In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding

Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease with important functions in DNA replication and DNA repair. It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro. The PCNA interaction site is located close to the C terminus of Fen1 and is flanked by a conserved basic region of 35-38 amino acids in eukaryotic species but not in archaea. We have constructed two deletion mutants of human Fen1 that lack either the PCNA interaction motif or a part of its adjacent C-terminal region and analyzed them in a variety of assays. Remarkably, deletion of the basic C-terminal region did not affect PCNA interaction but resulted in a protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe defect in substrate binding. Our results suggest that the C terminus of eukaryotic Fen1 consists of two functionally distinct regions that together might form an important regulatory domain.     

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.     

Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates

Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5′ flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5′ ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.     

Regulation of DNA replication and repair proteins through interaction with the front side of proliferating cell nuclear antigen.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase delta, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase delta/PCNA. We demonstrate that PCNA must be located below a 5' flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis.     

The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits <5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k(cat)) and site of incision, and is as efficient (k(cat)/K(m)) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.     

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.     

Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region

Interaction between human flap endonuclease-1 (hFEN-1) and proliferating cell nuclear antigen (PCNA) represents a good model for interactions between multiple functional proteins involved in DNA metabolic pathways. A region of 9 conserved amino acid residues (residues Gln-337 through Lys-345) in the C terminus of human FEN-1 (hFEN-1) was shown to be responsible for the interaction with PCNA. Our current study indicates that 4 amino acid residues in hFEN-1 (Leu-340, Asp-341, Phe-343, and Phe-344) are critical for human PCNA (hPCNA) interaction. A conserved PCNA interaction motif in various proteins from assorted species has been defined as Q(1)X(2)X(3)(L/I)(4)X(5)X(6)F(7)(F/Y)(8), although our results fail to implicate Q(1) (Gln-337 in hFEN-1) as a crucial residue. Surprisingly, all hFEN-1 mutants, including L340A, D341A, F343A, and F344A, retained hPCNA-mediated stimulation of both exo- and flap endonuclease activities. Furthermore, our in vitro assay showed that hPCNA failed to bind to the scRad27 (yeast homolog of FEN-1) nuclease. However, its nuclease activities were significantly enhanced in the presence of hPCNA. Four additional Saccharomyces cerevisiae scRad27 mutants, including multiple alanine mutants and a deletion mutant of the entire PCNA binding region, were constructed to confirm this result. All of these mutants retained PCNA-driven nuclease activity stimulation. We therefore conclude that stimulation of eukaryotic hFEN-1 nuclease activities by PCNA is independent of its in vitro interaction via the PCNA binding region.     

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.     

Stimulation of 3'-->5' exonuclease and 3'-phosphodiesterase activities of yeast apn2 by proliferating cell nuclear antigen

The Apn2 protein of Saccharomyces cerevisiae contains 3'-->5' exonuclease and 3'-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3'-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3'-->5' exonuclease and 3'-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.     

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.     

Coordination of multiple enzyme activities by a single PCNA in archaeal Okazaki fragment maturation

Chromosomal DNA replication requires one daughter strand-the lagging strand-to be synthesised as a series of discontinuous, RNA-primed Okazaki fragments, which must subsequently be matured into a single covalent DNA strand. Here, we describe the reconstitution of Okazaki fragment maturation in vitro using proteins derived from the archaeon Sulfolobus solfataricus. Six proteins are necessary and sufficient for coupled DNA synthesis, RNA primer removal and DNA ligation. PolB1, Fen1 and Lig1 provide the required catalytic activities, with coordination of their activities dependent upon the DNA sliding clamp, proliferating cell nuclear antigen (PCNA). S. solfataricus PCNA is a heterotrimer, with each subunit having a distinct specificity for binding PolB1, Fen1 or Lig1. Our data demonstrate that the most efficient coupling of activities occurs when a single PCNA ring organises PolB1, Fen1 and Lig1 into a complex.     

Disruption of the FEN-1/PCNA interaction results in DNA replication defects, pulmonary hypoplasia, pancytopenia, and newborn lethality in mice

The interaction between flap endonuclease 1 (FEN-1) and proliferation cell nuclear antigen (PCNA) is critical for faithful and efficient Okazaki fragment maturation. In a living cell, this interaction is probably important for PCNA to load FEN-1 to the replication fork, to coordinate the sequential functions of FEN-1 and other enzymes, and to stimulate its enzyme activity. The FEN-1/PCNA interaction is mediated by the motif (337)QGRLDDFFK(345) of FEN-1, such that an F343AF344A (FFAA) mutant cannot bind to PCNA but retains its nuclease activities. To determine the physiological roles of the FEN-1/PCNA interaction in a mammalian system, we knocked the FFAA Fen1 mutation into the Fen1 gene locus of mice. FFAA/FFAA mouse embryo fibroblasts underwent DNA replication and division at a slower pace, and FFAA/FFAA mutant embryos displayed significant defects in growth and development, particularly in the lung and blood systems. All newborn FFAA mutant pups died at birth, likely due to pulmonary hypoplasia and pancytopenia. Collectively, our data demonstrate the importance of the FEN-1/PCNA complex in DNA replication and in the embryonic development of mice.     

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.     

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5′–3′-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5′-overhang. A 3′-single-stranded tail arising from the same end of the duplex as the 5′-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5′-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5′-terminal overhangs as well as to remove nucleotides from the 5′-termini enables it to effectively process the 5′-termini of Okazaki fragments before they are ligated.     

The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication

During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5′ DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen (PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway.     

Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG)n and (GAA)n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG)n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for single-stranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.     

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.     

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.