In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway.
Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. 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 biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.
Alleva
J L
JL
Department of Biochemistry, Graduate Program in Genetics and Molecular Biology, and Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Zuo
S
S
Hurwitz
J
J
Doetsch
P W
PW
D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D045585:Flap Endonucleases; D005656:Fungal Proteins / Q000378:metabolism; D006868:Hydrolysis; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D013379:Substrate Specificity / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C073998:RAD2 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; D043211:Exodeoxyribonuclease V; C105073:uve1 protein, S pombe