Cleavage of Substrates with Mismatched Nucleotides by Flap Endonuclease-1
1999; Elsevier BV; Volume: 274; Issue: 21 Linguagem: Inglês
10.1074/jbc.274.21.14602
ISSN1083-351X
AutoresJeffrey A. Rumbaugh, Leigh A. Henricksen, Michael S. DeMott, Robert A. Bambara,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoFlap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5′-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase α/primase. Because the fidelity of DNA polymerase α is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5′-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5′-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5′-end of the fragment. Flap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5′-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase α/primase. Because the fidelity of DNA polymerase α is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5′-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5′-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5′-end of the fragment. Mammalian DNA replication proceeds by two distinct processes occurring at replication forks (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman & Co., New York1992Google Scholar). The leading strand is made as a large continuous segment, whereas the lagging strand is synthesized as a series of discontinuous segments called Okazaki fragments. Each segment is individually initiated by a RNA primer. Later, these primers are removed and replaced with DNA, and the fragments are joined into one continuous strand. DNA polymerase α/primase synthesizes every RNA primer (2Wang T.S. Annu. Rev. Biochem. 1991; 60: 513-552Crossref PubMed Scopus (451) Google Scholar, 3Tsurimoto T. Melendy T. Stillman B. Nature. 1990; 346: 534-539Crossref PubMed Scopus (319) Google Scholar) and the adjacent 10–20 nucleotides of DNA (4Nethanel T. Zlotkin T. Kaufmann G. J. Virol. 1992; 66: 6634-6640Crossref PubMed Google Scholar). It is then replaced by either DNA polymerase δ or ε via a process called polymerase switching (5Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1961-1968Abstract Full Text PDF PubMed Google Scholar, 6Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (496) Google Scholar, 7Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar). DNA polymerase α lacks the proofreading 3′-5′ exonuclease activity present in polymerases δ and ε (2Wang T.S. Annu. Rev. Biochem. 1991; 60: 513-552Crossref PubMed Scopus (451) Google Scholar). Because DNA polymerase α has no mechanism to remove erroneously inserted nucleotides, it can continue synthesis only by mismatch extension. DNA polymerase α incorporates an incorrect nucleotide at a rate of approximately 1 in every 10,000 nucleotides, whereas the error rate of polymerases δ and ε is at most 1 in 50,000–200,000 nucleotides (8Kunkel T.A. Bioessays. 1992; 14: 303-308Crossref PubMed Scopus (89) Google Scholar). Initiator RNA removal is accomplished by a eukaryotic 5′ to 3′ exonuclease/endonuclease before Okazaki fragment joining (9Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Most frequently called flap endonuclease-1 (FEN1), 1The abbreviations used are: FEN1, flap endonuclease-1. 1The abbreviations used are: FEN1, flap endonuclease-1. this enzyme is also known as RAD2 homologue nuclease (10Murante R.S. Huang L. Turchi J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 11Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (372) Google Scholar, 12Murray J.M. Tavassoli M. Al-Harithy R. Sheldrick K.S. Lehmann A.R. Carr A.M. Watts F.Z. Mol. Cell. Biol. 1994; 14: 4878-4888Crossref PubMed Scopus (145) Google Scholar, 13Hiraoka L.R. Harrington J.J. Gerhard D.S. Lieber M.R. Hsieh C.L. Genomics. 1995; 25: 220-225Crossref PubMed Scopus (63) Google Scholar, 14Sommers 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, 15Reagan M.S. Pittenger C. Siede W. Friedberg E.C. J. Bacteriol. 1995; 177: 364-371Crossref PubMed Google Scholar). In one proposed pathway, RNase HI-directed cleavage removes almost the entire RNA primer but leaves a single RNA residue. FEN1 exonucleolytically removes this last ribonucleotide (16Turchi 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 (168) Google Scholar). However, genetic evidence suggests that alternative pathways exist. Yeast cells are still viable after deletion of the gene that accounts for 75% of RNase H activity (17Frank P. Reiter C.B. Wintersberger U. FEBS Lett. 1998; 421: 23-26Crossref PubMed Scopus (43) Google Scholar). In a second proposed pathway, FEN1 alone removes the initiator RNA (18Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) by its favored endonucleolytic activity (10Murante R.S. Huang L. Turchi J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 19Murante R.S. Rust L. Bambara R.A. J. Biol. Chem. 1995; 270: 30377-30383Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). FEN1 endonucleolytic cleavage requires that the substrate has a downstream primer with an unannealed 5′-tail, or flap. FEN1 appears to recognize the 5′-end, slide over the entire length of the tail, and cleave near the junction of the tail with the template. This releases the tail as an intact segment (10Murante R.S. Huang L. Turchi J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 19Murante R.S. Rust L. Bambara R.A. J. Biol. Chem. 1995; 270: 30377-30383Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Sometimes, the presence of an upstream primer is also required for FEN1 activity. In fact, for both exonucleolytic and endonucleolytic substrates, the upstream primer is often stimulatory (10Murante R.S. Huang L. Turchi J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 20Siegal G. Turchi J.J. Myers T.W. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9377-9381Crossref PubMed Scopus (61) Google Scholar, 21Turchi J.J. Bambara R.A. J. Biol. Chem. 1993; 268: 15136-15141Abstract Full Text PDF PubMed Google Scholar) but can sometimes be inhibitory (18Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 22Huang L. Rumbaugh J.A. Murante R.S. Lin R.J.R. Rust L. Bambara R.A. Biochemistry. 1996; 35: 9266-9277Crossref PubMed Scopus (31) Google Scholar). For endonucleolytic removal, the RNA must be within a displaced tail. An upstream primer may or may not be present, as appropriate for the particular cleavage site. Displacement of the RNA may be accomplished by a DNA helicase with or without DNA polymerase-directed displacement synthesis from an upstream fragment (18Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 23Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 1995; 270: 26766-26769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Depending on how far the tail is displaced, FEN1 can cleave within the RNA, at the RNA-DNA junction, or within the DNA beyond the RNA (18Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). FEN1 may also participate in DNA repair. It is a member of the RAD2 family of repair nucleases (24Lehmann A.R. Mutat. Res. 1996; 363: 147-161Crossref PubMed Scopus (31) Google Scholar). This family includes XPG in mammals, RAD2 in Saccharomyces cerevisiae, and RAD13 inSchizosaccharomyces pombe. These homologous nucleases are important components of the nucleotide excision repair pathway and are responsible for the incision 3′ to the damage site (24Lehmann A.R. Mutat. Res. 1996; 363: 147-161Crossref PubMed Scopus (31) Google Scholar). FEN1 is homologous to the family members called RAD27 or RAD2 homologue inS. cerevisiae and RAD2 in S. pombe. These family members are smaller than the XPG nucleases and are believed to function in other repair processes (24Lehmann A.R. Mutat. Res. 1996; 363: 147-161Crossref PubMed Scopus (31) Google Scholar). A third class of members includes exonuclease I of S. pombe, for which genetic studies suggest a role in mismatch repair (24Lehmann A.R. Mutat. Res. 1996; 363: 147-161Crossref PubMed Scopus (31) Google Scholar). The FEN1 class enzymes are believed to participate in base excision repair because the null mutant in yeast is sensitive to methylmethane sulfonate (14Sommers 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, 15Reagan M.S. Pittenger C. Siede W. Friedberg E.C. J. Bacteriol. 1995; 177: 364-371Crossref PubMed Google Scholar). In vitro, FEN1 can remove abasic sites (25DeMott 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) and can function in reconstitutions of base excision repair using purified enzymes (26Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (660) Google Scholar, 27Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 28DeMott M.S. Zigman S. Bambara R.A. J. Biol. Chem. 1998; 273: 27492-27498Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). FEN1 can also remove a more diverse group of flap adducts such as cisplatin derivatives (29Barnes C.J. Wahl A.F. Shen B. Park M.S. Bambara R.A. J. Biol. Chem. 1996; 271: 29624-29631Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Additionally, it may also participate in the removal of monoribonucleotides embedded in chromosomal DNA (30Rumbaugh J.A. Murante R.S. Shi S. Bambara R.A. J. Biol. Chem. 1997; 272: 22591-22599Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Finally, FEN1 has been implicated as a part of the mismatch repair pathway (31Johnson R.E. Kovvali G.K. Prakash L. Prakash S. Science. 1995; 269: 238-240Crossref PubMed Scopus (194) Google Scholar). In the current report, we provide evidence that FEN1 participates in the repair of mismatches produced during the priming and synthesis of Okazaki fragments. We show that a single mismatch in an otherwise fully annealed DNA primer changes the pattern of FEN1 cleavage. Typically, on a fully annealed substrate, FEN1 will exonucleolytically cleave the 5′ most nucleotide. We find that a single nucleotide mismatch up to 15 nucleotides in from the 5′-end promotes endonucleolytic cleavages. This constitutes a 5′ proofreading process in which the mismatch promotes the nuclease action that leads to its removal. Unlabeled nucleotides were purchased from Amersham Pharmacia Biotech, and [γ-32P]ATP (3,000 or 6,000 mCi/mmol) and [α-32P]dATP (3,000 mCi/mmol) were from NEN Life Science Products. Oligonucleotides were synthesized by Genosys Biotechnologies (The Woodlands, TX). T4 polynucleotide kinase and SequenaseTM version 2.0 were obtained from U. S. Biochemical Corp. RNase inhibitor and snake venom phosphodiesterase were from Roche Molecular Biochemicals. All other reagents were from Sigma Chemical Co. Recombinant human FEN1 was purified as described previously (18Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29Barnes C.J. Wahl A.F. Shen B. Park M.S. Bambara R.A. J. Biol. Chem. 1996; 271: 29624-29631Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The final preparation was >95% pure as determined by silver-stained SDS-polyacrylamide gel electrophoresis. The final specific activity was 65,000 units/mg, with 1 unit defined as the amount of nuclease required to exonucleolytically cleave 4,000 fmol in 30 min at 37 °C of a standard exonucleolytic test substrate consisting of a downstream primer (5′-TTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA-3′) annealed to a template (3′-GAGTGATTTCCCTTGTTTTCGAACGTACGGACGTCCAGCTGAGATCTCCTAGGGGCCCATT-5′). The sequences of the primers and the structures of the substrates used in this study are described in Table I and in the figure legends. TableI depicts template T1 and the downstream primers used in this study. The primers are divided into four groups, depending on the location of the 5′ most nucleotide; the 5′-end of all primers in a given group is located at the same place on the template. The first digit of the primer name indicates the group: 1, 2, 3, or 4. To generate a mismatch, each primer has a single base change that forms a single mismatch with the template, T1. The remaining digits in the name indicate the distance of this mismatch in nucleotides from the 5′-end of the primer. For all experiments, the substrate name reflects the use of a specific downstream primer. For example, substrate 2.11 indicates that a primer from group 2 was annealed to the template to create a mismatch at position 11. Control substrates 1.0, 2.0, and 4.0 have no mismatch. Mismatches were made so that nucleotides were paired with themselves (e.g. G was paired with G). The 5′-end of the template extends beyond the downstream primer to permit 3′-end labeling. In substrates 1.T and 1.A, the mismatch is not internal but is, in fact, the 5′ most nucleotide, which is mispaired as T-T or A-A, respectively. Substrate 3.6 uses a primer that lacks the three 5′ most nucleotides of the primer in substrate 2.9. Thus, both the location of the 5′-end and the position of mismatch relative to the 5′-end have changed. Substrates made from primers in group 4 are similar to those made from primers in group 2, except that 25 nucleotides were added to the 5′-end to form an unannealed 5′-tail, the sequence of which is shown in TableI.Table IOligonucleotide sequencesDownstream primers (5′-3′)1.0(35-mer)ATCCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA1.2aUnderlined nucleotides indicate the position of a mismatched base pair.(35-mer)AACCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA1.3(35-mer)ATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA1.TbSubstrate 1.A uses primer 1.0 with template T2 (which differs from T1) so that the 5′ most nucleotide of the primer forms an A-A mismatch.(35-mer)TTCCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA2.0(39-mer)TTGCATCCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA2.9(38-mer)TTGCATCCGTGCAGGTCGACTCTAGAGGATCCCCGGGT2.11(38-mer)TTGCATCCCTCCAGGTCGACTCTAGAGGATCCCCGGGT2.13(38-mer)TTGCATCCCTGCTGGTCGACTCTAGAGGATCCCCGGGT2.15(38-mer)TTGCATCCCTGCAGCTCGACTCTAGAGGATCCCCGGGT3.6(36-mer)CATCCGTGCAGGTCGACTCTAGAGGATCCCCGGGTA4.Xc25 nucleotides added to the 5′-end to substrates 2.0, 2.7, and 2.9 to generate primers 4.0, 4.7, and 4.9, respectively, are shown.CGTACGGACGTAGAGCTGTTTCCAAUpstream primer (5′-3′)UP(25-mer)TAAAGGGAACAAAAGCTTGCATCCCTemplate (3′-5′)T1dThe underlined nucleotide in the template sequence is the annealing position of the 5′ nucleotide of primers in group 1; the boldfaced nucleotide is the annealing position of the 5′ nucleotide of primers in group 2.(61-mer)GAGTGATTTCCCTTGTTTTCGAACGTAGGGACGTCCAGCTGAGATCTCCTAGGGGCCCATTa Underlined nucleotides indicate the position of a mismatched base pair.b Substrate 1.A uses primer 1.0 with template T2 (which differs from T1) so that the 5′ most nucleotide of the primer forms an A-A mismatch.c 25 nucleotides added to the 5′-end to substrates 2.0, 2.7, and 2.9 to generate primers 4.0, 4.7, and 4.9, respectively, are shown.d The underlined nucleotide in the template sequence is the annealing position of the 5′ nucleotide of primers in group 1; the boldfaced nucleotide is the annealing position of the 5′ nucleotide of primers in group 2. Open table in a new tab For 5′-end labeling, downstream primers were incubated with T4 polynucleotide kinase and [γ-32P]ATP according to the manufacturer's protocol and then annealed to the template in annealing buffer (50 mm Tris, pH 8, 10 mm magnesium acetate, 50 mm NaCl, and 1 mm dithiothreitol). For 3′-end-labeled substrates, downstream primers were first phosphorylated using T4 polynucleotide kinase and ATP. Then, after annealing to template T1, they were extended by addition to the 3′ terminus using [α-32P]dATP and SequenaseTMversion 2.0. Substrates were isolated via 12% native gel electrophoresis (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar), eluted from the gel using elution buffer (0.5m ammonium acetate, 0.1% SDS, and 0.1 mmEDTA), precipitated in ethanol, and resuspended in annealing buffer or TE, pH 8.0. Assays to monitor cleavage by FEN1 were performed in FEN1 buffer containing 60 mm BisTris (pH 7.0), 5% glycerol, 0.1 mg/ml bovine serum albumin, 5 mmβ-mercaptoethanol, 10 mm MgCl2, and 10 fmol of substrate/reaction in a volume of 25 μl/reaction. Reactions were initiated by the addition of 15 ng (340 fmol) of FEN1/reaction and incubated at 37 °C for 30 min. Reactions were stopped with 25 μl of 2× formamide loading dye (98% formamide, 10 mm EDTA (pH 8.0), 0.01% (w/v) each of xylene cyanole and bromphenol blue) and heated at 95 °C for 5 min. Controls lacking enzyme were also assayed. Products were separated on either 12% or 18% polyacrylamide, 7 m urea denaturing gel electrophoresis (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar) and visualized by autoradiography. DNA size markers were generated by digesting 5′-end-labeled primers with snake venom phosphodiesterase. Any adjustments to the above are noted in the figure legends. We hypothesized that FEN1 participates in a 5′ proofreading reaction linked to DNA replication. In this reaction, FEN1 would remove mismatches generated by DNA polymerase α in the region of DNA just beyond the initiator RNA. On a fully annealed DNA primer, the action of FEN1 is exonucleolytic, whereas on a flap, it is endonucleolytic. Our initial experiments were designed to determine whether the presence of a mismatch would destabilize a primer so that it would appear as a flap to the nuclease. If so, we wanted to measure the distance over which the mismatch could influence cleavage activity. We first compared the cleavage of a fully annealed control substrate (substrate 1.0) to that of substrates containing a single mismatch either two or three nucleotides from the 5′-end (substrates 1.2 and 1.3, respectively) (Fig. 1A). For this and all other experiments in this study, control substrates were identical to the mismatch substrates, except that they were without a mismatch. As expected, on these 5′-end-labeled substrates, FEN1 cleavage in the absence of mismatch resulted in the release of a single nucleotide (Fig. 1A, lane 2). Cleavage of fully annealed substrates most often results in exonucleolytic cleavage (10Murante R.S. Huang L. Turchi J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 11Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (372) Google Scholar, 21Turchi J.J. Bambara R.A. J. Biol. Chem. 1993; 268: 15136-15141Abstract Full Text PDF PubMed Google Scholar, 22Huang L. Rumbaugh J.A. Murante R.S. Lin R.J.R. Rust L. Bambara R.A. Biochemistry. 1996; 35: 9266-9277Crossref PubMed Scopus (31) Google Scholar, 30Rumbaugh J.A. Murante R.S. Shi S. Bambara R.A. J. Biol. Chem. 1997; 272: 22591-22599Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 33Longley M.J. Bennett S.E. Mosbaugh D.W. Nucleic Acids Res. 1990; 18: 7317-7322Crossref PubMed Scopus (59) Google Scholar), although with some sequences, additional products from cleavage at internal sites are observed. Apparently, FEN1 has the capacity to invade a small proportion of fully annealed substrates to perform endonucleolytic cleavage. Such minor internal cleavage products were seen with substrate 1.0. When a mismatch was present at the second or third nucleotide, the proportion of products resulting from endonucleolytic cleavage (trimers and pentamers) was greatly enhanced (Fig. 1A, lanes 3 and4). The mechanism of FEN1 was shifted from predominantly exonucleolytic cleavage to predominantly endonucleolytic cleavage simply by the presence of a single mismatched base pair. For consistency, mismatched substrates were produced by pairing nucleotides with themselves (i.e. G with G). However, we have noticed that the sequence of the mismatch affects the distribution of internal cleavages, as shown in Fig. 1B. Lane 1 shows the cleavage of control substrate 1.0, as in Fig. 1A. For lane 2, the sequence of the primer was modified to generate a T-T mispair with the template at the 5′-end of the primer (substrate 1.T). For lane 3, the template sequence was modified so that the 5′-end nucleotide of the original primer formed an A-A mismatch with the new template (substrate 1.A). In comparing lanes 2 and 3, the identity of the mismatch clearly alters the ratio of the three major products. There is a higher percentage of pentamer released with the A-A mismatch. These results suggest that the specific nucleotides of the mismatch affect the internal cleavage capacity of FEN1, perhaps by altering the exact amount of primer destabilization or the shape of helical distortion. On the other hand, the pattern of products is similar to those generated when the mismatch was at position 2 or 3 from the 5′-end (Fig. 1, compare A with B). This suggests that the mismatch alters the structure of the substrate around it to favor endonucleolytic cleavage but does not define the position of cleavage. Instead, the nuclease appears to cleave at sites that it favors, irrespective of the exact location of the mismatch. As the mismatch was moved further into the strand, the effect on the distribution of 5′ products decreased. Mismatches at 7, 9, 11, 13, and 15 nucleotides from the 5′-end produced a product distribution that differed little from the distribution seen with no mismatch (data not shown). A primer template in which the primer has an unannealed 5′-tail is the preferred substrate of FEN1 (10Murante R.S. Huang L. Turchi J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 18Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19Murante R.S. Rust L. Bambara R.A. J. Biol. Chem. 1995; 270: 30377-30383Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). We wanted to determine whether a mismatch in the annealed region downstream of an unannealed 5′-tail would alter the pattern of cleavage. We thought the mismatch might effectively lengthen the tail, enabling FEN1 to cleave further internally than it otherwise would. FEN1 can remove initiator RNA endonucleolytically when it has been displaced into a tail (18Murante R.S. Rumbaugh J.A. Barnes C.J. Norton J.R. Bambara R.A. J. Biol. Chem. 1996; 271: 25888-25897Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The helical disruption caused by a mismatch might allow FEN1 to remove the initiator RNA and a mismatch simultaneously, with an appropriately placed endonucleolytic cut. Fig. 2 clearly shows no difference in cleavage on endonucleolytic substrates with no mismatch (lane 1), a mismatch seven nucleotides into the annealed region (lane 2), and a mismatch nine nucleotides into the annealed region (lane 3). Cleavage of each substrate results in the production of two major products of ∼26 and 10 nucleotides. The expected product is ∼26 nucleotides, given that the unannealed 5′-tail is 25 nucleotides. The smaller product is the result of cleavage within the tail. This might occur from either transient annealing between the tail and the upstream portion of the template or the secondary structure within the unannealed tail. The presence of the tail might have facilitated the binding of FEN1 and movement of the nuclease to the site of the mismatch to make an initial cleavage. However, these data provide no evidence that the two features collaborate to favor an altered position of cleavage. Presumably, after the tail has been removed, the mismatch will promote further internal cleavages as suggested by the results shown below. To perform effective 5′ proofreading, FEN1 must cleave beyond the mismatch. It would appear that if the mismatch is sufficiently far from the 5′-end, it cannot be removed by a single cut. However, because the primers examined to this point had been 5′-end-labeled, only the first cleavage event could be observed. A mismatch that is initially deep within a primer becomes increasingly close to the 5′-end as FEN1 cleaves the substrate. As this occurs, the mismatch would be expected to have an increasingly greater influence on the cleavage events. Although the mismatch is not removed in a single cut, FEN1 may do so through a series of cleavage events. In Fig. 3, substrate 2.9 has a mismatched base pair at position 9. Lane 1 demonstrates that FEN1 can release a monomer, trimer, or hexamer on this substrate. In each event, the mismatch is not removed in a single cut. After FEN1 removes the first three nucleotides, the mismatched base pair at position 9 is now at position 6. We used a second substrate, 3.6, to model this intermediate and examine FEN1 activity. Using substrate 3.6, we can observe the results of subsequent cuts. Cleavage of substrate 3.6 released monomers, dimers, and tetramers (Fig. 3, lane 2). The mobilities of products in lanes 2 and 3 differ from those in lane 1 because of the sequence difference between substrates 2.9 and 3.6. In particular, released monomer dC migrates farther in lane 2than does monomer dT in lane 1. To verify the exact lengths of the products from both substrates, we made molecular weight ladders of each primer by digestion with snake venom phosphodiesterase. Because substrate 3.6 already has three nucleotides removed compared with substrate 2.9, the release of a monomer, dimer, etc. in lane 2 is equivalent to cleavage beyond the fourth, fifth, etc. nucleotide in lane 1. Removal of the original mismatch nine nucleotides into the substrate requires the release of a hexamer inlanes 2 and 3. The monomers, dimers, and tetramers released in lane 2 correspond to a second cut 3′ of the fourth, fifth, and seventh nucleotides of the original substrate 2.9. Even this second cut falls short of the goal of mismatch removal. However, the process has now placed the mismatch as little as two nucleotides from the 5′-end. The fact that cleavage did not reach the mismatch was surprising. In substrate 3.6, the mismatch is only six nucleotides from the 5′-end, a distance reached on substrate 2.9 (lane 1). One reason we failed to observe cleavage beyond the mismatch could be because of its distance from the 5′-end. Alternatively, failure to cleave beyond the mismatch may reflect the influence of specific sequences. In many sequence contexts, FEN1 requires an upstream primer for stimulation of cleavage (10Murante R.S. Huang L. Turchi J.J. Bambara R.A. J. Biol. Chem. 1994; 269: 1191-1196Abstract Full Text PDF PubMed Google Scholar, 20Siegal G. Turchi J.J. Myers T.W. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9377-9381Crossref PubMed Scopus (61) Google Scholar, 21Turchi J.J. Bambara R.A. J. Biol. Chem. 199
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