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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

AutorPicher, Ángel J.
DirectorBlanco, Luis
Palabras claveReplicación de ADN
ADN polimerasas
Fecha de publicación2007
EditorUniversidad Autónoma de Madrid
ResumenPol 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.
DescripciónTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-03-2007
URIhttp://hdl.handle.net/10261/8047
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