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.     

Rate-determining Step of Flap Endonuclease 1 (FEN1) Reflects a Kinetic Bias against Long Flaps and Trinucleotide Repeat Sequences

Flap endonuclease 1 (FEN1) is a structure-specific nuclease responsible for removing 5′-flaps formed during Okazaki fragment maturation and long patch base excision repair. In this work, we use rapid quench flow techniques to examine the rates of 5′-flap removal on DNA substrates of varying length and sequence. Of particular interest are flaps containing trinucleotide repeats (TNR), which have been proposed to affect FEN1 activity and cause genetic instability. We report that FEN1 processes substrates containing flaps of 30 nucleotides or fewer at comparable single-turnover rates. However, for flaps longer than 30 nucleotides, FEN1 kinetically discriminates substrates based on flap length and flap sequence. In particular, FEN1 removes flaps containing TNR sequences at a rate slower than mixed sequence flaps of the same length. Furthermore, multiple-turnover kinetic analysis reveals that the rate-determining step of FEN1 switches as a function of flap length from product release to chemistry (or a step prior to chemistry). These results provide a kinetic perspective on the role of FEN1 in DNA replication and repair and contribute to our understanding of FEN1 in mediating genetic instability of TNR sequences.     

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.     

An alternative pathway for Okazaki fragment processing: resolution of fold-back flaps by Pif1 helicase

Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo.     

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.     

Components of the Secondary Pathway Stimulate the Primary Pathway of Eukaryotic Okazaki Fragment Processing

Reconstitution of eukaryotic Okazaki fragment processing implicates both one- and two-nuclease pathways for processing flap intermediates. In most cases, FEN1 (flap endonuclease 1) is able to efficiently cleave short flaps as they form. However, flaps escaping cleavage bind replication protein A (RPA) inhibiting FEN1. The flaps must then be cleaved by Dna2 nuclease/helicase before FEN1 can act. Pif1 helicase aids creation of long flaps. The pathways were considered connected only in that the products of Dna2 cleavage are substrates for FEN1. However, results presented here show that Dna2, Pif1, and RPA, the unique proteins of the two-nuclease pathway from Saccharomyces cerevisiae, all stimulate FEN1 acting in the one-nuclease pathway. Stimulation is observed on RNA flaps representing the initial displacement and on short DNA flaps, subsequently displaced. Neither the RNA nor the short DNA flaps can bind the two-nuclease pathway proteins. Instead, direct interactions between FEN1 and the two-nuclease pathway proteins have been detected. These results suggest that the proteins are either part of a complex or interact successively with FEN1 because the level of stimulation would be similar either way. Proteins bound to FEN1 could be tethered to the flap base by the interaction of FEN1 with PCNA, potentially improving their availability when flaps become long. These findings also support a model in which cleavage by FEN1 alone is the preferred pathway, with the first opportunity to complete cleavage, and is stimulated by components of the backup pathway.     

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.     

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.     

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.     

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.     

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.     

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.     

Reconstituted Okazaki fragment processing indicates two pathways of primer removal

Eukaryotic Okazaki fragments are initiated by an RNA/DNA primer and extended by DNA polymerase delta (pol delta) and the replication clamp proliferating cell nuclear antigen (PCNA). Joining of the fragments by DNA ligase I to generate the continuous double-stranded DNA requires complete removal of the RNA/DNA primer. Pol delta extends the upstream Okazaki fragment and displaces the downstream RNA/DNA primer into a flap removed by nuclease cleavage. One proposed pathway for flap removal involves pol delta displacement of long flaps, coating of those flaps by replication protein A (RPA), and sequential cleavage of the flap by Dna2 nuclease followed by flap endonuclease 1 (FEN1). A second pathway involves reiterative single nucleotide or short oligonucleotide displacement by pol delta and cleavage by FEN1. We measured the length of FEN1 cleavage products on flaps strand-displaced by pol delta in an oligonucleotide system reconstituted with Saccharomyces cerevisiae proteins. Results showed that in the presence of PCNA and FEN1, pol delta displacement synthesis favors formation and cleavage of primarily short flaps, up to eight nucleotides in length; still, a portion of flaps grows to 20-30 nucleotides. The proportion of long flaps can be altered by mutations in the relevant proteins, sequence changes in the DNA, and reaction conditions. These results suggest that FEN1 is sufficient to remove a majority of Okazaki fragment primers. However, some flaps become long and require the two-nuclease pathway. It appears that both pathways, operating in parallel, are required for processing of all flaps.     

Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1.Eukaryotic cellular DNA is replicated semi-conservatively in the 5′ to 3′ direction. A leading strand is synthesized by DNA polymerase ϵ in a continuous manner in the direction of opening of the replication fork (1, 2). A lagging strand is synthesized by DNA polymerase δ (pol δ)³ in the opposite direction in a discontinuous manner, producing segments called Okazaki fragments (3). These stretches of ∼150 nucleotides (nt) must be joined together to create the continuous daughter strand. DNA polymerase α/primase (pol α) initiates each fragment by synthesizing an RNA/DNA primer consisting of ∼1-nt of RNA and ∼10–20 nt of DNA (4). The sliding clamp proliferating cell nuclear antigen (PCNA) is loaded on the DNA by replication factor C (RFC). pol δ then complexes with PCNA and extends the primer. When pol δ reaches the 5′-end of the downstream Okazaki fragment, it displaces the end into a flap while continuing synthesis, a process known as strand displacement (5, 6). These flap intermediates are cleaved by nucleases to produce a nick for DNA ligase I (LigI) to seal, completing the DNA strand.In one proposed mechanism for flap processing, the only required nuclease is flap endonuclease 1 (FEN1). pol δ displaces relatively short flaps, which are cleaved by FEN1 as they are created, leaving a nick for LigI (7–9). FEN1 binds at the 5′-end of the flap and tracks down the flap cleaving only at the base (5, 10, 11). Because pol δ favors the displacement of RNA-DNA hybrids over DNA-DNA hybrids, strand displacement generally is limited to that of the initiator RNA of an Okazaki fragment (12). In addition, the tightly coordinated action of pol δ and FEN1 also tends to keep flaps short. However, biochemical reconstitution studies demonstrate that some flaps can become long (13, 14). Once these flaps reach ∼30 nt, they can be bound by the eukaryotic single strand binding protein replication protein A (RPA) (15). Binding by RPA to a flap substrate inhibits cleavage by FEN1 (16). The RPA-bound flap would then require another mechanism for proper processing.This second mechanism is proposed to utilize Dna2 (16) in addition to FEN1. Dna2 is both a 5′-3′ helicase and an endonuclease (17, 18). Like FEN1, Dna2 recognizes 5′-flap structures, binding at the 5′-end of the flap and tracking downward toward the base (19, 20). Unlike FEN1, Dna2 cleaves the flap multiple times but not all the way to the base, such that a short flap remains (20). RPA binding to a flap has been shown to stimulate Dna2 cleavage (16). Therefore, if a flap becomes long enough to bind RPA, Dna2 binds and cleaves it to a length of 5–10 nucleotides from which RPA dissociates (21). FEN1 can then enter the flap, displace the Dna2, and then cleave at the base to make the nick for ligation (16, 18, 22). The need for this mechanism may be one reason why DNA2 is an essential gene in Saccharomyces cerevisiae (23, 24). It has been proposed that, in the absence of Dna2, flaps that become long enough to bind RPA cannot be properly processed, leading to genomic instability and cell death (23).In reconstitution of Okazaki fragment processing with purified proteins, even though some flaps became long enough to bind RPA, FEN1 was very effective at cleaving essentially all of the generated flaps (13, 14). Evidently, FEN1 could engage the flaps before binding of RPA. However, these reconstitution assays did not include the 5′-3′ helicase Pif1 (25, 26). Pif1 is involved in telomeric and mitochondrial DNA maintenance (26) and was first implicated in Okazaki fragment processing from genetic studies in S. cerevisiae. Deletion of PIF1 rescued the lethality of dna2Δ, although the double mutant was still temperature-sensitive (27). The authors of this report proposed that Pif1 creates a need for Dna2 by promoting longer flaps. Further supporting this conclusion, deletion of POL32, which encodes the subunit of pol δ that interacts with PCNA, rescued the temperature sensitivity of the dna2Δpif1Δ double mutant (12, 27). Importantly, pol δ exhibited reduced strand displacement activity when POL32 was deleted (12, 28, 29). The combination of pif1Δ and pol32Δ is believed to create a situation in which virtually no long flaps are formed, eliminating the requirement for Dna2 flap cleavage (27).We recently performed reconstitution assays showing that Pif1 can assist in the creation of long flaps. Inclusion of Pif1, in the absence of RPA, increased the proportion of flaps that lengthened to ∼28–32 nt before FEN1 cleavage (14). With the addition of RPA, the appearance of these long flap cleavage products was suppressed. Evidently, Pif1 promoted such rapid flap lengthening that RPA bound some flaps before FEN1 and inhibited cleavage. The RPA-bound flaps would presumably require cleavage by Dna2 for proper processing.Only a small fraction of flaps became long with Pif1. However, there are hundreds of thousands of Okazaki fragments processed per replication cycle (30). Therefore, thousands of flaps are expected to be lengthened by Pif1 in vivo, a number significant enough that improper processing of such flaps could lead to cell death.Our goal here was to determine whether Pif1 can influence the flow of Okazaki fragments through the two proposed pathways. We first questioned whether Pif1 stimulates strand displacement synthesis by pol δ. Next, we asked whether Pif1 lengthens short flaps so that Dna2 can bind and cleave. Finally, we used a complete reconstitution system to determine whether Pif1 promotes creation of RPA-bound flaps that require cleavage by both Dna2 and FEN1 before they can be ligated. Our results suggest that Pif1 promotes the two-nuclease pathway, and reveal the mechanisms involved.     

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ～ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.     

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.     

Flap Endonuclease 1 Mechanism Analysis Indicates Flap Base Binding Prior to Threading

FEN1 cleaves 5′ flaps at their base to create a nicked product for ligation. FEN1 has been reported to enter the flap from the 5′-end and track to the base. Current binding analyses support a very different mechanism of interaction with the flap substrate. Measurements of FEN1 binding to a flap substrate show that the nuclease binds with similar high affinity to the base of a long flap even when the 5′-end is blocked with biotin/streptavidin. However, FEN1 bound to a blocked flap is more sensitive to sequestration by a competing substrate. These results are consistent with a substrate interaction mechanism in which FEN1 first binds the flap base and then threads the flap through an opening in the protein from the 5′-end to the base for cleavage. Significantly, when the unblocked flap length is reduced from five to two nucleotides, FEN1 can be sequestered from the substrate to a similar extent as a blocked, long flap substrate. Apparently, interactions related to threading occur only when the flap is greater than two to four nucleotides long, implying that short flaps are cleaved without a threading requirement.