©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DNA Structural Elements Required for FEN-1 Binding (*)

(Received for publication, November 3, 1994; and in revised form, December 27, 1994)

John J. Harrington (1) Michael R. Lieber (1) (2)(§)

From the  (1)Department of Pathology and Program in Cancer Biology, Stanford University School of Medicine, Stanford, California 94305 and the (2)Departments of Pathology, Medicine, and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110-8118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In eukaryotic cells, a 5`-flap DNA endonuclease and a double-stranded DNA 5`-exonuclease activity reside within a 42-kDa enzyme called FEN-1 (flap endonuclease-1 and 5(five)`-exonuclease). This endo/exonuclease has been shown to be highly homologous to human XP-G, Saccharomyces cerevisiae RAD2, and S. cerevisiae YKL510. Like FEN-1, these related structure-specific nucleases recognize and cleave a branched DNA structure called a DNA flap and its derivative, called a pseudo Y-structure. To dissect the important structural components of the DNA flap structure, we have developed a mobility shift assay. We find that the F strand (located adjacent to the displaced flap strand) is necessary for efficient binding and cleavage of flap structures by FEN-1. When this strand is absent or when it is present, but recessed from the elbow of the flap strand, binding efficiency drops. Further investigation of the role of the F strand using double flap structures reveals that the F strand is necessary to provide a double-stranded template upon which FEN-1 can bind near the elbow of the flap strand. These results provide a basis for understanding how this structure-specific nuclease recognizes a variety of DNA substrates.


INTRODUCTION

DNA flap structures have been proposed to be important intermediates in many DNA metabolic reactions. During DNA replication, for example, displacement of an upstream primer by the incoming polymerase results in the formation of a 5`-flap structure(1) . The formation and resolution of this structure may allow certain types of DNA damage to be removed from the DNA duplex. Likewise, other repair processes may also occur via flap intermediates. DNA flaps have been proposed in homologous recombination(2) , double-strand break repair (3, 4) , and nucleotide excision repair(5) .

A structure-specific endonuclease capable of resolving DNA flap structures has been purified and characterized(4) . This enzyme, called FEN-1 (flap endonuclease-1), cleaves 5`-flap structures endonucleolytically and has a double strand-specific 5`-3`-exonuclease activity. Because these activities are similar to the activities of exonuclease VI (the 5`-3`-exonuclease domain of Escherichia coli DNA polymerase I) (1) and identical to an activity necessary for in vitro DNA replication by mammalian cell extracts(6, 7) , it has been hypothesized that FEN-1 plays a role in replication(4, 5) . It is still unclear whether FEN-1 also plays a role in DNA recombination and repair.

The murine gene that encodes FEN-1 has been identified and shown to be highly homologous to the RAD2 gene family(5) . Within this family, FEN-1 is most homologous in sequence and structure to Saccharomyces cerevisiae YKL510 and Schizosaccharomyces pombe rad2. Although these gene products have no known biological function at this time, YKL510 and FEN-1 have been shown to have identical enzymatic activities. This suggests that YKL510 (and presumably S. pombe Rad2) is the yeast analog of FEN-1 and likely plays a role in DNA replication. Other members of this family include the nucleotide excision repair genes RAD2 (S. cerevisiae), RAD13 (S. pombe), and XP-G (human). Analysis of enzymatic specificity confirms that a truncated form of S. cerevisiae Rad2 is a structure-specific endonuclease that cleaves flap and pseudo Y-structures(5) . This suggests that these structures may be important intermediates in nucleotide excision repair. Furthermore, the relationship between XP-G and FEN-1 raises the possibility that mutation of the FEN-1 gene may result in a disease phenotype similar to xeroderma pigmentosum. The recent cloning and mapping of the human FEN-1 gene indicate that FEN-1 does not map to any known congenital disease locus(8) , although alterations in malignant somatic cells remain a possibility.

As the role of flap structures in DNA metabolism is becoming more clear, it is necessary to understand the important components of this structure that are necessary for recognition by this family of structure-specific nucleases. Although the substrate specificity of FEN-1 has been characterized using a variety of nuclease assays, many questions related to the recognition of DNA structures by FEN-1 can not be answered by this approach. Specifically, the important structural components of many nucleic acid substrates cannot be dissected because small changes in structure result in an inability of FEN-1 to cleave the substrate. To answer questions regarding substrate recognition by FEN-1, we have developed a mobility shift assay using a labeled DNA flap structure as a probe. The study described here is aimed at providing a foundation upon which a better understanding of the substrate recognition by structure-specific nucleases can be reached.


EXPERIMENTAL PROCEDURES

Purification of FEN-1

FEN-1 was overexpressed in E. coli and purified as described previously(5) . The final purity of FEN-1 was >95%.

Oligonucleotides

All DNA oligonucleotides were purchased from Operon Technologies, Inc. (Alameda, CA). The sequence of each is shown after its name and is written 5` to 3`: SC1, CAGCAACGCAAGCTTG; SC3, GTCGACCTGCAGCCCAAGCTTGCGTTGCTG; SC5, ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC; AI4, GGCCGTATCTGGGTTCGAATTCATCAAGGGACATCTCCTAC; HJ46, CCTATCGTTGCATGGTGCCAGAACCCAGATACGGC; HJ47, GTAGGAGATGTCCCTTGATGAATTC; HJ73, GTAGGAGATGTCCCTTGATGAATT; HJ74, GTAGGAGATGTCCCTTGATGAA; HJ75, GTAGGAGATGTCCCTTGATG; HJ77, GTAGGAGAT GTCCCTTGATGAATTCC; HJ78, GTAGGAGATGTCCCTTGATGAATTCCTTGTGTTTC; CLH2, GTAGGAGATGTCCCTTGATGAATT; CLH3, CGAACCCAGATACGGC; and CLH6, GTAGGAGATGTCCCTTGATGAATTCGAACCCAGATACGGC.

Flap Endonuclease Assay

The standard endonuclease assay has been described previously(4) . Briefly, FEN-1 endonuclease activity was measured in a 15-µl reaction containing 50 mM Tris (pH 8), 10 mM MgCl(2), 50 µg/ml bovine serum albumin, and 10 fmol of labeled substrate. Reactions were carried out at 30 °C for 30 min and terminated by adding 15 µl of 95% formamide, 10 mM EDTA, 1 mg/ml bromphenol blue, 1 mg/ml xylene cyanol. The tubes were heated to 95 °C for 5 min, loaded onto a 10% polyacrylamide gel containing 7 M urea and 1 times TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA (pH 8)), and run for 90 min at 75 watts. One unit of FEN-1 activity produces 1 fmol of cleaved product under these standard reaction conditions.

Mobility Shift Assay for FEN-1

Binding reactions were carried out in 20 µl containing 50 mM Tris (pH 8), 10 mM NaCl, 5 mM EDTA, 10% glycerol, 50 µg/ml bovine serum albumin, 0.25 ng (10 fmol) of labeled oligonucleotide probe, and the indicated amount of FEN-1. Following a 5-min incubation at 20 °C, the reactions were loaded onto a 5% polyacrylamide gel containing 0.5 times TBE and run at 125 V for 90 min. The gels were dried and exposed to PhosphorImager screens to allow quantitation of bands. When more than one oligonucleotide composes a multi-oligonucleotide competitor structure (e.g. AI4/HJ46/HJ47 in a flap structure) in any of the figures, the mass given in the figure is only for the oligonucleotide AI4, and an equimolar quantity of the other oligonucleotides listed in the name of the structure is added in addition to constitute the complete competitor structure.


RESULTS

The recognition of unusual DNA structures by endonucleases is poorly understood at this time. In part, this is due to the lack of purified and cloned structure-specific nucleases. The structure-specific nuclease FEN-1 has now been cloned, overexpressed, and purified and therefore is a good candidate for carrying out substrate binding studies. To characterize the substrate binding characteristics of FEN-1, we have developed a mobility shift assay. Because FEN-1 is a potent nuclease in the presence of divalent metal ions, EDTA was included in the binding reaction to remove residual metal ions. Under these conditions, FEN-1 was found to bind to the DNA flap structure in a dose-dependent fashion (Fig. 1). This dose response was linear between 10 and 60 ng of FEN-1 under the standard binding conditions. In this range of FEN-1 concentrations, a single shifted species was observed. Although we cannot rule out Mg being involved in the binding, we believe this to be unlikely because the substrate binding properties of purified FEN-1 in the absence of Mg are entirely consistent with the endonucleolytic activity of FEN-1 in the presence of Mg (see below).


Figure 1: Effect of FEN-1 concentration in the mobility shift assay. Various amounts of purified FEN-1 were incubated with 10 fmol of labeled flap substrate SC1/SC3/SC5 as described under ``Experimental Procedures.'' Lanes 1-5, 0, 10 (0.2 pmol), 20 (0.4 pmol), 40 (0.8 pmol), and 80 (1.6 pmol) ng of purified FEN-1, respectively. The structure of the labeled binding substrate (SC1/SC3/SC5) is shown at the top. Individual strands are illustrated as solidlines with a half-arrow at the 3`-end and are designated F strand (top), flap strand (bottomleft), and F strand (bottomright). The position of the radioactive end label is indicated by an asterisk. On the right, the positions of free and complexed labeled substrates are shown. The gradual shift in the free probe is due to the binding of labeled substrate by FEN-1, followed by release during electrophoresis, which is more apparent with increasing FEN-1 concentration.



To determine the specificity of FEN-1 for the 5`-flap structure in the mobility shift assay, we titrated various nucleic acid competitors into the binding reaction (Fig. 2). Double- and single-stranded DNAs compete inefficiently in this assay. An 1000-fold molar excess of these competitors was needed to fully compete the binding of FEN-1 away from the labeled flap structure. tRNA failed to compete for FEN-1 binding even when present in a 1000-fold molar excess over labeled substrate. These results are consistent with the inhibition profiles of these nucleic acid competitors in the standard flap endonuclease assay(4) .


Figure 2: Effect of nonspecific nucleic acid competitors on FEN-1 binding. Binding reactions were carried out as described under ``Experimental Procedures.'' Each reaction contained 60 ng (1.2 pmol) of purified FEN-1 and the indicated amounts of the following nucleic acid competitors: lanes 1 and 2, no competitor; lanes 3-6, AI4/CLH6; lanes 7-10, AI4; lanes 11-14, tRNA. The competitor structure and concentration are indicated below their respective lanes. When more than one oligonucleotide composes a structure (e.g. AI4/CLH6 in a double-stranded configuration) in this or any of the other figures, the mass given is only for the oligonucleotide AI4, and an equimolar quantity of the other oligonucleotides listed in the name of the structure is added in addition to constitute the complete competitor structure. The structure of the labeled substrate (SC1/SC3/SC5) is illustrated at the top (see the legend to Fig. 1for details). The positions of free and complexed labeled substrates are indicated on the right.



The binding specificity of FEN-1 was further characterized by titrating derivatives of the flap structure into the reaction (Fig. 3). Unlike double- and single-stranded DNAs, a 40-fold excess of unlabeled 5`-flap structure was sufficient to fully compete the binding of FEN-1 away from the labeled flap substrate. In addition, we found that the entire flap structure was required for optimal competition to occur. The pseudo Y-structure, which was missing the F strand, competed 3-fold less efficiently than the 5`-flap structure. Furthermore, the 5`-overhang, which was missing the flap strand, competed 4-fold less efficiently than the 5`-flap structure, but 5-fold more efficiently than single-stranded DNA. This indicates that the presence of the F strand enhances the binding of FEN-1 to this overhang structure. Thus, the reduction in binding of FEN-1 to the pseudo Y-structure (compared with the 5`-flap structure) and the enhancement in binding of FEN-1 to the 5`-overhang (compared with single-stranded DNA) demonstrate the importance of the F strand in the binding step of this reaction.


Figure 3: Competition of FEN-1 binding activity with DNA flap structure derivatives. Binding reactions were carried out as described under ``Experimental Procedures'' and contained 60 ng (1.2 pmol) of purified FEN-1. In addition, the indicated amounts of the following nucleic acid competitors were present: lanes1 and 2, no competitor; lanes 3-6, AI4/HJ46/HJ47; lanes 7-10, AI4/HJ46; and lanes 11-14, AI4/HJ47. The structures of the labeled substrate (SC1/SC3/SC5) and the unlabeled competitors are illustrated above and below the figure, respectively. Structure designations are as described in the legend to Fig. 1. On the right, the positions of free and complexed substrates are shown.



In addition to binding, we have shown that the F strand is required for efficient cleavage of the flap strand. The efficiency of cleavage correlated with the proximity of the 3`-end of the F strand to the elbow of the flap strand(4) . We were interested in determining whether these recessed F strands were cleaved inefficiently due to a reduced ability of FEN-1 to bind to these substrates. To test this, we carried out the binding reaction in the presence of unlabeled 5`-flap structures with various gap sizes between the F strand and the flap strand elbow (Fig. 4). We found that the ability of FEN-1 to bind to the flap structure efficiently was dependent upon the proximity of the F strand to the elbow of the flap strand. This result suggests that the inability of FEN-1 to efficiently cleave 5`-flap structures with large gaps between the F strand and the flap strand elbow is due, at least in part, to the failure of FEN-1 to bind to this substrate. Interestingly, FEN-1 binds to a 5nucleotide gap more efficiently than to a pseudo Y-structure. This indicates that the 5-nucleotide recessed F strand is capable of partially stabilizing FEN-1 binding, thereby suggesting that FEN-1 contacts the F strand at >5 nucleotides from the flap strand elbow.


Figure 4: Competition with 5`-flap structures containing recessed F strands. Binding reactions were carried out as described under ``Experimental Procedures'' and contained 40 ng (0.8 pmol) of purified FEN-1. In addition, the indicated amounts of the following nucleic acid competitors was present: lanes 1 and 2, no competitor; lanes 3-7, AI4/HJ46/HJ47; lanes 8-12, AI4/HJ46/HJ73; lanes 13-17, AI4/HJ46/HJ74; lanes 18-22, AI4/HJ46/HJ75; and lanes 23-27, AI4/HJ46. Note that the 5`-flap structure competitor was titrated from 2 to 32 ng, while all other competitors were titrated from 4 to 64 ng. The structures of the labeled substrate (SC1/SC3/SC5) and the unlabeled competitors are illustrated above and below the figure, respectively. Structure designations are as described in the legend to Fig. 1. On the right, the positions of free and complexed substrates are shown. nt, nucleotide.



We have demonstrated the importance of the F strand for the binding and cleavage of a 5`-flap structure by FEN-1. The exact role that the F strand plays in the recognition of this structure by FEN-1, however, is not clear. For example, does FEN-1 require a fully base-paired 3`-terminus located in juxtaposition to the elbow of the flap strand, or does FEN-1 simply require that the region near the elbow of the flap strand be double-stranded? To determine which of these two possibilities is correct, we designed a flap substrate that contained an F strand that had either 1 nucleotide or 10 nucleotides of extra sequence at the 3`-end. Thus, the base that contained the 3`-terminus was neither base-paired nor located in immediate juxtaposition to the elbow of the flap strand. This substrate, called a double flap structure, contained both a 5`- and a 3`-flap strand. Previously, we have shown that FEN-1 is not capable of cleaving 3`-flap strands(4) . Here, we were interested in determining whether a displaced 3`-flap strand can serve as an F strand to allow efficient binding to this structure by FEN-1. In the mobility shift assay, we found that FEN-1 could bind a double flap efficiently (Fig. 5A). Double Flap 1 and the 5`-flap structure competed 3-5-fold more efficiently than Double Flap 2, which, in turn, competed 2-fold more efficiently compared with the pseudo Y-structure. This indicates that both a 1nucleotide and a 10-nucleotide 3`-flap strand can serve as an F strand in the binding step of this reaction. Furthermore, this result indicates that the F strand is required to create a double-stranded region next to the elbow of the flap strand rather than to supply a base-paired 3`-terminus.


Figure 5: 3`-Flap strands can serve as an F strand. A, the ability of FEN-1 to bind to double flap structures was tested in the mobility shift assay. Binding reactions were carried out as described under ``Experimental Procedures'' and contained 40 ng (0.8 pmol) of purified FEN-1. In addition, the indicated amounts of the following nucleic acid competitors were present: lanes 1 and 2, no competitor; lanes 3-6, AI4/HJ46/HJ47; lanes 7-10, AI4/HJ46; lanes 11-14, AI4/HJ46/HJ77; and lanes 15-18, AI4/HJ46/HJ78. The structures of the labeled substrate (SC1/SC3/SC5) and the unlabeled competitors are illustrated above and below the figure, respectively. Structure designations are as described in the legend to Fig. 1. On the right, the positions of free and complexed substrates are shown. B, the ability of FEN-1 to cleave the 5`-flap strand of a double flap structure was tested in the standard flap endonuclease assay. Various amounts of FEN-1 were incubated with the labeled flap structures shown below their respective lanes. The nucleotide composition of each substrate structure is indicated above. Lanes1, 6, 11, and 16, 0 units of FEN-1; lanes2, 7, 12, and 17, 3 units of FEN-1; lanes3, 8, 13, and 18, 1 unit of FEN-1; lanes4, 9, 14, and 19, 0.33 units of FEN-1; lanes5, 10, 15, and 20, 0.11 units of FEN-1. The locations of substrate and product bands are indicated on the right. nts, nucleotides.



The ability of FEN-1 to bind to a double flap structure suggests that FEN-1 may be capable of cleaving the 5`-flap strand on this structure. To test this, we carried out the standard flap endonuclease assay. As determined previously(4) , FEN-1 cleaves the flap strand of a 5`-flap structure efficiently; however, it cleaves a pseudo Y-structure 100-fold less efficiently. When Double Flaps 1 and 2 were tested in this cleavage assay, we found that the 1- and 10-nucleotide 3`-flap strands served as an F strand, allowing FEN-1 to cleave the 5`-flap strand efficiently (Fig. 5B). Furthermore, cleavage of the double flap structures by FEN-1 was more efficient than that of the standard 5`-flap structure. The ability of FEN-1 to cleave the double flap structure raises important questions regarding the types of structures FEN-1 cleaves in vivo.

In addition to flap cleavage activity, FEN-1 has been shown to have a 5`-3`-exonuclease activity that is dependent upon double-stranded DNA. Like the flap endonuclease, the 5`-3`-exonuclease is stimulated by the presence of an F strand. Thus, the FEN-1 exonuclease is optimally active at a nick. To test the relative binding efficiency of FEN-1 on 5`-flap structures versus nicks, we compared the ability of these structures to compete in the mobility shift assay using a labeled flap substrate. We found that in this assay, a 5`-flap structure competed 2-4 times more efficiently than a nick structure (data not shown). In the cleavage assay, we found that FEN-1 cleaved flap structures severalfold more efficiently than nicks; however, there appears to be considerable variation among nicked DNA substrates. Substrate preference has been observed previously with CCA exonuclease (9) , an enzyme that we believe to be the same as FEN-1. The basis for variability in substrate preference is not known at this time.

The ability of nicked DNA to compete with FEN-1 binding to a 5`-flap structure suggests that we could use the mobility shift assay to dissect the important substrate features necessary for FEN-1 binding to DNA nicks. To test this, we labeled a nicked DNA substrate and carried out the mobility shift assay under standard binding and electrophoresis conditions. In this assay, FEN-1 did bind to a labeled nicked DNA substrate and retard its mobility on a native gel (Fig. 6, lanes 1 and 2). Upon addition of unlabeled nicked DNA, the shifted substrate was no longer detectable in the presence of an 80-fold molar excess of competitor. In contrast, titration of unlabeled double-stranded DNA lacking a nick but containing the same nucleotide sequence failed to compete FEN-1 away from the labeled substrate completely, even in the presence of a 250-fold molar excess. This indicates that FEN-1 is recognizing some component of the nicked DNA structure. Analysis of the individual contribution of the F strand and the 5`-recessed strand demonstrates that the F strand is important in the binding step of this reaction. This conclusion is consistent with the observation that the calf thymus 5`-3`-exonuclease, the bovine analog of mouse FEN-1, requires the F strand for efficient exonuclease activity(10) .


Figure 6: Important substrate elements required for efficient FEN-1 binding to nicked duplex DNA. Forty nanograms of purified FEN-1 (0.8 pmol) was incubated with 0.2 ng (8 fmol) of labeled nicked duplex DNA (AI4/CLH2/CLH3) and the indicated amounts of each unlabeled competitor. Lanes1 and 2, no competitor; lanes 3-6, AI4/CLH2/CLH3; lanes 7-10, AI4/CLH6; lanes 11-14, AI4/CLH2; lanes 15-18, AI4/CLH3. The structures of the substrate and competitors are shown schematically with designations as described in the legend to Fig. 1.




DISCUSSION

The experiments described here have identified the structural components of DNA flap structures that are recognized by FEN-1 in the binding portion of the cleavage reaction. The role of the F strand, in particular, appears to play a pivotal role in the binding and cleavage steps of this reaction. When this strand is absent, FEN-1 binds to the 5`-flap structure 3-fold less efficiently. Because cleavage of the pseudo Y-structure is reduced by 100-fold, it appears that FEN-1 also requires the F strand during the catalysis step of this reaction. In addition to the pseudo Y-structure, we found that FEN-1 binding to 5`-flap structures with recessed F strands decreased to an extent proportional to the gap length between the 3`-terminus of the F strand and the flap strand elbow. This effect correlated well with the reduction in FEN-1 cleavage using similar substrates reported previously(4) . Taken together, these results demonstrate the importance of the F strand in the binding portion (and probably the cleavage portion) of the nucleolytic reaction.

Recently, it has been reported that FEN-1, referred to as DNase IV, cleaves pseudo Y-structures, but not bubble structures(11) . In this study, flap structures were not analyzed. Although we have observed cleavage of pseudo Y-structures by FEN-1, cleavage was reduced by 20-100-fold compared with the flap structure(4) . In addition, we observed that while cleavage of flap structures with different sequences was equally efficient, the same substrates that lacked the F strand were cleaved with variable efficiency and at different cleavage sites. One explanation for this observation is that the single-stranded region of the F strand (see the legend to Fig. 1for nomenclature) snaps back to form a transient F strand near the elbow of the flap strand, allowing FEN-1 to cleave. One would expect that the efficiency of snap-back would increase proportional to the length of the single-stranded region of the F strand. In addition, one might expect snap-back within the single-stranded region of a bubble structure to be less efficient. Thus, it is possible that in cases where a low level of pseudo Y-structure cleavage is observed, a transient F strand may be present. Further experiments, including detailed kinetic analysis, must be carried out to elucidate the mechanism of FEN-1 cleavage.

Finally, the observation that FEN-1 cleaves the 5`-flap strand on a double flap structure raises an interesting question: do double flap structures form during normal cellular processes, and if so, is FEN-1 responsible for their resolution? Although a definitive answer is uncertain at this time, double flap structures have been hypothesized as intermediates in DNA end joining. Additionally, double flaps may form in some types of homologous recombination pathways that do not proceed through the classical Holliday junction. It is possible that FEN-1 resolves the 5`-flap and that some other enzyme cleaves the 3`-flap. It has been suggested that RAD1/RAD10 may be capable of cleaving 3`-flap and pseudo Y-structures(5) , and genetic evidence supports this hypothesis(12) . Recently, this has been definitively demonstrated(13) .

The ultimate answers regarding the details of FEN-1/flap structure interactions will come upon co-crystallization of this complex. In the meantime, many important questions can be answered regarding enzyme/substrate interactions using a combination of mutagenesis, cleavage assays, and binding assays including the mobility shift assay described here.


FOOTNOTES

*
This work was supported in part by National Institutes of Health grants (to M. R. L.) and by a grant from the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Cancer Research Institute Investigator and Scholar of the Leukemia Society of America. To whom correspondence should be addressed. Tel.: 314-362-4227; Fax: 314-362-8756; lieber{at}visar.wustl.edu.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.