derivadas de pVP2 del virus causante de la enfermedad de la bursitis infecciosa (IBDV). Las cápsidas vacías del virus causante de la enfermedad de la bursitis infecciosa (IBDV) están constituidas por ensamblaje de proteínas derivadas de la proteína pVP2 de IBDV, de distinto tamaño y tienen aplicación en la producción de vacunas y en la elaboración de vectores para terapia génica.Peer reviewe

Caracterización de los genes implicados en la biosíntesis del antibiótico A201A en Streptomyces capreolus NRRL 3817

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-10-200

Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus

Journal now known as FEBS Journal.-- Open Access content older than 1 year.A novel cosmid (pABC6.5) whose DNA insert from Streptomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identification of a DNA stretch of 19 kb between ard1 and ard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster. The sequence of the 7 kb upstream of ard1 towards ard2 reveals seven consecutive open reading frames: ataP3, ataP5, ataP4, ataP10, ataP7, ata12 and ataPKS1. Except for the last two, their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6,N6-dimethyl-3′-amino-3′-deoxyadenosine moiety of puromycin. Because A201A contains this chemical moiety, these ataP genes are most likely implicated in its biosynthesis. Accordingly, the ataP4, ataP5 and ataP10 genes complemented specific puromycin nonproducing Δpur4, Δpur5 and Δpur10 mutants of S. alboniger, respectively. Amino acid sequence comparisons suggest that ata12 and ataPKS1 could be implicated in the biosynthesis of the D-rhamnose and α-p-coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ard1 has disclosed a region which might contain one end of the ata cluster.This work was supported by grants BIO096-1168-C02-02 and BIO1999-0959 of the Comisión Interministerial de Ciencia y Tecnología.Peer Reviewe

Subnuclear Relocalization of Structure-Specific Endonucleases in Response to DNA Damage

Structure-specific endonucleases contribute to the maintenance of genome integrity by cleaving DNA intermediates that need to be resolved for faithful DNA repair, replication, or recombination. Despite advances in the understanding of their function and regulation, it is less clear how these proteins respond to genotoxic stress. Here, we show that the structurespecific endonuclease Mus81-Mms4/EME1 relocalizes to subnuclear foci following DNA damage and colocalizes with the endonucleases Rad1-Rad10 (XPF-ERCC1) and Slx1-Slx4. Recruitment takes place into a class of stress foci defined by Cmr1/WDR76, a protein involved in preserving genome stability, and depends on the E2-ubiquitin-conjugating enzyme Rad6 and the E3-ubiquitin ligase Bre1. Foci dynamics show that, in the presence of DNA intermediates that need resolution by Mus81-Mms4, Mus81 foci persist until this endonuclease is activated by Mms4 phosphorylation. Our data suggest that subnuclear relocalization is relevant for the function of Mus81-Mms4 and, probably, of the endonucleases that colocalize with itSpanish Ministry of Economy, Industry, and Competitiveness (MINECO); CBMSO receives an institutional grant from Fundación Ramón ArecesPeer Reviewe

Rad5 plays a major role in the cellular response to DNA damage during chromosome replication

© 2014 The Authors. The RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired lesions that block replication forks. It is subdivided into two branches: translesion DNA synthesis, which is frequently error prone, and the error-free DNA-damage-avoidance subpathway. Here, we show that Rad5HLTF/SHPRH, which mediates the error-free branch, has a major role in the response to DNA damage caused by methyl methanesulfonate (MMS) during chromosome replication, whereas translesion synthesis polymerases make only a minor contribution. Both the ubiquitin-ligase and the ATPase/helicase activities of Rad5 are necessary for this cellular response. We show that Rad5 is required for the progression of replication forks through MMS-damaged DNA. Moreover, supporting its role during replication, this protein reaches maximum levels during S phase and forms subnuclear foci when replication occurs in the presence of DNA damage. Thus, Rad5 ensures the completion of chromosome replication under DNA-damaging conditions while minimizing the risk of mutagenesis, thereby contributing significantly to genome integrity maintenance.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; grants BFU2010-16989, BFU2013-43766Peer Reviewe

Partículas quiméricas vacías similares a virus derivadas del virus de la enfermedad de la bursitis infecciosa (ibdv), proceso para su producción y aplicaciones

Partículas quiméricas vacías similares a virus derivadas del virus de la enfermedad de la bursitis infecciosa(IBDV), proceso para su producción y aplicaciones.Peer reviewedConsejo Superior de Investigaciones Científicas (España), BIONOSTRA S.L.A1 Solicitud de patente con informe sobre el estado de la técnic

Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus

Antibiotic A201A produced by Saccharothrix mutabilis subsp. capreolus NRRL3817 contains an aminonucleoside (N 6, N 6 -dimethyl-3′-amino-3′-deoxyadenosyl), a polyketide (α-methyl-p-coumaric acid) and a disaccharide moiety. The heterologous expression in Streptomyces lividans and Streptomyces coelicolor of a S. mutabilis genomic region of ∼34 kb results in the production of A201A, which was identified by microbiological, biochemical and physicochemical approaches, and indicating that this region may contain the entire A201A biosynthetic gene cluster (ata). The analysis of the nucleotide sequence of the fragment reveals the presence of 32 putative open reading frames (ORF), 28 of which according to boundary gene inactivation experiments are likely to be sufficient for A201A biosynthesis. Most of these ORFs could be assigned to the biosynthesis of the antibiotic three structural moieties. Indeed, five ORFs had been previously implicated in the biosynthesis of the aminonucleoside moiety, at least nine were related to the biosynthesis of the polyketide (ata-PKS1-ataPKS4, ata18, ata19, ata2, ata4 and ata7) and six were associated with the synthesis of the disaccharide (ata12, ata13, ata16, ata17, ata5 and ata10) moieties. In addition to AtaP5, three putative methyltransferase genes are also found in the ata cluster (Ata6, Ata8 and Ata11), and no regulatory genes were found.BIO2003-03314 and BIO2013-48779-C4-4 from the Spanish CICYT. We also thank the Fundació Ramón Areces for an institutional grant to the CBMSO (CSIC-UAM).Peer Reviewe

The 2.6-Angstrom Structure of Infectious Bursal Disease Virus-Derived T=1 Particles Reveals New Stabilizing Elements of the Virus Capsid

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a double-stranded RNA virus that causes a highly contagious disease in young chickens leading to significant economic losses in the poultry industry. The VP2 protein, the only structural component of the IBDV icosahedral capsid, spontaneously assembles into T=1 subviral particles (SVP) when individually expressed as a chimeric gene. We have determined the crystal structure of the T=1 SVP to 2.60 Å resolution. Our results show that the 20 trimeric VP2 clusters forming the T=1 shell are further stabilized by calcium ions located at the threefold icosahedral axes. The structure also reveals a new unexpected domain swapping that mediates interactions between adjacent trimers: a short helical segment located close to the end of the long C-terminal arm of VP2 is projected toward the threefold axis of a neighboring VP2 trimer, leading to a complex network of interactions that increases the stability of the T=1 particles. Analysis of crystal packing shows that the exposed capsid residues, His253 and Thr284, determinants of IBDV virulence and the adaptation of the virus to grow in cell culture, are involved in particle-particle interactions