Distinct roles of two Mg2+ binding sites in regulation of murine flap endonuclease-1 activities

Removal of flap DNA intermediates in DNA replication and repair by flap endonuclease-1 (FEN-1) is essential for mammalian genome integrity. Divalent metal ions, Mg(2+) or Mn(2+), are required for the active center of FEN-1 nucleases. However, it remains unclear as to how Mg(2+) stimulates enzymatic activity. In the present study, we systemically characterize the interaction between Mg(2+) and murine FEN-1 (mFEN-1). We demonstrate that Mg(2+) stimulates mFEN-1 activity at physiological levels but inhibits the activity at concentrations higher than 20 mM. Our data suggest that mFEN-1 exists as a metalloenzyme in physiological conditions and that each enzyme molecule binds two Mg(2+) ions. Binding of Mg(2+) to the M1 binding site coordinated by the D86 residue cluster enhances mFEN-1's capability of substrate binding, while binding of the metal to the M2 binding site coordinated by the D181 residue cluster induces conformational changes. Both of these steps are needed for catalysis. Weak, nonspecific Mg(2+) binding is likely responsible for the enzyme inhibition at high concentrations of the cation. Taken together, our results suggest distinct roles for two Mg(2+) binding sites in the regulation of mFEN-1 nuclease activities in a mode different from the "two-metal mechanism".     

Arginine residues 47 and 70 of human flap endonuclease-1 are involved in DNA substrate interactions and cleavage site determination

Flap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively charged amino acid residues, Arg-47 and Arg-70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the Arg-70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of Arg-47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Although the effect of the single Arg-47 mutation on binding activities is not as severe as R70A, its double mutation with Asp-181 had a synergistic effect. Furthermore the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally data were provided to indicate that the observed negative effects of a high concentration of Mg(2+) on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate since mutants were found to be less tolerant.     

Physical and functional interaction between human oxidized base-specific DNA glycosylase NEIL1 and flap endonuclease 1

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.     

Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks

Restarting stalled replication forks partly depends on the break-induced recombination pathway, in which a DNA double-stranded break (DSB) is created on the stalled replication fork to initiate the downstream recombination cascades. Single-stranded DNA gaps accumulating on stalled replication forks are potential targets for endonucleases to generate DSBs. However, it is unclear how this process is executed and which nucleases are involved in eukaryotic cells. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork. In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Replication protein A facilitates FEN-1 interaction with DNA bubble structures. Human FEN-1, but not the GEN-deficient mutant, E178A, was shown to rescue the defect in resistance to UV and camptothecin in a yeast FEN-1 null mutant.     

Interaction of flap endonuclease-1 and replication protein A with photoreactive intermediates of DNA repair

A new method for enzymatic synthesis of radioactive DNA flapped structures containing a photoreactive dCMP moiety at a branch point with 4-(4-azido-2,3,5,6-tetrafluorobenzylidene-hydrazinocarbonyl)butylcarbamoyl group attached at exo-N-position of cytosine was developed. The formation of complexes of flap endonuclease-1 (FEN-1) with flapped DNA was shown by photoaffinity modification and gel retardation assays. The substrate properties of the flapped structures with different flap lengths were studied in the reaction of endonuclease cleavage catalyzed by FEN-1. It was demonstrated that inhibition of FEN-1 activity by replication protein A (RPA) depends on the length of the single-stranded part of the flapped substrate. A significant inhibition of cleavage was observed when the flap length was sufficient for effective RPA binding, while for structures with short single-stranded part the efficiency of cleavage was independent of the presence of RPA. FEN-1 and RPA were modified by photoaffinity labeling using flap structures with single-stranded parts consisting of 8 and 21 nucleotides. Products of DNA photoattachment to FEN-1 were observed in both cases, while the covalent adducts with RPA were obtained only with the 21-nucleotide-long flap. Photoaffinity modification demonstrated that FEN-1 and RPA compete for the binding of the flapped substrates with long single-stranded parts.     

Interaction of replication protein A and flap endonuclease 1 with DNA duplexes containing a nick or flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA. Using Pol beta, a photoreactive dTTP analog was added to the 3' end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5' end of a nick or a flap. Photoaffinity labeling of Pol beta substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol beta-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol beta during long-patch base excision repair.     

Biochemical characterization of the WRN-FEN-1 functional interaction

Werner Syndrome is a premature aging disorder characterized by chromosomal instability. Recently we reported a novel interaction of the WRN gene product with human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in pathways of DNA metabolism that are important for genomic stability. To characterize the mechanism for WRN stimulation of FEN-1 cleavage, we have determined the effect of WRN on the kinetic parameters of the FEN-1 cleavage reaction. WRN enhanced the efficiency of FEN-1 cleavage rather than DNA substrate binding. WRN effectively stimulated FEN-1 cleavage on a flap DNA substrate with streptavidin bound to the terminal 3' nucleotide at the end of the upstream duplex, indicating that WRN does not require a free upstream end to stimulate FEN-1 cleavage of the 5' flap substrate. These results indicate that the mechanism whereby WRN stimulates FEN-1 cleavage is distinct from that proposed for the functional interaction between proliferating cell nuclear antigen and FEN-1. To understand the potential importance of the WRN-FEN-1(1) interaction in DNA replication, we have tested the effect of WRN on FEN-1 cleavage of several DNA substrate intermediates that may arise during Okazaki fragment processing. WRN stimulated FEN-1 cleavage of flap substrates with a terminal monoribonucleotide, a long 5' ssDNA tract, and a pseudo-Y structure. The ability of WRN to facilitate FEN-1 cleavage of DNA replication/repair intermediates may be important for the role of WRN in the maintenance of genomic stability.     

Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity

Werner syndrome (WS) is a human premature aging disorder characterized by chromosomal instability. The cellular defects of WS presumably reflect compromised or aberrant function of a DNA metabolic pathway that under normal circumstances confers stability to the genome. We report a novel interaction of the WRN gene product with the human 5' flap endonuclease/5'-3' exonuclease (FEN-1), a DNA structure-specific nuclease implicated in DNA replication, recombination and repair. WS protein (WRN) dramatically stimulates the rate of FEN-1 cleavage of a 5' flap DNA substrate. The WRN-FEN-1 functional interaction is independent of WRN catalytic function and mediated by a 144 amino acid domain of WRN that shares homology with RecQ DNA helicases. A physical interaction between WRN and FEN-1 is demonstrated by their co-immunoprecipitation from HeLa cell lysate and affinity pull-down experiments using a recombinant C-terminal fragment of WRN. The underlying defect of WS is discussed in light of the evidence for the interaction between WRN and FEN-1.     

Functional analysis of point mutations in human flap endonuclease-1 active site.

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.     

The characterization of a mammalian DNA structure-specific endonuclease.

The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.     

Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways

Flap endonuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease that plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair, resolution of DNA secondary structures and stalled DNA replication forks, and apoptotic DNA fragmentation. How FEN-1 is involved in multiple pathways, of which some are seemingly contradictory, is of considerable interest. To date, at least 20 proteins are known to interact with FEN-1; some form distinct complexes that affect one or more FEN-1 activities presumably to direct FEN-1 to a particular DNA metabolic pathway. FEN-1 consists of a nuclease core domain and a C-terminal extension. While the core domain harbors the nuclease activity, the C-terminal extension may be important for protein-protein interactions. Here, we have truncated or mutated the C-terminus of FEN-1 to identify amino acid residues that are critical for interaction with five proteins representing roles in different DNA replication and repair pathways. We found with all five proteins that the C-terminus is important for binding and that each protein uses a subset of amino acid residues. Replacement of one or more residues with an alanine in many cases leads to the complete loss of interaction, which may consequently lead to severe biological defects in mammals.     

Defective flap endonuclease 1 activity in mammalian cells is associated with impaired DNA repair and prolonged S phase delay.

Flap endonuclease 1 (FEN-1) is a 5'-3' flap exo-/endonuclease that plays an important role in Okazaki fragment maturation, nonhomologous end joining of double-stranded DNA breaks, and long patch base excision repair. Here, we demonstrate that the wild type FEN-1 binds tightly to chromatin in conjunction with proliferating cell nuclear antigen (PCNA) recruitment after MMS treatment, and the nuclease-defective FEN-1 increased the sensitivity of the cells to methylmethane sulfonate (MMS) and to UV light but not to ionizing radiation. In contrast, the cells expressing the nuclease-defective and PCNA binding-defective double mutant FEN-1 exhibited sensitivities similar to those in the cells expressing the wild type FEN-1. MMS treatment caused a prolonged delay of S phase progression and impairment in colony-forming activity of cells expressing nuclease-defective FEN-1. A comet assay demonstrated that DNA repair after MMS or UV treatment was impaired in the cells expressing nuclease-deficient FEN-1 but not in the cells with double-mutated FEN-1. Taken together, these findings suggest that FEN-1 plays an essential role in the DNA repair processes in mammalian cells and that this activity of FEN-1 is PCNA-dependent.     

Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA.

In eukaryotic cells, a 5' flap DNA endonuclease activity and a ds DNA 5'-exonuclease activity exist within a single enzyme called FEN-1 [flap endo-nuclease and 5(five)'-exo-nuclease]. This 42 kDa endo-/exonuclease, FEN-1, is highly homologous to human XP-G, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1. These structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap, and its derivative called a pseudo Y-structure. FEN-1 is essential for lagging strand DNA synthesis in Okazaki fragment joining. FEN-1 also appears to be important in mismatch repair. Here we find that human PCNA, the processivity factor for eukaryotic polymerases, physically associates with human FEN-1 and stimulates its endonucleolytic activity at branched DNA structures and its exonucleolytic activity at nick and gap structures. Structural requirements for FEN-1 and PCNA loading provide an interesting picture of this stimulation. PCNA loads on to substrates at double-stranded DNA ends. In contrast, FEN-1 requires a free single-stranded 5' terminus and appears to load by tracking along the single-stranded DNA branch. These physical constraints define the range of DNA replication, recombination and repair processes in which this family of structure-specific nucleases participate. A model explaining the exonucleolytic activity of FEN-1 in terms of its endonucleolytic activity is proposed based on these observations.     

Interaction interface of human flap endonuclease-1 with its DNA substrates

Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. To map further the corresponding interactions between FEN-1 residues and DNA substrates, we performed biochemical assays employing a series of flap DNA substrates lacking some structural components and a series of binding-deficient point mutants of FEN-1. It was revealed that Arg(47), Arg(70), and Lys(326)-Arg(327) of FEN-1 interact with the upstream duplex of DNA substrates, whereas Lys(244)-Arg(245) interact with the downstream duplex. This result indicates the orientation of the FEN-1-DNA interaction. Moreover, Arg(70) and Arg(47) were determined to interact with the sites around the 2nd nucleotide (Arg(70)) or the 5th/6th nucleotide (Arg(47)) of the template strand in the upstream duplex portion counting from the nick point of the flap substrate. Together with previously published data and the crystallographic ainformation from the FEN-1.DNA complex that we published recently (Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J. A. (2004) Cell 116, 39-50) we are able to propose a reasonable model for how the human FEN-1 protein interacts with its DNA substrates.     

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.     

Interaction and stimulation of human FEN-1 nuclease activities by heterogeneous nuclear ribonucleoprotein A1 in alpha-segment processing during Okazaki fragment maturation

High-fidelity DNA replication depends on both accurate incorporation of nucleotides in the newly synthesized strand and the maturation of Okazaki fragments. In eukaryotic cells, the latter is accomplished by a series of coordinated actions of a set of structure-specific nucleases, which, with the assistance of accessory proteins, recognize branched RNA/DNA configurations. In the current model of Okazaki fragment maturation, displacement of a 27-nucleotide or longer flap is envisioned to attract replication protein A (RPA), which inhibits flap endonuclease-1 (FEN-1) but stimulates Dna2 nuclease for cleavage. Dna2 cleavage generates a short flap of 5-7 nucleotides, which resists binding by RPA and further cleavage by Dna2. FEN-1 then removes the remaining flap to produce a suitable substrate for ligation. However, FEN-1 is not efficient in cleaving the short flap, and we therefore set out to identify cellular factors that might regulate FEN-1 activity. Through co-immunoprecipitation experiments, we have isolated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which forms a direct complex with FEN-1 and stimulates its enzymatic activities. The stimulation by hnRNP A1 is most dramatic using DNA substrates with short flaps. With longer flap substrates the hnRNP A1 effect is more modest and is suppressed by the addition of RPA. A model is provided to explain the possible in vivo role of this interaction and activity in Okazaki fragment maturation.     

Multiple but dissectible functions of FEN-1 nucleases in nucleic acid processing, genome stability and diseases

Flap EndoNuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease involved in nucleic acid processing pathways. It plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair and resolution of dinucleotide and trinucleotide repeat secondary structures. In addition to its flap endonuclease (FEN) and nick exonuclease (EXO) activities, a new gap endonuclease (GEN) activity has been characterized. This activity may be important in apoptotic DNA fragmentation and in resolving stalled DNA replication forks. The multiple functions of FEN-1 are regulated via several means, including formation of complexes with different protein partners, nuclear localization in response to cell cycle or DNA damage and post-translational modifications. Its functional deficiency is predicted to cause genetic diseases, including Huntington's disease, myotonic dystrophy and cancers. This review summarizes the knowledge gained through efforts in the past decade to define its structural elements for specific activities and possible pathological consequences of altered functions of this multirole player.     

CRN-1, a Caenorhabditis elegans FEN-1 homologue,cooperates with CPS-6/EndoG to promote apoptotic DNA degradation

Oligonucleosomal fragmentation of chromosomes in dying cells is a hallmark of apoptosis. Little is known about how it is executed or what cellular components are involved. We show that crn-1, a Caenorhabditis elegans homologue of human flap endonuclease-1 (FEN-1) that is normally involved in DNA replication and repair, is also important for apoptosis. Reduction of crn-1 activity by RNA interference resulted in cell death phenotypes similar to those displayed by a mutant lacking the mitochondrial endonuclease CPS-6/endonuclease G. CRN-1 localizes to nuclei and can associate and cooperate with CPS-6 to promote stepwise DNA fragmentation, utilizing the endonuclease activity of CPS-6 and both the 5'-3' exonuclease activity and a previously uncharacterized gap-dependent endonuclease activity of CRN-1. Our results suggest that CRN-1/FEN-1 may play a critical role in switching the state of cells from DNA replication/repair to DNA degradation during apoptosis.     

Stimulation of flap endonuclease-1 by the Bloom's syndrome protein

Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5'-flap endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.     

DmGEN shows a flap endonuclease activity, cleaving the blocked-flap structure and model replication fork

Drosophila melanogaster XPG-like endonuclease (DmGEN) is a new category of nuclease belonging to the RAD2/XPG family. The DmGEN protein has two nuclease domains (N and I domains) similar to XPG/class I nucleases; however, unlike class I nucleases, in DmGEN these two nuclease domains are positioned close to each other as in FEN-1/class II and EXO-1/class III nucleases. To confirm the properties of DmGEN, we characterized the active-site mutant protein (E143A E145A) and found that DmGEN had flap endonuclease activity. DmGEN possessed weak nick-dependent 5'-3' exonuclease activity. Unlike XPG, DmGEN could not incise the bubble structure. Interestingly, based on characterization of flap endonuclease activity, DmGEN preferred the blocked-flap structure as a substrate. This feature is distinctly different from FEN-1. Furthermore, DmGEN cleaved the lagging strand of the model replication fork. Immunostaining revealed that DmGEN was present in the nucleus of actively proliferating Drosophila embryos. Thus, our studies revealed that DmGEN belongs to a new class (class IV) of the RAD2/XPG nuclease family. The biochemical properties of DmGEN and its possible role are also discussed.