Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication

Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase β with various DNA structures imitating intermediates of DNA repair and replication were investigated by gel retardation and photoaffinity labeling. Photoaffinity labeling of APE1 and DNA polymerase β was accomplished by DNA containing photoreactive group at the 3′-end in mouse embryonic fibroblast (MEF) cell extract or for purified proteins. On the whole, modification efficiency was the same for MEF-extract proteins and for purified APE1 and DNA polymerase β depending on the nature of the 5′-group of a nick/gap in the DNA substrate. Some of DNA duplexes used in this work can be considered as short-patch (DNA with the 5′-phosphate group in the nick/gap) or long-patch (DNA containing 5′-sugar phosphate or 5′-flap) base excision repair (BER) intermediates. Other DNA duplexes (3′-recessed DNA and DNA with the 5′-hydroxyl group in the nick/gap) have no relation to intermediates forming in the course of BER. As shown by both methods, APE1 binds with the highest efficiency to DNA substrate containing 5′-sugar phosphate group in the nick/gap, whereas DNA polymerase β binds to DNA duplex with a mononucleotide gap flanked by the 5′-p group. When APE1 and DNA polymerase β are both present, a ternary complex APE1-DNA polymerase β-DNA is formed with the highest efficiency with DNA product of APE1 endonuclease activity and with DNA containing 5′-flap or mononucleotide-gapped DNA with 5′-p group. It was found that APE1 stimulates DNA synthesis catalyzed by DNA polymerase β, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3′–5′ exonuclease activity on 3′-recessed DNA duplex.