Implicación de la dna polimerasa lambda en reparación de dna y tumorogénesis

• Autores: Gloria Terrados Aguado
• Directores de la Tesis: Luis Blanco Dávila (dir. tes.), Margarita Salas Falgueras (codir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2010
• Idioma: español
• Tribunal Calificador de la Tesis: José Fernández Piqueras (presid.), José Antonio Tercero Orduña (secret.), José Francisco Ruiz Pérez (voc.), Antonio Bernad Miana (voc.), Vicente Larraga Rodríguez de Vera (voc.), Enrique Viguera Mínguez (voc.), Ana Alonso (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • This Doctoral Thesis reports the purification of Leishmania infantum DNA polymerase beta (LiPolß), as well as its characterization as a DNA repair enzyme, with a metal preference for Mn2+ over Mg2+. LiPolß prefers gapped-DNA substrates having a 5¿-P end, in agreement with its role in DNA repair reactions. Purified LiPolß also displayed a 5¿-deoxyribose-5-phosphate (dRP) lyase activity, consistent with a ßelimination mechanism. The concerted action of dRP lyase and DNA polymerization activities of LiPolß on a uracil-containing DNA suggests its participation in ¿singlenucleotide¿ base excision repair (BER). Analysis of LiPolß DNA polymerization activity at different stages of the L. infantum infective cycle supports a role for LiPolß in parasite DNA repair, counteracting the oxidative damage occurring inside the macrophage.

    DNA polymerase lambda (Pol¿), a recently described Family X DNA polymerase, has been also studied in this work regarding its in vivo role in DNA repair and tolerance of oxidative damage. Analysis of the BER activity in tissue extracts from ¿knock-out¿ lambda mice (KO¿) indicated that Pol¿ is not essential for the BER of 8oxoG:dC pairs. However, Pol¿ has a role in the correction of 8oxoG:dA to 8oxoG:dC, thus minimizing 8oxoG associated mutagenesis. Moreover, Pol¿ does not contribute to BER of either dU:dG or 5FU:dG pairs in adult testis. However, our analysis suggests its participation in the BER of 5FU:dG in brain. In agreement with the participation of Family X in translesion DNA synthesis (TLS) of 8oxoG and 6mG and the possible role in BER of 5FU, we demostrated that hPolß, hPol¿ and hPol¿ are able to tolerate 5FU in DNA, mainly inserting dA in front of the lesion. Likewise, these three enzymes proficiently extended the pair 5FU:dA, comparable to normal pairs dT:dA y dU:dA, whereas they were very inefficient to extend the pair 5FU:dG. Moreover, they incorporated 5FdUTP opposite dA much better than opposite dG in the template.

    We have carried out the identification of two hPol¿ splicing variants, that are highly expressed in different tumors. hPol¿¿6 purification and enzymatic characterization suggests that the omision of exon 6 results in a poorly active variant, that could act as a dominant negative by sequestering NHEJ factors via interaction with its intact BRCT domain.

    We have identified and characterized a single nucleotide polymorphism (SNP), of hPol¿, having a 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 Pol¿ variant revealed that it retained both DNA polymerization and dRP lyase activities, but had reduced base substitution fidelity. In agreement with this in vitro mutator phenotype, ectopic expression of the W438 hPol¿variant in mammalian cells increased mutation frequency, affecting the DSB repair NHEJ pathway, and generating chromosome aberrations. All these phenotypes were dependent upon the catalytic activity of the W438 hPol¿. RFLP analysis in normal versus cancer patients demonstrated an association of the W438 polymorphism and rectal cancer.