RESUMO
Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.
Assuntos
DNA Polimerase beta/metabolismo , Replicação do DNA , Mutagênese/genética , Animais , Antígenos Virais de Tumores/metabolismo , Sequência de Bases , Calibragem , Bovinos , Extratos Celulares , Sistema Livre de Células , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Viral/biossíntese , DNA Viral/genética , DNA Viral/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Didesoxinucleotídeos , Células HeLa , Humanos , Dados de Sequência Molecular , Vírus 40 dos Símios/genética , Especificidade por Substrato , Moldes GenéticosRESUMO
We report here that DNA polymerase beta (pol beta), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure. To investigate the potential role of pol beta in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol beta. We firstly demonstrated that mammalian cells overexpressing pol beta are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). By using in vitro primer extension reactions with purified pol beta, we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed. pol beta mostly incorporates the correct dATP opposite the 3'-terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively. In the case of CPD, efficient and error-prone extension of the correct dATP was found. These data support a biological role of pol beta in UV lesion bypass and suggest that deregulated pol beta may enhance UV-induced genetic instability.