Characterization of the Enzymatic Properties of the Yeast Dna2 Helicase/Endonuclease Suggests a New Model for Okazaki Fragment Processing

Abstract

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...

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

“…The preparation of substrates and their labeling at either 5Ј-or 3Ј-end were as described previously (2,15). The structure and position of radioisotopic labels (indicated by asterisks) in substrates are described in each figure.…”

“…Reactions were carried out at 37°C for 5 min unless indicated otherwise. The products were boiled, when necessary, followed by separation in low resolution gels (12% polyacrylamide gel containing 0.5% SDS and 7 M urea) or high resolution denaturing gels as described previously (15). Gels were dried on DEAE-cellulose paper and subjected to autoradiography.…”

“…Recently, it was shown that Dna2 contributes to the protection of stalled replication forks by inhibiting reversion of replication forks in a manner dependent on the intra-S-phase checkpoint in Schizosaccharomyces pombe (14). The enzymatic properties of Dna2 endonuclease are suited to remove flaps generated from 5Ј-ends of Okazaki fragments by DNA displacement activity of DNA polymerase (pol) ␦ (13,15). It was shown that the coordination of pol ␦ and flap endonuclease 1 (Fen1) is sufficient to remove initiating primers from 5Ј-ends of Okazaki fragments (16 -18).…”

The Transcriptome of Prematurely Aging Yeast Cells Is Similar to That of Telomerase-deficient Cells

“…RAD27, the homolog of the mammalian FEN-1, is a structure-specific nuclease implicated in Okazaki fragment maturation during DNA replication. These genetic interactions suggested that at least one specific role for Dna2p is also in the maturation of the 5Ј ends of Okazaki fragments (Budd and Campbell, 1997), and in vitro reconstitution studies indicate that Dna2 is required for efficient RNA removal and ligation when FEN-1 activity is impaired (Bae and Seo, 2000;Ayyagari et al, 2003; Kao, Campbell, and Bambara, unpublished data). With respect to aging, it is interesting that the human BLM gene, encoding a RecQ helicase defective in Bloom syndrome, can suppress the replication defect of dna2 mutants (Imamura and Campbell, 2003).…”

Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner

“…FEN-1 is highly conserved between yeast and human, and is a 5' flap specific endonuclease (Li et al, 1995). The 5'-flap structure can occur by strand displacement at 5' ends of Okazaki fragments during lagging strand synthesis, and can be removed by FEN-1 and other replication proteins, resulting in nicked double strand DNA suitable for ligation (Gordenin et al, 1997;Bae and Seo, 2000). On primertemplate substrates containing a flap structure, FEN1 employs unknown mechanism to cleave the 5'-tail.…”