334회 A Novel Model of Okazaki Fragment Processing In Eukaryotes; RPA-G…

334회

연사 : 서 연 수, 성균관대학교 의과대학

제목:  A Novel Model of Okazaki Fragment Processing In Eukaryotes; RPA-Governed Endonuclease Switching

Abstract

In vitro reconstituted DNA synthesis of simian virus 40 DNA contributed substantially to identification of replication proteins required for leading and lagging DNA synthesis in eukaryotes. Investigation of more specific enzymatic reactions and yeast genetic studies has uncovered additional proteins thought to be involved directly in eukaryotic chromosomal DNA synthesis. One such example is the essential Dna2 endonuclease/helicase that has been implicated in Okazaki fragment maturation on the basis of its genetic and physical interaction with proteins involved in Okazaki fragment metabolism. Close examination of biochemical properties revealed that Dna2 is well suited in removal of RNA-terminated single-stranded DNA of Okazaki fragments. In this report, we showed replication protein-A (RPA) and Dna2 form a ternary complex with flap DNA generated by incoming polymerase d. RPA markedly stimulates the cleavage of the RNA-containing primer ends by Dna2, resulting in the release of the bound RPA which in turn allows Fen1 to cleave the shortened flap into a ligatable nick. Therefore, the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA. The sequential action of these enzymes is governed by RPA. Our results demonstrate that the processing of Okazaki fragments in eukaryotes differs significantly from and is more complicated than that occurring in prokaryotes. We propose a novel biochemical mechanism for the maturation of eukaryotic Okazaki fragments.