Effect of Flap Modifications on Human FEN1 Cleavage

Bornarth, Carole J.; Ranalli, Tamara A.; Henricksen, Leigh A.; Wahl, and Alan F.; Bambara, Robert A.

doi:10.1021/bi991321u

Cited by 73 publications

(76 citation statements)

References 40 publications

(122 reference statements)

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“…However, some differences in the ratios of products generated by the wild type and mutants were observed, e.g., more of the 22-nt-long product is produced by the mutant nucleases when compared with the wild-type enzyme. In the latter case most of the radiolabeled products run as trimers or pentamers, whereas reactions catalyzed by the modified proteins produce predominantly endonucleolytic products (19,21, and 22 mers). The major cleavage product obtained with the 5Ј endlabeled 5OVH substrate arose from hydrolysis of the second base-paired nucleotide in the duplex region yielding a product 16 nt in length.…”

Section: Resultsmentioning

confidence: 99%

“…Bambara and coworkers (21) found that FEN1 is able to process substrates containing bulky adducts (even branched DNA structures) in the 5Ј tail. Our laboratory reported that T5 5Ј nuclease is able to digest double-stranded closed-circular DNA under conditions that promote duplex melting (20).…”

Section: Discussionmentioning

confidence: 99%

“…Single-stranded circular DNA (18,19) and double-stranded plasmid DNA can be cleaved under forcing conditions (20), implying that a 5Ј end is not an absolute requirement for activity. Bambara and coworkers (21) showed that flap endonuclease I (FEN1, a eukaryotic 5Ј nuclease) can effect endonucleolytic cleavage of substrates with 5Ј tails containing 2Ј-5Ј branch points (see Fig. 6, which is published as supporting information on the PNAS web site, www.pnas.org).…”

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confidence: 99%

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Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

Dervan

Feng²,

Patel³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Previous structural studies on native T5 5 nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5 overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter k cat appreciably. However, these mutant nucleases displayed significantly increased values for K d and Km. A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5 overhang suggests a better model for 5 nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.T he flap endonucleases, or 5Ј nucleases, are structure-specific endonucleases that also possess 5Ј-3Ј exonucleolytic activity. They are involved in processing substrates with 5Ј singlestranded tails such as those that arise during nick translation and replication and in some DNA damage-repair pathways (1, 2). These enzymes bind substrates containing a single-stranded 5Ј end and a duplex region such as 5Ј overhangs (5OVHs), 5Ј flaps, and pseudoY (Ps-Y) substrates (Fig. 1;. It has been suggested that the single-stranded 5Ј tail of such substrates threads through the 5Ј nuclease (4). Several prokaryotic and archaeal flap endonuclease structures have been solved (6-10). Because none of the reported structures contained bound DNA substrates, the precise mode of nucleic acid binding is unclear. These nucleases share significant primary sequence conservation as well as extensive structural similarities (11)(12)(13)(14). A central ␤-sheet carrying many of the core metal-binding ligands is a striking feature of all 5Ј-nuclease structures reported to date. This core region contains acidic residues responsible for binding the two divalent metal ions required for nuclease activity in vitro. A series of helices and loops comprise the enzyme's core. The most variable region seems to be that comprising a helical arch in T5 5Ј nuclease, the observation of which led us to develop a model of substrate binding (ref. 8; Fig. 2). In T5 5Ј nuclease, the archway delineates a hole able to accommodate a threaded single-stranded nucleic acid. Although a similar hole is present in the Methanococcus jannaschii homologue (9), this region often appears disordered (6, 7) or as a folded loop in the structure of the Pyrococcus furiosus homologue (10). The conceptual model for flap endonuclease-DNA binding has been widely accepted (1,7,9,10,15,16). Nevertheless, this model suffers f...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

Dervan

Feng²,

Patel³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…The model predominantly accepted for mammalian FEN1 proteins has long been the threading or tracking model, wherein the protein recognizes the free 5Ј-ssDNA tail and threads the ssDNA strand through the helical arch. Upon reaching the junction, the protein is hypothesized to cleave the appropriate scissile phosphate (62,66,67). Based on work with the 5Ј-3Ј exonuclease domain of Escherichia coli DNA polymerase I, Joyce and co-workers proposed that FEN1 proteins initially recognize or capture the two-way junction and then thread the helical arch with the ssDNA 5Ј-flap (40).…”

Section: The Addition Of Monovalent Salt Does Not Inhibit Hfen1-catalmentioning

confidence: 99%

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Finger¹,

Blanchard²,

Theimer

et al. 2009

Journal of Biological Chemistry

145

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Flap endonuclease 1 (FEN1) proteins, which are present in all kingdoms of life, catalyze the sequence-independent hydrolysis of the bifurcated nucleic acid intermediates formed during DNA replication and repair. How FEN1s have evolved to preferentially cleave flap structures is of great interest especially in light of studies wherein mice carrying a catalytically deficient FEN1 were predisposed to cancer. Structural studies of FEN1s from phage to human have shown that, although they share similar folds, the FEN1s of higher organisms contain a 3-extrahelical nucleotide (3-flap) binding pocket. When presented with 5-flap substrates having a 3-flap, archaeal and eukaryotic FEN1s display enhanced reaction rates and cleavage site specificity. To investigate the role of this interaction, a kinetic study of human FEN1 (hFEN1) employing well defined DNA substrates was conducted. The presence of a 3-flap on substrates reduced K m and increased multiple-and single turnover rates of endonucleolytic hydrolysis at near physiological salt concentrations. Exonucleolytic and fork-gap-endonucleolytic reactions were also stimulated by the presence of a 3-flap, and the absence of a 3-flap from a 5-flap substrate was more detrimental to hFEN1 activity than removal of the 5-flap or introduction of a hairpin into the 5-flap structure. hFEN1 reactions were predominantly rate-limited by product release regardless of the presence or absence of a 3-flap. Furthermore, the identity of the stable enzyme product species was deduced from inhibition studies to be the 5-phosphorylated product. Together the results indicate that the presence of a 3-flap is the critical feature for efficient hFEN1 substrate recognition and catalysis.In eukaryotic DNA replication and repair, various bifurcated nucleic acid structure intermediates are formed and must be processed by the appropriate nuclease. Two examples of biological processes that create bifurcated DNA intermediates are Okazaki fragment maturation (1, 2) and long patch excision repair (3). In both models, a polymerase executes strand-displacement synthesis to create a double-stranded DNA (dsDNA) 6 two-way junction from which a 5Ј-flap structure protrudes. The penultimate step of both pathways is the cleavage of this flap structure to create a nicked DNA that is then ligated. Because the bifurcated DNA structures that are formed in the aforementioned processes can theoretically occur anywhere in the genome, the nuclease associated with the cleavage of 5Ј-flap structures in eukaryotic cells, which is called flap endonuclease 1 (FEN1), must be capable of cleavage regardless of sequence. Therefore, FEN1 nucleases, which are found in all kingdoms of life (4), have evolved to recognize substrates based upon nucleic acid structure and strand polarity (5, 6).The Okazaki fragment maturation pathway of yeast has become a paradigm of eukaryotic lagging strand DNA synthesis. In the yeast model, bifurcated intermediates with large single-stranded DNA (ssDNA) 5Ј-flap structures are imprecisely cleaved by DNA2 ...

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“…FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. (Bornarth et al, 1999). Therefore, the investigation on the FEN-1 protein functions on CTG/CAG repeat DNA is crucial for understanding the mechanism of triplet repeat instability.…”

Section: Introductionmentioning

confidence: 99%

Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner

Lee

Park²

2002

Exp Mol Med

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Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.

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Effect of Flap Modifications on Human FEN1 Cleavage

Cited by 73 publications

References 40 publications

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner

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