Fen1 mutations that specifically disrupt its interaction with PCNA cause aneuploidy-associated cancer

DNA replication and repair are critical processes for all living organisms to ensure faithful duplication and transmission of genetic information. Flap endonuclease 1 (Fen1), a structure-specific nuclease, plays an important role in multiple DNA metabolic pathways and maintenance of genome stability. Human FEN1 mutations that impair its exonuclease activity have been linked to cancer development. FEN1 interacts with multiple proteins, including proliferation cell nuclear antigen (PCNA), to form various functional complexes. Interactions with these proteins are considered to be the key molecular mechanisms mediating FEN1's key biological functions. The current challenge is to experimentally demonstrate the biological consequence of a specific interaction without compromising other functions of a desired protein. To address this issue, we established a mutant mouse model harboring a FEN1 point mutation (F343A/F344A, FFAA), which specifically abolishes the FEN1/PCNA interaction. We show that the FFAA mutation causes defects in RNA primer removal and long-patch base excision repair, even in the heterozygous state, resulting in numerous DNA breaks. These breaks activate the G2/M checkpoint protein, Chk1, and induce near-tetraploid aneuploidy, commonly observed in human cancer, consequently elevating the transformation frequency. Consistent with this, inhibition of aneuploidy formation by a Chk1 inhibitor significantly suppressed the cellular transformation. WT/FFAA FEN1 mutant mice develop aneuploidy-associated cancer at a high frequency. Thus, this study establishes an exemplary case for investigating the biological significance of protein-protein interactions by knock-in of a point mutation rather than knock-out of a whole gene.     

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases. Both WRN and BLM bind to the extreme C-terminal 18 amino acid tail of FEN-1 that is adjacent to the PCNA binding site of FEN-1. The importance of the WRN/BLM physical interaction with the FEN-1 C-terminal tail was confirmed by functional interaction studies with catalytically active purified recombinant FEN-1 deletion mutant proteins that lack either the WRN/BLM binding site or the PCNA interaction site. The distinct binding sites of WRN and PCNA and their combined effect on FEN-1 nuclease activity suggest that they may coordinately act with FEN-1. WRN was shown to facilitate FEN-1 binding to its preferred double-flap substrate through its protein interaction with the FEN-1 C-terminal binding site. WRN retained its ability to physically bind and stimulate acetylated FEN-1 cleavage activity to the same extent as unacetylated FEN-1. These studies provide new insights to the interaction of WRN and BLM helicases with FEN-1, and how these interactions might be regulated with the PCNA–FEN-1 interaction during DNA replication and repair.     

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.     

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.     

Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair

Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.     

Two modes of FEN1 binding to PCNA regulated by DNA

The FEN1 nuclease functions during Okazaki fragment maturation in the eukaryotic cell. Like many other proliferating cell nuclear antigen (PCNA)-binding proteins, FEN1 interacts with the interdomain connector loop (IDCL) of PCNA, and PCNA greatly stimulates FEN1 activity. A yeast IDCL mutant pcna-79 (IL126,128AA) failed to interact with FEN-1, but, surprisingly, pcna-79 was still very active in stimulating FEN1 activity. In contrast, a C-terminal mutant pcna-90 (PK252,253AA) showed wild-type binding to FEN1 in solution, but poorly stimulated FEN1 activity. When PCNA was loaded onto a DNA substrate coupled to magnetic beads, it stabilized retention of FEN1 on the DNA. In this DNA-dependent binding assay, pcna-79 also stabilized retention of FEN1, but pcna-90 was inactive. Therefore, in the absence of DNA, FEN1 interacts with PCNA mainly through the IDCL. However, when PCNA encircles the DNA, the C-terminal domain of PCNA rather than its IDCL is important for binding FEN1. An FF-->GA mutation in the PCNA-interaction domain of FEN1 severely decreased both modes of interaction with PCNA and resulted in replication and repair defects in vivo.     

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.     

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.     

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.     

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.     

The FEN-1 family of structure-specific nucleases in eukaryotic dna replication,recombination and repair

Unlike the most well-characterized prokaryotic polymerase, E. Coli DNA pol I, none of the eukaryotic polymerases have their own 5′ to 3′ exonuclease domain for nick translation and Okazaki fragment processing. In eukaryotes, FEN-1 is an endo-and exonuclease that carries out this function independently of the polymerase molecules. Only seven nucleases have been cloned from multicellular eukaryotic cells. Among these, FEN-1 is intriguing because it has complex structural preferences; specifically, it cleaves at branched DNA structures. The cloning of FEN-1 permitted establishment of the first eukaryotic nuclease family, predicting that S. cerevisiae RAD2 (S. pombe Rad13) and its mammalian homolog, XPG, would have similar structural specficity. The FEN-1 nuclease family includes several similar enzymes encoded by bacteriophages. The crystal structures of two enzymes in the FEN-1 nuclease family have been solved and they provide a structural basis for the interesting steric requirements of FEN-1 substrates. Because of their unique structural specificities, FEN-1 and its family members have important roles in DNA replication, repair and, potentially, recombination. Recently, FEN-1 was found to specifically associate with PCNA, explaining some aspects of FEN-1 function during DNA replication and potentially in DNA repair.     

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.     

Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications

Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.     

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.     

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.     

PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins

In DNA replication, the leading strand is synthesized continuously, but lagging strand synthesis requires the complex, discontinuous synthesis of Okazaki fragments, and their subsequent joining. We have used a combination of in situ extraction and dual color photobleaching to compare the dynamic properties of three proteins essential for lagging strand synthesis: the polymerase clamp proliferating cell nuclear antigen (PCNA) and two proteins that bind to it, DNA Ligase I and Fen1. All three proteins are localized at replication foci (RF), but in contrast to PCNA, Ligase and Fen1 were readily extracted. Dual photobleaching combined with time overlays revealed a rapid exchange of Ligase and Fen1 at RF, which is consistent with de novo loading at every Okazaki fragment, while the slow recovery of PCNA mostly occurred at adjacent, newly assembled RF. These data indicate that PCNA works as a stationary loading platform that is reused for multiple Okazaki fragments, while PCNA binding proteins only transiently associate and are not stable components of the replication machinery.     

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.     

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.     

Genetic instability induced by overexpression of DNA ligase I in budding yeast

Recombination and microsatellite mutation in humans contribute to disorders including cancer and trinucleotide repeat (TNR) disease. TNR expansions in wild-type yeast may arise by flap ligation during lagging-strand replication. Here we show that overexpression of DNA ligase I (CDC9) increases the rates of TNR expansion, of TNR contraction, and of mitotic recombination. Surprisingly, this effect is observed with catalytically inactive forms of Cdc9p protein, but only if they possess a functional PCNA-binding site. Furthermore, in vitro analysis indicates that the interaction of PCNA with Cdc9p and Rad27p (Fen1) is mutually exclusive. Together our genetic and biochemical analysis suggests that, although DNA ligase I seals DNA nicks during replication, repair, and recombination, higher than normal levels can yield genetic instability by disrupting the normal interplay of PCNA with other proteins such as Fen1.     

Human flap endonuclease-1: conformational change upon binding to the flap DNA substrate and location of the Mg2+ binding site

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is a key enzyme in DNA replication and repair. FEN-1 recognizes the 5'-flap DNA structure and cleaves it, a specialized endonuclease function essential for the processing of Okazaki fragments during DNA replication and for the repair of 5'-end single-stranded tails from nicked double-stranded DNA substrates. Magnesium is a cofactor required for nuclease activity. We have used Fourier transform infrared (FTIR) spectroscopy to better understand how Mg2+ and flap DNA interact with human FEN-1. FTIR spectroscopy provides three fundamentally new insights into the structural changes induced by the interaction of FEN-1 with substrate DNA and Mg2+. First, FTIR difference spectra in the amide I vibrational band (1600-1700 cm(-1)) reveal a change in the secondary structure of FEN-1 induced by substrate DNA binding. Quantitative analysis of the FTIR spectra indicates a 4% increase in helicity upon DNA binding or about 14 residues converted from disordered to helical conformations. The observation that the residues are disordered without DNA strongly implicates the flexible loop region. The conversion to helix also suggests a mechanism for locking the flexible loop region around the bound DNA. This is the first direct experimental evidence for a binding mechanism that involves a secondary structural change of the protein. Second, in contrast with DNA binding, no change is observed in the secondary structure of FEN-1 upon Mg2+ binding to the wild type or to the noncleaving D181A mutant. Third, the FTIR results provide direct evidence (via the carboxylate ligand band at 1535 cm(-1)) that not only is D181 a ligand to Mg2+ in the human enzyme but Mg2+ binding does not occur in the D181A mutant which lacks this ligand.