Analysis of Human Flap Endonuclease 1 Mutants Reveals a Mechanism to Prevent Triplet Repeat Expansion
2003; Elsevier BV; Volume: 278; Issue: 16 Linguagem: Inglês
10.1074/jbc.m212061200
ISSN1083-351X
Autores Tópico(s)Cancer Genomics and Diagnostics
ResumoFlap endonuclease 1 (FEN1), involved in the joining of Okazaki fragments, has been proposed to restrain DNA repeat sequence expansion, a process associated with aging and disease. Here we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity. Introduction of these mutants into yeast led to sequence expansions. Reconstituting triplet repeat expansion in vitro, we previously found that DNA ligase I promotes expansion, but FEN1 prevents the ligation that forms expanded products. Here we show that among the intermediates that could generate sequence expansion, a bubble is necessary for ligation to produce the expansion product. Severe exonuclease defects in the mutant FEN1 suggested that the inability to degrade bubbles exonucleolytically leads to expansion. However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated. Instead, we propose that FEN1 suppresses sequence expansion by degrading flaps that equilibrate with bubbles, thereby reducing bubble concentration. In this way FEN1 employs endonuclease rather than exonuclease to prevent expansions. A model is presented describing the roles of DNA structure, DNA ligase I, and FEN1 in sequence expansion. Flap endonuclease 1 (FEN1), involved in the joining of Okazaki fragments, has been proposed to restrain DNA repeat sequence expansion, a process associated with aging and disease. Here we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity. Introduction of these mutants into yeast led to sequence expansions. Reconstituting triplet repeat expansion in vitro, we previously found that DNA ligase I promotes expansion, but FEN1 prevents the ligation that forms expanded products. Here we show that among the intermediates that could generate sequence expansion, a bubble is necessary for ligation to produce the expansion product. Severe exonuclease defects in the mutant FEN1 suggested that the inability to degrade bubbles exonucleolytically leads to expansion. However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated. Instead, we propose that FEN1 suppresses sequence expansion by degrading flaps that equilibrate with bubbles, thereby reducing bubble concentration. In this way FEN1 employs endonuclease rather than exonuclease to prevent expansions. A model is presented describing the roles of DNA structure, DNA ligase I, and FEN1 in sequence expansion. Repeats of simple sequences occur commonly in the human genome (1Debrauwere H. Gendrel C.G. Lechat S. Dutreix M. Biochimie (Paris). 1997; 79: 577-586Crossref PubMed Scopus (90) Google Scholar,2Paulson H.L. Fischbeck K.H. Annu. Rev. Neurosci. 1996; 19: 79-107Crossref PubMed Scopus (301) Google Scholar). They are highly polymorphic in populations, allowing them to be widely used as markers for gene mapping and medical purposes (3Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar). Within the last decade it has become more evident that these simple repeats can lead to a characteristic type of mutation that causes human diseases (3Sutherland G.R. Richards R.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3636-3641Crossref PubMed Scopus (301) Google Scholar). Many studies have shown that the expansion of dinucleotide repeats is associated with human cancers including those of colon, esophagus, stomach, and lung (4Thibodeau S.N. Bren G. Schaid D. Science. 1993; 260: 816-819Crossref PubMed Scopus (2812) Google Scholar, 5Ionov Y. Peinado M.A. Malkhosyan S. Shibata D. Perucho M. Nature. 1993; 363: 558-561Crossref PubMed Scopus (2416) Google Scholar, 6Mironov N.M. Aguelon M.A. Potapova G.I. Omori Y. Gorbunov O.V. Klimenkov A.A. Yamasaki H. Cancer Res. 1994; 54: 41-44PubMed Google Scholar, 7Merlo A. Mabry M. Gabrielson E. Vollmer R. Baylin S.B. Sidransky D. Cancer Res. 1994; 54: 2098-2101PubMed Google Scholar). The expansion of trinucleotide repeats (TNR) 1The abbreviations used are: TNRtrinucleotide repeatFEN1flap endonulease 1PCNAproliferating cell nuclear antigen5-FOA5-fluoro-orotic acidMMRmismatch repair is now known to be responsible for at least 15 hereditary neurological diseases in humans (8Mirkin S.M. Smirnova E.V. Nat. Genet. 2002; 31: 5-6Crossref PubMed Scopus (40) Google Scholar). However, among all the possible triplet repeats, only three of them are capable of expanding: (CAG)n·(CTG)n, (CGG)n·(CCG)n, and (GAA)n·(TTC)n. The propensity of these sequences to form secondary structures such as hairpins, tetraplexes, and triplexes is thought to inhibit the activities of DNA replication and repair proteins and lead to sequence expansion (9Gacy A.M. Goellner G. Juranic N. Macura S. McMurray C.T. Cell. 1995; 81: 533-540Abstract Full Text PDF PubMed Scopus (522) Google Scholar, 10Fry M. Loeb L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4950-4954Crossref PubMed Scopus (316) Google Scholar, 11Sakamoto N. Chastain P.D. Parniewski P. Ohshima K. Pandolfo M. Griffith J.D. Wells R.D. Mol. Cell. 1999; 3: 465-475Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). trinucleotide repeat flap endonulease 1 proliferating cell nuclear antigen 5-fluoro-orotic acid mismatch repair Several models have been proposed to explain the mechanism of TNR expansion. Some of them are based on DNA replication (12Jakupciak J.P. Wells R.D. J. Biol. Chem. 2000; 275: 40003-40013Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Others are derived from the mechanisms of recombination, including gene conversion and DNA repair (13Kovtun I.V. McMurray C.T. Nat. Genet. 2001; 27: 407-411Crossref PubMed Scopus (242) Google Scholar). Most current information relates to the DNA replication-based models. In the DNA slippage model, the DNA polymerase is forced to pause by the secondary structures formed within the TNR sequence. The delay in replication allows nascent Okazaki fragments to transiently dissociate and reanneal in misaligned configurations, creating hairpin and bubble structures. Elongation and ligation of adjacent Okazaki fragments with such structures produce a sequence expansion (14Wells R.D. J. Biol. Chem. 1996; 271: 2875-2878Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). The positive correlation among stabilities of secondary structures, inhibition of replication proteins, and propensity for TNR expansion supports the model (15Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). It is also consistent with the finding that both CTG and CGG repeats are prone to expand when they are replicated as the lagging strand (16Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 17Hirst M.C. White P.J. Nucleic Acids Res. 1998; 26: 2353-2358Crossref PubMed Scopus (51) Google Scholar). The sequence expansion model proposed by Gordenin, Kunkel, and Resnick (19Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar) is based on the fact that inefficient cleavage of a flap by flap endonuclease 1 (FEN1) mainly leads to DNA sequence duplication mutations (18Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). In this model, DNA strand displacement synthesis creates a TNR flap. Internal complimentarity of the repeats in the flap allow it to form various secondary structures such as foldbacks and hairpins. These structures inhibit FEN1 cleavage so that the enzyme fails to resolve them. Subsequently the unresolved structures reanneal to the template in a misaligned configuration to form a bubble intermediate. The bubble is ligated with the upstream Okazaki fragment by DNA ligase, generating triplet repeat expansion (19Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar, 20Henricksen L.A. Tom S. Liu Y. Bambara R.A. J. Biol. Chem. 2000; 275: 16420-16427Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). FEN1 is a structure-specific endo/exonuclease involved in removing initiator RNA primers on Okazaki fragments (21Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar, 22Ishimi Y. Claude A. Bullock P. Hurwitz J. J. Biol. Chem. 1988; 263: 19723-19733Abstract Full Text PDF PubMed Google Scholar, 23Robins P. Pappin D.J. Wood R.D. Lindahl T. J. Biol. Chem. 1994; 269: 28535-28538Abstract Full Text PDF PubMed Google Scholar, 24Turchi J.J. Huang L. Murante R.S. Kim Y. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9803-9807Crossref PubMed Scopus (169) Google Scholar). In addition, it has been proposed to participate in DNA long patch base excision repair (25Matsumoto Y. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 129-138Crossref PubMed Google Scholar, 26Matsumoto Y. Kim K. Hurwitz J. Gary R. Levin D.S. Tomkinson A.E. Park M.S. J. Biol. Chem. 1999; 274: 33703-33708Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 27DeMott M.S. Shen B. Park M.S. Bambara R.A. Zigman S. J. Biol. Chem. 1996; 271: 30068-30076Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The name of FEN1 derives from its preference for substrates with a primer having an unannealed 5′-flap (21Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (373) Google Scholar). The mechanism of RNA primer removal involves creation of a flap by strand displacement synthesis. By the current understanding of this process, part of the flap is removed by an endonuclease named Dna2 protein. The remainder of the flap is removed by FEN1, leaving a nick that can be ligated (28Bae S.H. Bae K.H. Kim J.A. Seo Y.S. Nature. 2001; 412: 456-461Crossref PubMed Scopus (284) Google Scholar). The importance of FEN1 in DNA replication and repair has been implicated by studies in vivo. In S. cerevisiae, the FEN1 homologue is called RAD27/RTH1 (29Reagan M.S. Pittenger C. Siede W. Friedberg E.C. J. Bacteriol. 1995; 177: 364-371Crossref PubMed Google Scholar). Therad27/RTH1Δ mutant displayed phenotypes indicative of defects in DNA replication and repair (29Reagan M.S. Pittenger C. Siede W. Friedberg E.C. J. Bacteriol. 1995; 177: 364-371Crossref PubMed Google Scholar, 30Vallen E.A. Cross F.R. Mol. Cell. Biol. 1995; 15: 4291-4302Crossref PubMed Scopus (95) Google Scholar). At a restrictive temperature (37 °C), the growth and cell division of therad27Δ mutant stop. Morphological examination of the mutant cells has shown that most of the cells arrest during division as two large partially divided cells, with a blocked nuclear division, indicating cell cycle arrest. This phenotype resembles that ofcdc2 mutants known to be defective in DNA replication. The mutant is highly sensitive to alkylating agents, such as methyl methanesulfonate, but moderately sensitive to x-ray and UV irradiation, suggesting that the enzyme is important in DNA base excision repair (29Reagan M.S. Pittenger C. Siede W. Friedberg E.C. J. Bacteriol. 1995; 177: 364-371Crossref PubMed Google Scholar, 31Sommers C.H. Miller E.J. Dujon B. Prakash S. Prakash L. J. Biol. Chem. 1995; 270: 4193-4196Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In mice, the absence of FEN1 function results in embryonic lethality, demonstrating that FEN1 is essential in mammals (32Kucherlapati M. Yang K. Kuraguchi M. Zhao J. Lia M. Heyer J. Kane M. Fan K. Russell R. Brown A. Kneitz B. Edelmann W. Kolodner R. Lipkin M. Kucherlapati R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9924-9929Crossref PubMed Scopus (213) Google Scholar). Knocking out one copy of each FEN1 and adenomatous polyposis coli genes in mice leads to progression of adenocarcinomas. These phenotypes arise because of the partial loss of FEN1 function in mammalian DNA replication and repair (32Kucherlapati M. Yang K. Kuraguchi M. Zhao J. Lia M. Heyer J. Kane M. Fan K. Russell R. Brown A. Kneitz B. Edelmann W. Kolodner R. Lipkin M. Kucherlapati R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9924-9929Crossref PubMed Scopus (213) Google Scholar). FEN1 activity is also critical in maintaining genome stability. Genetic studies have shown that functional defects in the 5′ to 3′ exonuclease domain of Escherichia coli DNA polymerase I, a homologue of mammalian FEN1 and Saccharomyces cerevisiae RAD27, enhances dinucleotide repeat expansion and spontaneous mutation (33Morel P. Reverdy C. Michel B. Ehrlich S.D. Cassuto E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10003-10008Crossref PubMed Scopus (42) Google Scholar, 34Nagata Y. Mashimo K. Kawata M. Yamamoto K. Genetics. 2002; 160: 13-23PubMed Google Scholar). Most of these mutations are caused by sequence duplications. The rad27Δ mutant also allows frequent repeat sequence expansion. The CAG and CGG repeat sequences associated with human neurogenetic diseases are readily expanded in therad27Δ mutant strain (35Schweitzer J.K. Livingston D.M. Hum. Mol. Genet. 1998; 7: 69-74Crossref PubMed Scopus (164) Google Scholar, 36White P.J. Borts R.H. Hirst M.C. Mol. Cell. Biol. 1999; 19: 5675-5684Crossref PubMed Scopus (85) Google Scholar). As with its bacterial counterpart, the deletion of RAD27 increases spontaneous forward mutation frequency by about 50-fold (18Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Even partially defective rad27 mutant strains show enhanced forward mutation frequency (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). Interestingly, most of the forward mutations resulting from the duplications of small direct repeats are primarily due to the increase in repeat length (18Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). These facts suggest that an unresolved flap resulting from a FEN1 functional defect reanneals to its template and forms a bubble intermediate that generates sequence expansion. In vitro studies have been performed to further examine the role of FEN1 in TNR expansion (20Henricksen L.A. Tom S. Liu Y. Bambara R.A. J. Biol. Chem. 2000; 275: 16420-16427Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These used various secondary structures that simulate the foldback and hairpin intermediates that are frequently found in TNR flaps (20Henricksen L.A. Tom S. Liu Y. Bambara R.A. J. Biol. Chem. 2000; 275: 16420-16427Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 38Spiro C. Pelletier R. Rolfsmeier M.L. Dixon M.J. Lahue R.S. Gupta G. Park M.S. Chen X. Mariappan S.V. McMurray C.T. Mol. Cell. 1999; 4: 1079-1085Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The results demonstrated that both foldback and hairpin flap structures inhibit FEN1 activity. Proliferating cell nuclear antigen (PCNA), a known stimulator of FEN1, and replication protein A both failed to assist FEN1 to resolve these structures (20Henricksen L.A. Tom S. Liu Y. Bambara R.A. J. Biol. Chem. 2000; 275: 16420-16427Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 39Wu X. Li J. Li X. Hsieh C.L. Burgers P.M. Lieber M.R. Nucleic Acids Res. 1996; 24: 2036-2043Crossref PubMed Scopus (198) Google Scholar, 40Tom S. Henricksen L.A. Bambara R.A. J. Biol. Chem. 2000; 275: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Recently, reconstituting expansion in vitro with purified replication and repair proteins and the substrates containing CTG repeats, we observed progressive inhibition of CTG repeat expansion by increasing amounts of FEN1 (41Henricksen L.A. Veeraraghavan J. Chafin D.R. Bambara R.A. J. Biol. Chem. 2002; 277: 22361-22369Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Overall these results suggest that secondary structures in TNR flaps resist the mechanism by which FEN1 suppresses expansions. However, in vivo, wild type FEN1 is highly effective at inhibiting expansion. In addition, FEN1 is involved in suppressing recombination. Therad27Δ mutant displays high recombination rates (18Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 30Vallen E.A. Cross F.R. Mol. Cell. Biol. 1995; 15: 4291-4302Crossref PubMed Scopus (95) Google Scholar,42Gary R. Park M.S. Nolan J.P. Cornelius H.L. Kozyreva O.G. Tran H.T. Lobachev K.S. Resnick M.A. Gordenin D.A. Mol. Cell. Biol. 1999; 19: 5373-5382Crossref PubMed Scopus (91) Google Scholar). One possible mechanism is that the unresolved flaps inrad27 mutant strains accumulate and further misalign into loops and nicks. These flap and loop structures, if not removed by other repair proteins, are susceptible to double-strand breaks. The presence of such breaks is known to promote recombination (18Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). In fact, RAD27 has been found to be essential for viability of yeast strains having a defective double-strand break repair function (18Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Furthermore, fragmented DNA was identified in rad27mutant strains (30Vallen E.A. Cross F.R. Mol. Cell. Biol. 1995; 15: 4291-4302Crossref PubMed Scopus (95) Google Scholar). The observations indicate that production of double-strand DNA breaks is likely a mechanism that leads to increased DNA recombination in rad27 mutants, presumably because of the failure of resolving flap structures in these strains. Another consideration is that the persistence of flap and loop structures expected in rad27 mutants provides single-stranded regions that could serve as strand invasion sites for recombination. Therefore, one can readily envision how prompt resolution of the FEN1 flap substrate would prevent accumulation of recombinogenic lesions. A recent study has found that expression of human FEN1 in yeast restricts recombination between short sequences, implying that the nuclease participates the removal of 5′ overhangs that mediate short sequence recombination (43Negritto M.C. Qiu J. Ratay D.O. Shen B. Bailis A.M. Mol. Cell. Biol. 2001; 21: 2349-2358Crossref PubMed Scopus (44) Google Scholar). FEN1 is a highly conserved enzyme. Its homologues have been identified from bacteriophage, bacteria, archea, yeast, and mammals including mouse and human (44Shen B. Qiu J. Hosfield D. Tainer J.A. Trends Biochem. Sci. 1998; 23: 171-173Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Rad27p, the FEN1 homologue from S. cerevisiae, is highly homologous with human FEN1. Among the amino acids of the two FEN1 homologues, 79% are conserved, and 61% are identical (44Shen B. Qiu J. Hosfield D. Tainer J.A. Trends Biochem. Sci. 1998; 23: 171-173Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In addition, human FEN1 can complement most of the phenotypes resulting from the rad27Δ mutation in yeast demonstrating functional conservation (45Greene A.L. Snipe J.R. Gordenin D.A. Resnick M.A. Hum. Mol. Genet. 1999; 8: 2263-2273Crossref PubMed Scopus (39) Google Scholar). This makes therad27Δ strain a good model system to test the biological properties of human FEN1 mutants in vivo (45Greene A.L. Snipe J.R. Gordenin D.A. Resnick M.A. Hum. Mol. Genet. 1999; 8: 2263-2273Crossref PubMed Scopus (39) Google Scholar). Previously, we characterized two RAD27 mutant alleles,rad27-G67S and rad27-G240D isolated by genetic screening (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). These mutations are located on two highly conserved glycines (Gly67 and Gly240) within seven different species including bacteriophage, bacteria, yeast, mouse, and human (44Shen B. Qiu J. Hosfield D. Tainer J.A. Trends Biochem. Sci. 1998; 23: 171-173Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Both mutant strains exhibit a high frequency of dinucleotide repeat expansion and spontaneous mutation. In vitro biochemical characterization has shown that both mutant proteins have reduced endonucleolytic activity, have a severe exonucleolytic defect, and failed to degrade bubbles (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). These results lead us to propose that loss of exonuclease function of FEN1 allows sequence expansions. Here we explore whether human FEN1 employs exonuclease to suppress sequence expansion to further understand the roles of this enzyme in suppressing TNR expansion directly associated with human diseases. We took advantage of high homology and functional conservation between Rad27 and FEN1 proteins to make point mutations in the analogous glycines (Gly66 and Gly242) of human FEN1. We show that the mutations cause defects that lead to sequence expansion when the human enzyme is assayed in yeast. However, our study suggests that FEN1 utilizes its endonuclease rather than exonuclease activity to suppress the sequence expansion, leading us to revise our previous hypothesis. Our results also have provided refinements to the expansion model proposed by Gordenin, Kunkel, and Resnick (19Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar). Based on this model, we suggest the pathways for generating triplet repeat expansion when FEN1 activity is compromised during DNA replication and roles of DNA structure, FEN1, and DNA ligase I in the process. Yeast rad27Δ strains, EAY614 (MATa leuΔ ura3-25 lys2BglII trp1Δ 63 his3Δ rad27::HIS3) and EAY615 (MATα leuΔ ura3-25 lys2BglII trp1Δ63 his3Δ rad27::HIS3) were obtained from Dr. Eric Alani at Cornell University and were grown in yeast extract-peptone-dextrose medium. The plasmid pRS315 (yeast centromere plasmid containing LEU2 selectable marker) and pSH44 ((GT)16G-URA3-ARSH4 CEN6 TRP1) were generous gifts from Dr. Fred Sherman at University of Rochester and Dr. Eric Alani, respectively. The strains bearing either RAD27or human FEN1 were grown in minimal selective medium (46Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 171-174Google Scholar). 5-Fluoro-orotic acid (5-FOA) was from Zymo Research (Orange, CA) and U.S. Biologicals (Swampscott, MA), respectively, and used as previously described (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). Radionucleotides [γ-32P]ATP (3000 Ci/mmol) and [α-32P]dCTP (3000 or 6000 Ci/mmol) were obtained from PerkinElmer Life Sciences. T4 polynucleotide kinase and Klenow fragment of DNA polymerase I (labeling grade) were from Roche Diagnostics. All of the other reagents were purchased from Sigma-Aldrich and were analytical grade. A protein expression plasmid, FCH-pET28b, containing wild type human FEN1 open reading frame (47Bornarth C.J. Ranalli T.A. Henricksen L.A. Wahl A.F. Bambara R.A. Biochemistry. 1999; 38: 13347-13354Crossref PubMed Scopus (69) Google Scholar) was utilized as a template for creating two point mutations, G66S and G242D in the protein coding region. PCR-based site-directed mutagenesis was performed using a QuikChangeTM site-directed mutagenesis kit. Oligonucleotides 5′-CCAGCCACCTGATGAGCATGTTCTACCGC-3′ and 5′-GCGGTAGAACATGCTCATCAGGTGGCTGG-3′ were used to generate the G66S mutation, and 5′-GTATCCGGGGTATTGACCCCAAGCGGGCTGTG-3′ and 5′-CACAGCCCGCTTGGGGTCAATACCCCGGATAC-3′ were used to generate the G242D mutation. The substituted codons are denoted in bold type for both primer sets, and the mutations were verified by DNA sequencing. To construct a plasmid that can constitutively express wild type and mutant human FEN1 in yeast, a standard PCR method was performed to generate fragments ofADH1 promoter and terminator using pGADT7 (Clontech, Palo Alto, CA) plasmid as a source of template sequences. Subsequently, ADH1 promoter was inserted into SacI and XbaI sites of pRS315, andADH1 terminator was inserted into XhoI andApaI sites of the plasmid. The encoding regions of wild type and mutant human FEN1 were amplified by PCR from plasmids FEN1-pET28b,fen1-G66S-pET28b, and fen1-G242D-pET28b as described above using the oligonucleotides 5′-GTCTAGAAAGCATATGGGAATTCAAGGCCTGGCC-3′ and 5′-ACGCTCGAGTTATTTTCCCCTTTTAAACTTCCCTGC-3′. TheRAD27 encoding region was amplified from plasmidRAD27-pET24b (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar) by PCR using the oligonucleotides 5′-TCTAGAAAGATGGGTATTAAAGGTTTGAATGC-3′, 5′-ACGCTCGAGTCATCTTCTTCCCTTTGTGACTTTATTC-3′. These PCR primers created XbaI and XhoI sites as underlined in the primers in the PCR products that subsequently were inserted in between ADH1 promoter and terminator to generate plasmids pYYL1 (RAD27-pRS315-ADH1promoter-terminator), pYYL4 (FEN1-pRS315-ADH1promoter-terminator), pYYL5 (fen1-G66S-pRS315-ADH1 promoter-terminator), and pYYL6 (fen1-G242D-pRS315-ADH1promoter-terminator). These plasmids possessing a centromere and aLEU2 selectable marker put either wild type or mutant FEN1 under the control of the ADH1 promoter and terminator. The plasmids were subsequently introduced into yeast EAY614 strain for expression of mutant forms of FEN1 to measure their effects on repeat tract instability. This created strains YL1 (RAD27), YL4 (FEN1), YL5 (fen1-G66S), and YL6 (fen1-G242D). The strains were subsequently transformed by an indicator plasmid, pSH44, for measuring dinucleotide repeat expansion. In pSH44, an in-frame poly(GT)16G tract is inserted upstream of the URA3 gene (48Henderson S.T. Petes T.D. Genetics. 1993; 134: 57-62Crossref PubMed Google Scholar), resulting in a Ura+ phenotype. The poly(GT)16 repeat expansions resulting from FEN1 functional defects can create the frameshifts that disrupt the URA3 gene product leading to 5-FOA resistance in the strains having pSH44. The sequence expansion rate (see Table II) was calculated from frequency of 5-FOA-resistant colonies by utilizing the method of the median (49Lea D.E. Coulson C.A. J. Genet. 1949; 49: 264-285Crossref PubMed Scopus (1092) Google Scholar). The frequency data were obtained by counting the number of surviving cells of a million (106) either wild type or mutant cells plated on the minimal selective medium containing 5-FOA. The experimental procedure was the same as described before (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). Previously, we found that all of the frameshifts in poly(GT)16G tract of pSH44 created byrad27Δ, rad27-G67S, and rad27-G240Dmutations are sequence expansions (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). Thus, the frameshift events of poly(GT)16G occurring in FEN1 mutant strains represent the sequence expansions created by FEN1 mutations. The genetic data shown in Table II were statistically analyzed using the Mann-Whitney test, where p values of <0.05 are considered significant (50Pfaffenberger R.C. Peterson J.H. Statitical Methods for Business and Economics. Richard D. Irwin Inc., Homewood, IL1981: 665-668Google Scholar).Table IIRate of dinucleotide repeat expansion in wild type and mutant FEN1 strainsStrainAverage rate of dinucleotide repeat expansion (10−6)Relative ratioRAD271.11FEN13.03rad27Δ5046fen1-G66S2725fen1-G242D3230 Open table in a new tab Recombinant wild type and mutant FEN1 were expressed in E. coli BL21(DE3) strain utilizing pET-28b containing either wild type or mutant FEN1 encoding region, which generates a C-terminal His-tagged FEN1 as described before (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). The pellet was resuspended in lysis buffer, and bacterial cells were lysed by three passes through a French press. The cell lysate was subsequently sedimented for 30 min at 30,000 ×g. The supernatant was loaded onto a 10-ml nickel resin column (Qiagen, Inc.), and the proteins were purified using a fast protein liquid chromatography system (Amersham Biosciences) according to the procedure described by Xie et al. (37Xie Y. Liu Y. Argueso J.L. Henricksen L.A. Kao H.I. Bambara R.A. Alani E. Mol. Cell. Biol. 2001; 21: 4889-4899Crossref PubMed Scopus (57) Google Scholar). The peak fractions were pooled and subsequently loaded onto a Mono S column (Amersham Biosciences) at 0.5 ml/min. The column was washed with three volumes of HI buffer (30 mm HEPES and 0.5% inositol) with 30 mm KCl, and the protein was eluted with a gradient of HI buffer with 200–500 mm KCl. The peak fractions were divided into aliquots a
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