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Título : Papel de la ADN polimerasa [lambda] humana en reparación de daño oxidativo y roturas de doble cadena en el ADN
Autor : Picher, Ángel J.
Director: Blanco, Luis
Palabras clave : Replicación de ADN
ADN polimerasas
Fecha de publicación : 2007
Editor: Universidad Autónoma de Madrid
Resumen: Pol λ, a recently described Family X DNA polymerase, is studied in this work. This enzyme shares 32% amino acid identity with Pol β, an enzyme involved in nuclear DNA repair in eukaryotic cells. Early studies revealed that Pol λ posseses both DNA polymerase and dRP lyase activities, consistent with a possible role in the base excision repair (BER) pathway. Unlike Pol β, Pol λ contains a BRCT domain and a Serine/Proline rich domain in its N-terminal region. Pol λ is a distributive and template dependent DNA polymerase, which lacks proofreading activity and shows high affinity for dNTPs. In this work we performed studies that suggest a role of Pol λ in base excision repair in vivo. Evaluation of the base excision repair activity in extracts derived from a variety of tissues and mouse embryonic fibroblasts representing wild-type and null genotypes for Pol λ, and also from a cell line overproducing Pol λ, supported a role of Pol λ in testis BER during post-natal development and in brain from adult animals. Moreover, over-production of Pol λ produces an increase in overall BER levels in NIH-3T3 cells. The post-translational regulation of the dRP lyase activity of Pol β has been previously demonstrated. Pol β is acetylated by p300 and this process provokes the specific inhibition of Pol β dRP lyase activity. In order to explore the regulation of Pol β and Pol λ during BER, we examined and demonstrated the acetylation of Pol λ by p300. However, unlike Pol β, the acetylation of Pol λ does not inactivate its dRP lyase activity, suggesting that acetylation acts as a regulatory mechanism affecting the activity balance of both DNA polymerases during BER. To further study the implication of Pol λ in various DNA repair mechanisms, we evaluated the affinity of Pol λ for different DNA substrates mimicking intermediates of various DNA synthesis events. Pol λ was able to stably bind “open” template/primer molecules, suggesting a role in processes related to DNA replication. In the same manner, Pol λ bound gapped molecules with high affinity, consistent with its role in BER, being critical the presence of a phosphate group at the 5´ end of the gap. Finally, Pol λ was able to bind template/downstream molecules with a 5´ phosphate group, a substrate related to the nonhomologous end joining (NHEJ) repair pathway. We have studied the effect on Pol λ polymerase activity of the presence of a phosphate group at the 5´ end of a gap. Pol λ increased its activity in presence of a phosphate group. Site directed mutagenesis allowed us to identify important residues for recognition of the phosphate group located at the 5´ end of a gap.
The observation that Pol λ has an extraordinary ability to generate frameshift errors suggested an ability to use DNA intermediates generated during NHEJ repair pathway. Moreover, gap-filling synthesis during NHEJ may require extending misaligned substrates that could include mismatched primer-termini. Here, we demonstrated that Pol λ efficiently extends DNA/DNA and DNA/RNA mismatches, either on “open” template/primer substrates, or on its preferred substrate, a 1-nucleotide gapped-DNA molecule having a 5´ phosphate. A crystal structure of Pol λ in complex with a single-nucleotide gap containing a dG·dGMP mismatch at the primer terminus suggested that, at least for certain mispairs, Pol λ is unable to differentiate between matched and mismatched termini during the DNA binding step. This property of Pol λ suggested a potential role as a “mismatch extender” during NHEJ and possibly during translesion DNA synthesis (TLS). Finally, the reported interaction between Pol λ and PCNA, together with the mismatch extension ability of Pol λ, suggests a possible role of Pol λ in TLS. Here, we demonstrated that Pol λ is able to replicate efficiently through 7,8-dihydro-8-oxoguanine (8oxoG), inserting dC and dA with similar frequency and extending proficiently from the error-free pair 8oxoG·dCMP, showing the highest efficiency and fidelity of DNA polymerases studied to date. Moreover, Pol λ also extends more efficiently the error-free pair formed by the lesion O6- methylguanine (6mG) and dC. These results suggest a possible role of Pol λ in error-free TLS, as well as in NHEJ repair reactions that involve modified bases.
Descripción : Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-03-2007
URI : http://hdl.handle.net/10261/8047
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