RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes

TLDR: It is shown that the endonucleases Dna2 and Fen1 act sequentially to facilitate the complete removal of the primer RNA in Okazaki fragments, governed by a single-stranded DNA-binding protein, replication protein-A (RPA).

Abstract: Extensive work on the maturation of lagging strands during the replication of simian virus 40 DNA suggests that the initiator RNA primers of Okazaki fragments are removed by the combined action of two nucleases, RNase HI and Fen1, before the Okazaki fragments join1,2,3,4,5 Despite the well established in vitro roles of these two enzymes6, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal7,8,9, suggesting that an additional enzymatic activity may be required for the removal of RNA One such enzyme is the Saccharomyces cerevisiae Dna2 helicase10,11,12/endonuclease12, which is essential for cell viability13,14 and is well suited to removing RNA primers of Okazaki fragments15 In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments10,16 Here we show that 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 a single-stranded DNA-binding protein, replication protein-A (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