Long Patch Base Excision Repair with Purified Human Proteins

Abstract

Among the different base excision repair pathways known, the long patch base excision repair of apurinic/ apyrimidinic sites is an important mechanism that re-quires proliferating cell nuclear antigen. We have recon-stituted this pathway using purified human proteins. Our data indicated that efficient repair is dependent on six components including AP endonuclease, replication factor C, proliferating cell nuclear antigen, DNA polym-erases ␦ or ⑀, flap endonuclease 1, and DNA ligase I. Fine mapping of the nucleotide replacement events showed that repair patches extended up to a maximum of 10 nucleotides 3؅ to the lesion. However, almost 70% of the repair synthesis was confined to 2– 4-nucleotide patches and DNA ligase I appeared to be responsible for limiting the repair patch length. Moreover, both proliferating cell nuclear antigen and flap endonuclease 1 are re-quired for the production and ligation of long patch repair intermediates suggesting an important role of this complex in both excision and resynthesis steps.