In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway

Schizosaccharomyces pombe alternative excision repair has been shown geneti cally and biochemically to be involved in the repair of a wide variety of D NA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and plat inum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesi on. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the bioch emical reconstitution of the AER pathway. A base mispair-containing substra te is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymeras e delta, replication factor C, proliferating cell nuclear antigen, and T4 D NA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exon uclease activity of Rad2p and not its "flap endonuclease" activity and is a bsolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uv e1p cleavage site.