ABSTRACT
DNA polymerase beta (Pol beta) is important for the base excision repair (BER) pathway. Overexpression of Pol beta is frequently found in cancer cells and is thought to be associated with tumorigenesis. In this study, we examined BER fidelity in extracts derived from a human lymphoblastoid cell line that over expresses Pol beta compared to normal control cells. Using an in vitro mutagenesis assay, we found an increased rate of frameshift mutations arising during DNA repair in whole-cell extracts derived from the Pol beta-overexpressing cells. We demonstrate that the addition of excess Pol beta to a control cell extract enhances the mutagenic potential of the extract. Furthermore, using cell extracts and purified Pol beta, we demonstrate that the mechanism of frameshift formation involves slippage of Pol beta during the one-nucleotide gap-filling step of BER and that this slippage is fixed by strand-displacement synthesis stimulated by an excess of Pol beta.
Subject(s)
DNA Polymerase beta/pharmacology , DNA Repair/physiology , Frameshift Mutation/drug effects , Base Sequence , Blotting, Western , Cell Line, Tumor , DNA Polymerase beta/metabolism , DNA Replication/physiology , Escherichia coli , Frameshift Mutation/physiology , Gene Expression Regulation, Enzymologic/physiology , Humans , Molecular Sequence Data , OligonucleotidesABSTRACT
The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3'-phosphodiesterase and 3'-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.
