Saturday, 18th November 2017

 

Genome Dynamics and Function

    Genome maintenance and variability: enzymology of DNA replication and repair

 


Grupo-400

 


Luis Blanco

ESciStaff

EPublications

Research summary:

Since the last 13 years, our group is focused in the study of two eukaryotic DNA polymerases involved in DNA double-strand break repair (DSBs) in humans: Polλ and Polμ, discovered in Blanco laboratory. Due to their role in the Non-Homologous-End-Joining (NHEJ) pathway of DSB repair, these two enzymes are crucial to maintain genome stability, but also to generate the required variability to specific genes, i.e. antigen receptors. Site-directed mutants and chimaeric versions of these two DNA polymerases are being analyzed to unravel the structural basis of their different specificity and catalytic efficiency. In vivo analysis of Polλ and Polμ function is being carried out by using cellular and mouse models of deficiency in one or both of these enzymes, particularly on their impact in genomic stability, aging and neurological disorders. In collaboration with Aidan Doherty (GSDC, Univ Sussex, UK) we have also characterized the polymerase involved in the NHEJ pathway of some bacteria as Mycobacterium tuberculosis, whose mechanism is convergent with that of Polλ y Polμ.

 Fig01-300

Model of the interaction of Polµ with the Ku heterodimer and the DNA substrate, through the BRCT domain.

 

Our lab has recently characterized a novel primase/polymerase, PrimPol, in human cells, able to tolerate lesions in DNA as those produced by oxidative damage and ultraviolet irradiation. As recently shown in collaboration with Ian Holt (MRC, UK), PrimPol has a role in mitochondrial DNA maintenance, and perhaps its deficiency could be at the heart of some human mitochondriopathies. In collaboration with Juan Méndez (CNIO, Spain) we showed that PrimPol also has a role in nuclear DNA replication, specifically in re-priming stalled replication forks that arise in response to replicative stress. Moreover, we have obtained a mouse model of PrimPol deficiency (KO) which is viable, and that will be characterized paying special attention at mitochondrial-dependent phenotypes, and at its value as a model for aging and tumorogenesis.

 

 

 


 

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A novel human DNA polymerase able to tolerate DNA lesions. PrimPol, is located at both DNA compartments: nucleus and mitochondria. The inset at the right shows alternative functions of PrimPol during DNA replication. When a replicative polymerase is blocked at a DNA lesion, PrimPol can act as a “classical” TLS polymerase, "reading" lesions as 8oxoG. If the lesion is "unreadable", PrimPol can reinitiate DNA synthesis ahead of the lesion, by means of its DNA primase activity. PrimPol would be recruited at the single stranded region generated by continued helix opening after the replicative DNA polymerase got stalled at a lesion. Such a “TLS primase” activity would allow replication fork progression, but a lesion-containing gap would be left behind for later repair.

 

 

Publications:

  • Brissett NC, Pitcher RS, Juarez R, Picher AJ, Green AJ, Dafforn TR, Fox GC, Blanco L* and Doherty AJ* (*corresponding author) (2007) Structure of a NHEJ polymerase-mediated DNA synaptic complex. Science 318, 456-459. PMID:17947582.
  • Andrade P, Martin MJ, Juarez R, Lopez de Saro F and Blanco L (2009) Limited terminal transferase in human DNA polymerase mu defines the required balance between accuracy and efficiency in NHEJ. PNAS106, 16203-16208. PMID:19805281.
  • Brissett NC, Martin MJ, Bartlett EJ, Bianchi J, Blanco L, Doherty AJ (2013) Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase.Cell Reports 5(4):1108-20.
  • García-Góméz S, Reyes A, Martínez-Jiménez MI, Chocrón ES, Mourón S, Terrados G, Powell C, Salido E, Méndez J, Holt IJ, and Blanco L (2013) PrimPol, an archaic primase-polymerase operating in human cells. Mol Cell 52(4):541-53.
  • Mourón S, Rodriguez-Acebes S, Martínez-Jiménez MI, García-Gómez S, Chocrón S, Blanco L, Méndez J. (2013) Repriming of DNA synthesis at stalled replication forks by human PrimPol. Nat Struct Mol Biol. 20(12):1383-9.

 

Other activities:

- Organization of the Cantoblanco Workshop: "Polymerases involved in DNA replication, repair and mutagenesis". Organized by Margarita Salas, Luis Blanco, Robert P. Fuchs and Miguel García-Díaz. Universidad Autónoma de Madrid. June 5-7, 2012.
- Coordinator of the module (BM6): "Estabilidad de la Información Genética: Replicación, Reparación y Mutagénesis", Master of Molecular and Celular Biology, Universidad Autónoma de Madrid (UAM).


 

Doctoral theses:

Maria José Martín Pereira (2011). Exclusive polymerases repairing DNA breaks, the same magics from bacteria to man. Universidad Autónoma de Madrid. Luis Blanco Dávila.

Sara García Gómez (2013). PrimPol, una nueva primasa/polimerasa en células humanas. Universidad Autónoma de Madrid. Luis Blanco Dávila.

Ana Gómez Bedoya (2013). Análisis estructura-función de la DNA polimerasa lambda humana y su implicación en la reparación del DNA mediante NHEJ. Universidad Autónoma de Madrid. Luis Blanco Dávila.

Ana Aza Montoya (2014). In vivo role of DNA polymerases lambda and mu in Genome Stability. Universidad Autónoma de Madrid. Luis Blanco Dávila.


 

Patents:

Inventors: Luis Blanco, Angel Picher. Title: "Method for Genotyping". Patent number: P056358WO S001. Priority country: España. Priority date: PCT sent on February 2012. Owner: X-Pol Biotech S.L.