Función de la DNA polimerasa lambda en reparación de DNA y tumorogénesis

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 11-06-201

DNA expansions generated by human Polμ on iterative sequences

Polm is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polm is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polm-mediated dinucleotide expansion as a function of the sequence context. In this model, Polm requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 50-P-containing DNA substrates, including those that mimic non-homologous endjoining (NHEJ) intermediates. Our mechanistic studies point at human Polm residues His329 and Arg387 as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polm at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalitiesMinisterio de Ciencia y Tecnologia [BFU2009-10085 and CONSOLIDER CSD2007-00015]; Fundacion Ramon Areces (to Centro de Biologia Molecular ‘‘Severo Ochoa’’); Ministerio de Educacion y Ciencia (to A.A.); Comunidad Autonoma de Madrid (to M.J.M.). Funding for open access charge: Comunidad de Madrid grant [S2011/BMD-2361

La globalización y el malestar en la democracia

El origen de este texto es una conferencia en el VII Congreso de la FES (Salamanca, 20-22 de septiembre de 2001) con el título "Estados, mercados y ciudadanía". Publicado en: Revista Internacional de Filosofía Política, 20: 5-24, 2002.En años recientes se ha convertido en un lugar común la idea de que los ciudadanos de los países democráticos, independientemente de que apoyen esta forma de gobierno por encima de cualquier otra, otorgan un nivel de confianza muy bajo a las instituciones de la democracia representativa, desde los partidos y los parlamentos hasta los gobiernos (Nye et al., 1997; Norris, 1999; Pharr y Putnam, 2000). En América Latina, además, los alarmantes resultados del Latinobarómetro de 2001 (Economist, 2001) hicieron temer que, ante la mala marcha de la economía, la insatisfacción de los ciudadanos pudiera conducir de forma imparable a la erosión del apoyo a la propia democracia.Proyecto Desconfianza Política y Gobernación Democrática (BSO2000- 1082) del Plan Nacional de I+D (Ministerio de Ciencia y Tecnología, España)Peer reviewe

Characterization of a Natural Mutator Variant of Human DNA Polymerase l which Promotes Chromosomal Instability by Compromising NHEJ

PLoS ONE 4(10): e7290.Background: DNA polymerase lambda (Poll) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB). Principal Findings: Here, we described a novel natural allelic variant of human Poll (hPoll) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Poll variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPoll variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPoll. Conclusions: The expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.This work was supported by Ministerio de Ciencia y Tecnologia Grants BFU2006-14390/BMC, CONSOLIDER CSD2007-00015 and Comunidad Autonoma de Madrid Grants P2006/BIO-0306 to L.B., by INCa ‘‘Checkpol’’, ARC, and ‘‘Ligue contre le Cancer (Region Midi-Pyrenees)’’ to J-S.H., by the Division of Intramural Research, NIEHS, NIH, DHHS to T.A.K., by SAF2002-02265 to A.V., and by an institutional grant to Centro de Biologia Molecular ‘‘Severo Ochoa’’ from Fundacion Ramon Areces. G.T. was recipient of a fellowship from the Ministerio de Educacion y Ciencia. A.V. is an Investigator of the Ramon y Cajal ProgramPeer reviewe

Altered Hematopoiesis in Mice Lacking DNA Polymerase μ Is Due to Inefficient Double-Strand Break Repair

Polymerase mu (Polμ) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polμ deficiency results in impaired Vκ-Jκ recombination and altered somatic hypermutation and centroblast development. In Polμ−/− mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body γ-irradiation revealed that Polμ also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polμ function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues

DNA expansions generated by human Polm on iterative sequences

Polm is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polm is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polm-mediated dinucleotide expansion as a function of the sequence context. In this model, Polm requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 5′-P-containing DNA substrates, including those that mimic non-homologous end-joining (NHEJ) intermediates. Our mechanistic studies point at human Polm residues His329 and Arg387 as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polο at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalities.Ministerio de Ciencia y Tecnologia [BFU2009-10085 and CONSOLIDER CSD2007-00015]; Fundacion Ramon Areces (to Centro de Biologia Molecular ‘‘Severo Ochoa’’); Ministerio de Educacion y Ciencia; Comunidad Autonoma de Madrid. Funding for open access charge: Comunidad de Madrid grant [S2011/BMD-2361]Peer Reviewe

Analysis of genetic instability in Mouse Embryonic Cells (MEFs) deficient in Pol and Polµ

Trabajo presentado en Gordon Research Conference: Mutagenesis, celebrada en Girona (España) del 15 al 20 de junio de 2014

Aphidicolin-resistant and -sensitive base excision repair in wild-type and DNA polymerase β-defective mouse cells

Several DNA polymerases (Pols) can add complementary bases at the gap created during the base excision repair (BER). To characterize the BER resynthesis step, the repair of a single abasic site by wild-type and Pol β-defective mouse cell extracts was analysed in the presence of aphidicolin, a specific inhibitor of replicative Pols. We show that there is a competition between distributive and processive Pols for the nucleotide addition at the primer terminus. In wild-type cell extracts, the initial nucleotide insertion involves mainly Pol β but the elongation step is carried out by a replicative Pol. Conversely, in Pol β-null cell extracts the synthesis step is carried out by a replicative Pol without any switching to an auxiliary polymerase. We present evidence that short-patch repair synthesis occurs even in the absence of both Pol β and replicative Pols. Exogeneously added purified human Pol λ was unable to stimulate this back-up synthesis.This work was partially supported by the Associazione Italiana Ricerca sul Cancro (A.I.R.C.) and by Compagnia di S. Paolo, Turin, Italy (Project coordinator: Dr. G. Frosina)

Are there mutator polymerases?

DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.This work was supported by DGES (PB97-1192), DGI (BMC2000-1138), and CAM (08.1/0044/98 and 08.5/0063/2000) and by an institutional grant from Fundación Ramón Areces