Characterization of the enzymatic properties of the yeast Dna2 helicase/endonuclease suggests a new model for Okazaki fragment processing

The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-speci fic endonuclease activity, interacts genetically and physically with Fen-l, a structure-specific endonuclease implicated in Okazaki fragment maturatio n during lagging strand synthesis. In this report, we investigated the prop erties of the Dna2 helicase/endonuclease activities in search of their in v ivo physiological functions in eukaryotes. We found that the Dnaa helicase activity translocates in the 5' to 3' direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage acti vity of Dna2 was markedly stimulated, by the presence of an RNA segment at the 5'-end of single-stranded DNA and occurred within the DNA, ensuring the complete removal of the initiator RNA segment on the Okazaki fragment. In addition, we demonstrated that the removal of pre-existing initiator 5'-ter minal RNA segments depended on a displacement reaction carried out during t he DNA polymerase delta -catalyzed elongation of the upstream Okazaki fragm ents. These properties indicate that Dna2 is well suited to remove the prim er RNA on the Okazaki fragment. Based op this information, we propose a new model in which Dna2 plays a direct role in Okazaki fragment maturation in conjunction with Fen-l.