Abstract
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.
Highlights
Exposure of cells to UV light results in the formation of a variety of lesions in their DNA, the most common being cyclobutane pyrimidine dimers (CPD)[1] and pyrimidine-pyrimidone (6-4) photoproducts ((6-4)PP) at adjacent pyrimidines (1)
We focused on a set of 11 primers, 8 primers representing the best incorporations opposite the dimers (primers ending with AA, AG, AT, AC, GA, GT, CA, CT for the CPD; primers ending with AT, CT, GT for the (6-4)TT), and 3 primers randomly chosen. pol  was able to extend efficiently a primer with two dA residues opposite the CPD, generating a full-size product
In the case of the (6-4)TT-containing template, we did not detect any significant primer extension with all the primers tested (Fig. 5, right part). These results suggest that pol  is able to extend efficiently mutagenic as well as correct nucleotides incorporated opposite the CPD
Summary
Western Blotting—Tissues from normal skin and metastasic melanoma, kindly given by Dr Voigt (ICR, Toulouse, France), were lysed. CHO cell lines overexpressing pol  were established previously after stable transfection of pUTPol plasmid (15). Primer Extension Assay—UV-modified 30-mer oligomers 5Ј-CTCGTCAGCATCTTCATCATACAGTCAGTG-3Ј were chemically synthesized as described previously (29, 30) They were hybridized to 5Ј-32P-labeled 16-mer (5Ј-CACTGACTGTATGATG-3Ј), 17-mer (5Ј-CACTGACTGTATGATGN-3Ј), or 18-mer (5Ј-CACTGACTGTATGATGNN-3Ј) primers at a molar ratio of 1:1 for 10 min at 70 °C in a buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 10 mM MgCl2 followed by slow cooling to room temperature. Replication reaction mixtures (25 l) contained 30 mM HEPES, pH 7.8, 7 mM MgCl2, 200 M each of CTP, GTP, and UTP, 4 mM ATP, 100 M each of dATP, dCTP, dTTP, 10 M dGTP, 40 mM creatine phosphate (Sigma), 100 g/ml creatine phosphokinase (Sigma), 100 ng of pBS-SvoriA(CPD) or oriB(CPD), 0.5 g of SV40 large T-antigen (Molecular Biology Resources), and 400 g of Hela cell extract. Quantification analysis of the resolved radioactive bands on the gel was achieved by PhosphorImager Storm-system analysis using ImageQuant software
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