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 join. Despite the well established in vitro roles of these two enzymes, genetic analyses in yeast revealed that null mutants of RNase HI and/or Fen1 are not lethal, suggesting that an additional enzymatic activity may be required for the removal of RNA. One such enzyme is the Saccharomyces cerevisiae Dna2 helicase/endonuclease, which is essential for cell viability and is well suited to removing RNA primers of Okazaki fragments. In addition, Dna2 interacts genetically and physically with several proteins involved in the elongation or maturation of Okazaki fragments. 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.

The Saccharomyces cerevisiae Dna2, which contains single-stranded DNA-specific endonuclease activity, interacts genetically and physically with Fen-1, a structure-specific endonuclease implicated in Okazaki fragment maturation during lagging strand synthesis. In this report, we investigated the properties of the Dna2 helicase/endonuclease activities in search of their in vivo physiological functions in eukaryotes. We found that the Dna2 helicase activity translocates in the 5 to 3 direction and uses DNA with free ends as the preferred substrate. Furthermore, the endonucleolytic cleavage activity 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-terminal RNA segments depended on a displacement reaction carried out during the DNA polymerase ␦-catalyzed elongation of the upstream Okazaki fragments. These properties indicate that Dna2 is well suited to remove the primer 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-1.Biochemical and genetic studies of DNA replication in viruses and lower eukaryotes have contributed substantially to our understanding of eukaryotic DNA synthesis (1-5). The various steps in eukaryotic DNA synthesis are basically similar to those in prokaryotes, requiring many common enzymatic functions. Despite these striking similarities, certain eukaryotic replication components differ from the prokaryotic counterparts. Most noteworthy is that three essential DNA polymerases (pol) 1 ␣, ␦, and ⑀ are required for DNA replication in eukaryotes (5-7). In contrast, pol III is the only replicative enzyme in prokaryotes involved in DNA synthesis. Unlike its prokaryotic counterpart, the eukaryotic primase is complexed with DNA pol ␣. Thus, the role of the pol ␣-primase complex appears to function solely in the synthesis of a short RNA-DNA primer (referred to as primer DNA in this paper). The primer DNAs are then utilized by pol ␦ for the initiation of leading strand synthesis and by pol ␦ or pol ⑀ for each Okazaki fragment synthesis for lagging strand DNA replication.The short and discontinuous Okazaki fragments at replication forks are processed and then joined to generate a continuous DNA strand through a series of complex enzymatic reactions that require a number of enzymes that include a 5Ј-to 3Ј-exonuclease (5, 8). However, none of the eukaryotic polymerases possess intrinsic 5Ј-to 3Ј-exonuclease activity for Okazaki fragment processing, unlike the well characterized prokaryotic polymerase, Escherichia coli DNA pol I (1). In the current model of replication in eukaryotes, Fen-1 provides the 5Ј-to 3Ј-exonuclease activity, and with the assistance of RNase HI removes the RNA segments on Okazaki fragments (9 -11). RNase HI hydrolyzes the initiator RNA o...

To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity. Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2. Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion. ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required. Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized. This finding implies that Dna2 unwinds only partially the 3-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity. A point mutation at the conserved ATPbinding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself. By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks.Maintaining the integrity of chromosomal DNA in eukaryotes is of critical importance to the cell and requires a series of complicated enzymatic processes. This is reflected in the complexity and redundancy of the enzyme systems that participate in DNA metabolism, such as replication, repair, and recombination (1, 2). In addition, DNA metabolism is tightly linked to cellular control pathways that regulate the cell division cycle (3-9). One of the enzymes required to achieve DNA replication, repair, or recombination is the DNA helicase, which uses the energy of ATP to translocate in a specific direction along a DNA strand melting the duplex regions it encounters (10 -14). The single-stranded DNA (ssDNA) 1 generated by the helicase is utilized by other enzymes that participate in the subsequent steps in DNA metabolic pathways. Recently, the DNA2 gene of Saccharomyces cerevisiae was implicated in chromosomal DNA replication (15, 16). DNA2 was originally identified by screening for cell division cycle mutants of S. cerevisiae and was shown to be essential for cell viability and to encode a 172-kDa protein with characteristic DNA helicase motifs (15). Analyses of a temperature-sensitive mutant of DNA2 demonstrated that the mutant cell arrested in the S phase of the cell cycle and was deficient in DNA synthesis but not RNA synthesis upon shift to the nonpermissive temperature (15). Immunoaffinity purified Dna2 fusion protein displayed a DNA-dependent ATPase activity as well as 3Ј to 5Ј DNA helicase activity specific for fork-structured substrates (15). In addition, a mutation in the ATP binding motif of DNA2 led to the inactivation of the ATPase and helicas...