Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing

Saccharomyces cerevisiae Dna2 possesses both helicase and endonuclease activities. Its endonuclease activity is essential and well suited to remove RNA-DNA primers of Okazaki fragments. In contrast, its helicase activity, although required for optimal growth, is not essential when the rate of cell growth is reduced. These findings suggest that DNA unwinding activity of Dna2 plays an auxiliary role in Okazaki fragment processing. To address this issue, we examined whether the Dna2 helicase activity influenced its intrinsic endonuclease activity using two mutant proteins, Dna2D657A and Dna2K1080E, which contain only helicase or endonuclease activity, respectively. Experiments performed with a mixture of Dna2D657A and Dna2K1080E enzymes revealed that cleavage of a single-stranded DNA by endonuclease activity of Dna2 occurs while the enzyme translocates along the substrate. In addition, DNA unwinding activity efficiently removed the secondary structure formed in the flap structure, which was further aided by replication protein A. Our results suggest that the Dna2 unwinding activity plays a role in facilitating the removal of the flap DNA by its intrinsic endonuclease activity.     

Helicase and nuclease activities of hyperthermophile Pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5'-end

Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophile Pyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae. Dna2Pho has single-stranded (ss) DNA-stimulated ATPase activity, DNA helicase activity (5' to 3' direction) requiring ATP, and nuclease activity, which prefers free 5'-ends of ssDNA as substrate. These activities depend on MgCl(2) concentrations. Dna2Pho requires a higher concentration of MgCl(2) for the nuclease than helicase activity. Both the helicase and nuclease activities of Dna2Pho were inhibited by substrates with RNA segments at the 5'-end of flap DNA, whereas the nuclease activity of Dna2 from S. cerevisiae was reported to be stimulated by RNA segments in the 5'-tail (Bae, S.-H., and Seo, Y. S. (2000) J. Biol. Chem. 38022-38031).     

Polymerase dynamics at the eukaryotic DNA replication fork

This review discusses recent insights in the roles of DNA polymerases (Pol) delta and epsilon in eukaryotic DNA replication. A growing body of evidence specifies Pol epsilon as the leading strand DNA polymerase and Pol delta as the lagging strand polymerase during undisturbed DNA replication. New evidence supporting this model comes from the use of polymerase mutants that show an asymmetric mutator phenotype for certain mispairs, allowing an unambiguous strand assignment for these enzymes. On the lagging strand, Pol delta corrects errors made by Pol alpha during Okazaki fragment initiation. During Okazaki fragment maturation, the extent of strand displacement synthesis by Pol delta determines whether maturation proceeds by the short or long flap processing pathway. In the more common short flap pathway, Pol delta coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase.     

Flap endonuclease disengages Dna2 helicase/nuclease from Okazaki fragment flaps

Okazaki fragments contain an initiator RNA/DNA primer that must be removed before the fragments are joined. In eukaryotes, the primer region is raised into a flap by the strand displacement activity of DNA polymerase delta. The Dna2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. Dna2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, Dna2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the Dna2. To address this, nuclease-inactive Dna2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type Dna2 from the flap. RNA can be cleaved by FEN1 but not by Dna2. Pre-bound wild type Dna2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the Dna2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing.     

Isolation of human Dna2 endonuclease and characterization of its enzymatic properties

In eukaryotes, the creation of ligatable nicks in DNA from flap structures generated by DNA polymerase δ-catalyzed displacement DNA synthesis during Okazaki fragment processing depends on the combined action of Fen1 and Dna2. These two enzymes contain partially overlapping but distinct endonuclease activities. Dna2 is well-suited to process long flaps, which are converted to nicks by the subsequent action of Fen1. In this report, we purified human Dna2 as a recombinant protein from human cells transfected with the cDNA of the human homologue of Saccharomyces cerevisiae Dna2. We demonstrated that the purified human Dna2 enzyme contains intrinsic endonuclease and DNA-dependent ATPase activities, but is devoid of detectable DNA helicase activity. We determined a number of enzymatic properties of human Dna2 including its substrate specificity. When both 5′ and 3′ tailed ssDNAs were present in a substrate, such as a forked-structured one, both single-stranded regions were cleaved by human Dna2 (hDna2) with equal efficiency. Based on this and other properties of hDna2, it is likely that this enzyme facilitates the removal of 5′ and 3′ regions in equilibrating flaps that are likely to arise during the processing of Okazaki fragments in human cells.     

On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing

Short DNA segments designated Okazaki fragments are intermediates in eukaryotic DNA replication. Each contains an initiator RNA/DNA primer (iRNA/DNA), which is converted into a 5'-flap and then removed prior to fragment joining. In one model for this process, the flap endonuclease 1 (FEN1) removes the iRNA. In the other, the single-stranded binding protein, replication protein A (RPA), coats the flap, inhibits FEN1, but stimulates cleavage by the Dna2p helicase/nuclease. RPA dissociates from the resultant short flap, allowing FEN1 cleavage. To determine the most likely process, we analyzed cleavage of short and long 5'-flaps. FEN1 cleaves 10-nucleotide fixed or equilibrating flaps in an efficient reaction, insensitive to even high levels of RPA or Dna2p. On 30-nucleotide fixed or equilibrating flaps, RPA partially inhibits FEN1. CTG flaps can form foldback structures and were inhibitory to both nucleases, however, addition of a dT(12) to the 5'-end of a CTG flap allowed Dna2p cleavage. The presence of high Dna2p activity, under reaction conditions favoring helicase activity, substantially stimulated FEN1 cleavage of tailed-foldback flaps and also 30-nucleotide unstructured flaps. Our results suggest Dna2p is not used for processing of most flaps. However, Dna2p has a role in a pathway for processing structured flaps, in which it aids FEN1 using both its nuclease and helicase activities.     

Acetylation of Dna2 Endonuclease/Helicase and Flap Endonuclease 1 by p300 Promotes DNA Stability by Creating Long Flap Intermediates

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5′–3′ endonuclease, the 5′–3′ helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase α.     

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

The removal of initiating primers from the 5′-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.     

Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation

The Dna2 protein is a multifunctional enzyme with 5'-3' DNA helicase, DNA-dependent ATPase, 3' exo/endonuclease, and 5' exo/endonuclease. The enzyme is highly specific for structures containing single-stranded flaps adjacent to duplex regions. We report here two novel activities of both the yeast and human Dna2 helicase/nuclease protein: single strand annealing and ATP-independent strand exchange on short duplexes. These activities are independent of ATPase/helicase and nuclease activities in that mutations eliminating either nuclease or ATPase/helicase do not inhibit strand annealing or strand exchange. ATP inhibits strand exchange. A model rationalizing the multiple catalytic functions of Dna2 and leading to its coordination with other enzymes in processing single-stranded flaps during DNA replication and repair is presented.     

Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal

Eukaryotic Okazaki fragment maturation requires complete removal of the initiating RNA primer before ligation occurs. Polymerase delta (Pol delta) extends the upstream Okazaki fragment and displaces the 5'-end of the downstream primer into a single nucleotide flap, which is removed by FEN1 nuclease cleavage. This process is repeated until all RNA is removed. However, a small fraction of flaps escapes cleavage and grows long enough to be coated with RPA and requires the consecutive action of the Dna2 and FEN1 nucleases for processing. Here we tested whether RPA inhibits FEN1 cleavage of long flaps as proposed. Surprisingly, we determined that RPA binding to long flaps made dynamically by polymerase delta only slightly inhibited FEN1 cleavage, apparently obviating the need for Dna2. Therefore, we asked whether other relevant proteins promote long flap cleavage via the Dna2 pathway. The Pif1 helicase, implicated in Okazaki maturation from genetic studies, improved flap displacement and increased RPA inhibition of long flap cleavage by FEN1. These results suggest that Pif1 accelerates long flap growth, allowing RPA to bind before FEN1 can act, thereby inhibiting FEN1 cleavage. Therefore, Pif1 directs long flaps toward the two-nuclease pathway, requiring Dna2 cleavage for primer removal.     

Flexibility of eukaryotic Okazaki fragment maturation through regulated strand displacement synthesis

Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase delta (Pol delta) with 5.-flap cutting by FEN1(RAD27) endonuclease. Excessive strand displacement is normally prevented by the 3.-exonuclease activity of Pol delta. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol delta) or PIF1 helicase genes can suppress lethality or growth defects of rad27Delta pol3-D520V mutants (defective for FEN1(RAD27) and the 3.-exonuclease of Pol delta) that produce long flaps and of dna2Delta mutants that are defective in cutting long flaps. On the contrary, pol32Delta or pif1Delta caused lethality of rad27Delta exo1Delta double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1(RAD27) and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol delta is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1(RAD27) during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol delta to FEN1(RAD27). Finally, RNA-DNA hybrids are more readily displaced by Pol delta than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.     

Characterization of the endonuclease and ATP-dependent flap endo/exonuclease of Dna2

Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5' single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5' single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn(2+). The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.     

The protein components and mechanism of eukaryotic Okazaki fragment maturation

An initiator RNA (iRNA) is required to prime cellular DNA synthesis. The structure of double-stranded DNA allows the synthesis of one strand to be continuous but the other must be generated discontinuously. Frequent priming of the discontinuous strand results in the formation of many small segments, designated Okazaki fragments. These short pieces need to be processed and joined to form an intact DNA strand. Our knowledge of the mechanism of iRNA removal is still evolving. Early reconstituted systems suggesting that the removal of iRNA requires sequential action of RNase H and flap endonuclease 1 (FEN1) led to the RNase H/FEN1 model. However, genetic analyses implied that Dna2p, an essential helicase/nuclease, is required. Subsequent biochemical studies suggested sequential action of RPA, Dna2p, and FEN1 for iRNA removal, leading to the second model, the Dna2p/RPA/FEN1 model. Studies of strand-displacement synthesis by polymerase delta indicated that in a reconstituted system, FEN1 could act as soon as short flaps are created, giving rise to a third model, the FEN1-only model. Each of the three pathways is supported by different genetic and biochemical results. Properties of the major protein components in this process will be discussed, and the validity of each model as a true representation of Okazaki fragment processing will be critically evaluated in this review.     

Dynamic removal of replication protein A by Dna2 facilitates primer cleavage during Okazaki fragment processing in Saccharomyces cerevisiae

Eukaryotic Okazaki fragments are initiated by a RNA/DNA primer, which is removed before the fragments are joined. Polymerase delta displaces the primer into a flap for processing. Dna2 nuclease/helicase and flap endonuclease 1 (FEN1) are proposed to cleave the flap. The single-stranded DNA-binding protein, replication protein A (RPA), governs cleavage activity. Flap-bound RPA inhibits FEN1. This necessitates cleavage by Dna2, which is stimulated by RPA. FEN1 then cuts the remaining RPA-free flap to create a nick for ligation. Cleavage by Dna2 requires that it enter the 5'-end and track down the flap. Because Dna2 cleaves the RPA-bound flap, we investigated the mechanism by which Dna2 accesses the protein-coated flap for cleavage. Using a nuclease-defective Dna2 mutant, we showed that just binding of Dna2 dissociates the flap-bound RPA. Facile dissociation is specific to substrates with a genuine flap, and will not occur with an RPA-coated single strand. We also compared the cleavage patterns of Dna2 with and without RPA to better define RPA stimulation of Dna2. Stimulation derived from removal of DNA folding in the flap. Apparently, coordinated with its dissociation, RPA relinquishes the flap to Dna2 for tracking in a way that does not allow flap structure to reform. We also found that RPA strand melting activity promotes excessive flap elongation, but it is suppressed by Dna2-promoted RPA dissociation. Overall, results indicate that Dna2 and RPA coordinate their functions for efficient flap cleavage and preparation for FEN1.     

The endonuclease activity of the yeast Dna2 enzyme is essential in vivo

Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA–DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function. Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable. Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity. Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced. Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress. In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested. These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing.     

Processing of G4 DNA by Dna2 helicase/nuclease and replication protein A (RPA) provides insights into the mechanism of Dna2/RPA substrate recognition

The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5'- to 3'-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3'- to 5'-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening.     

The Protein Components and Mechanism of Eukaryotic Okazaki Fragment Maturation

ABSTRACT

An initiator RNA (iRNA) is required to prime cellular DNA synthesis. The structure of double-stranded DNA allows the synthesis of one strand to be continuous but the other must be generated discontinuously. Frequent priming of the discontinuous strand results in the formation of many small segments, designated Okazaki fragments. These short pieces need to be processed and joined to form an intact DNA strand. Our knowledge of the mechanism of iRNA removal is still evolving. Early reconstituted systems suggesting that the removal of iRNA requires sequential action of RNase H and flap endonuclease 1 (FEN1) led to the RNase H/FEN1 model. However, genetic analyses implied that Dna2p, an essential helicase/nuclease, is required. Subsequent biochemical studies suggested sequential action of RPA, Dna2p, and FEN1 for iRNA removal, leading to the second model, the Dna2p/RPA/FEN1 model. Studies of strand-displacement synthesis by polymerase δ indicated that in a reconstituted system, FEN1 could act as soon as short flaps are created, giving rise to a third model, the FEN1-only model. Each of the three pathways is supported by different genetic and biochemical results. Properties of the major protein components in this process will be discussed, and the validity of each model as a true representation of Okazaki fragment processing will be critically evaluated in this review.     

Dna2p helicase/nuclease is a tracking protein, like FEN1, for flap cleavage during Okazaki fragment maturation

During cellular DNA replication the lagging strand is generated as discontinuous segments called Okazaki fragments. Each contains an initiator RNA primer that is removed prior to joining of the strands. Primer removal in eukaryotes requires displacement of the primer into a flap that is cleaved off by flap endonuclease 1 (FEN1). FEN1 employs a unique tracking mechanism that requires the recognition of the free 5' terminus and then movement to the base of the flap for cleavage. Abnormally long flaps are coated by replication protein A (RPA), inhibiting FEN1 cleavage. A second nuclease, Dna2p, is needed to cleave an RPA-coated flap producing a short RPA-free flap, favored by FEN1. Here we show that Dna2p is also a tracking protein. Annealed primers or conjugated biotin-streptavidin complex block Dna2p entry and movement. Single-stranded binding protein-coated flaps inhibit Dna2p cleavage. Like FEN1, Dna2p can track over substrates with a non-Watson Crick base, such as a biotin, or a missing base within a chain. Unlike FEN1, Dna2p shows evidence of a "threading-like" mechanism that does not support tracking over a branched substrate. We propose that the two nucleases both track, Dna2p first and then FEN1, to remove initiator RNA via long flap intermediates.     

Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism

In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2(+) of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2(+) was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2(+) led to chromosome fragmentation strikingly similar to that seen when cdc17(+), the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase delta (cdc1(+) and cdc27(+)), DNA ligase I (cdc17(+)), and Fen-1 (rad2(+)). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2(+) plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2(+) acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.