Title: Okazaki fragment maturation involves α‐segment error editing by the mammalian <scp>FEN</scp> 1/MutSα functional complex
Abstract: Article28 April 2015free access Source Data Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex Songbai Liu Songbai Liu Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Guojun Lu Guojun Lu Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Shafat Ali Shafat Ali Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Wenpeng Liu Wenpeng Liu Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Li Zheng Li Zheng Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Huifang Dai Huifang Dai Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Hongzhi Li Hongzhi Li Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Hong Xu Hong Xu Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Search for more papers by this author Yuejin Hua Yuejin Hua Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Search for more papers by this author Yajing Zhou Yajing Zhou Institute of Life Sciences, Jiangsu University, Zhen Jiang, Jiangsu, China Search for more papers by this author Janice Ortega Janice Ortega Graduate Center for Toxicology, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Guo-Min Li Guo-Min Li Graduate Center for Toxicology, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Thomas A Kunkel Thomas A Kunkel Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC, USA Search for more papers by this author Binghui Shen Corresponding Author Binghui Shen Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Songbai Liu Songbai Liu Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Guojun Lu Guojun Lu Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Shafat Ali Shafat Ali Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Wenpeng Liu Wenpeng Liu Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Li Zheng Li Zheng Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Huifang Dai Huifang Dai Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Hongzhi Li Hongzhi Li Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Hong Xu Hong Xu Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Search for more papers by this author Yuejin Hua Yuejin Hua Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China Search for more papers by this author Yajing Zhou Yajing Zhou Institute of Life Sciences, Jiangsu University, Zhen Jiang, Jiangsu, China Search for more papers by this author Janice Ortega Janice Ortega Graduate Center for Toxicology, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Guo-Min Li Guo-Min Li Graduate Center for Toxicology, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA Search for more papers by this author Thomas A Kunkel Thomas A Kunkel Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC, USA Search for more papers by this author Binghui Shen Corresponding Author Binghui Shen Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA Search for more papers by this author Author Information Songbai Liu1,2, Guojun Lu2,6, Shafat Ali2, Wenpeng Liu1,2, Li Zheng2, Huifang Dai2, Hongzhi Li2, Hong Xu1, Yuejin Hua1, Yajing Zhou3, Janice Ortega4, Guo-Min Li4, Thomas A Kunkel5 and Binghui Shen 2 1Colleges of Life Sciences and Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, China 2Departments of Radiation Biology and Molecular Medicine, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA, USA 3Institute of Life Sciences, Jiangsu University, Zhen Jiang, Jiangsu, China 4Graduate Center for Toxicology, Markey Cancer Center, University of Kentucky College of Medicine, Lexington, KY, USA 5Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC, USA 6Present address: Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, USA *Corresponding author. Tel: +1 626 301 8879; E-mail: [email protected] The EMBO Journal (2015)34:1829-1843https://doi.org/10.15252/embj.201489865 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5′ ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism. Synopsis Okazaki fragment synthesis during eukaryotic DNA replication is initiated by proofreading-deficient DNA polymerase α, requiring a dedicated repair process during Okazaki fragment maturation. This Pol alpha-segment error editing (AEE) involves the exonuclease activity of flap endonuclease (FEN1) in cooperation with MutSα mismatch repair protein. MutSα recognizes mismatches near the 5′ end of the DNA nick left after RNA primer flap cleavage. MutSα interacts with FEN1 and stimulates mismatch removal by FEN1 exonuclease activity. Defects in AEE lead to increased genome instability and cancer susceptibility. Introduction During replication of the eukaryotic nuclear genome, most of the nascent leading and lagging strands are synthesized by DNA polymerase ε (Pol ε) and Pol δ, respectively. These replicases are highly accurate, partly because their intrinsic 3′ exonucleases can proofread the rare mismatches they generate. In contrast, the third nuclear replicase, Pol α, lacks an intrinsic proofreading exonuclease (EXO) activity and is tenfold to 100-fold less accurate (Kunkel, 2009). Pol α's role in replication is to initiate Okazaki fragments by extending primers synthesized by its associated RNA primase. Thus, any mismatches made by Pol α will be near the 5′ ends of Okazaki fragments, in what can be called the α-segment. Genetic evidence suggests that some mismatches generated by Pol α are proofread by the EXO activity of Pol δ (Pavlov et al, 2006). Theoretically, mismatches generated by Pol α that escape proofreading could be removed during Pol δ-catalyzed strand displacement synthesis associated with normal Okazaki fragment maturation (OFM) (Burgers, 2009; Zheng & Shen, 2011; Balakrishnan & Bambara, 2013). However, studies of yeast strains encoding variant alleles of Pol α (Niimi et al, 2004; Nick McElhinny et al, 2008, 2010) indicate that OFM alone does not remove all mismatches from the α-segment. In fact, the most recent studies indicate that despite Okazaki fragment processing, DNA synthesized by Pol α is retained in vivo and DNA-binding proteins including histones and transcription factors that rapidly re-associate, post-replication, act as partial barriers to Pol-δ-mediated displacement of the α-segment, resulting in increased mutation rates in the region (Clausen et al, 2015; Daigaku et al, 2015; Koh et al, 2015; Reijns et al, 2015). Pol α variant strains have mild mutator phenotypes, resulting from DNA replication errors generated by Pol α, and their mutation rates are synergistically increased by loss of MSH2-dependent mismatch repair (MMR). Thus, MSH2-dependent MMR in yeast plays a major role in correcting mismatches in the α-segment. How is this done? A recent study (Liberti et al, 2013) indicated that some mismatches generated by yeast Pol α are excised by Exonuclease 1 (EXO1), a 5′ exonuclease involved in MMR (Tishkoff et al, 1997b; Wei et al, 2003). Interestingly, the loss of MMR resulting from deleting EXO1 is mild compared to the loss of MMR from deleting MSH2, implying the existence of a MSH2-dependent but EXO1-independent mechanism that removes mismatches from the α-segment. This mechanism could be the same as the mechanism used to repair more internal mismatches generated by Pols ε and δ. In addition, a non-exclusive possibility was previously suggested for mismatches generated by Pol α near the 5′ end of an Okazaki fragment, namely FEN1-dependent mismatch removal (Nick McElhinny et al, 2010). This possibility is supported by an earlier genetic study (Johnson et al, 1995), suggesting that yeast FEN1 participates in MMR in yeast, and it is consistent with a biochemical study of mammalian MMR in vitro, providing evidence for EXO1-independent MMR via strand displacement synthesis by Pol δ (Kadyrov et al, 2009). The present study investigates a role for FEN1 in MSH2-dependent removal of mismatches from the α-segment. We show that FEN1 and MSH2 interact and that FEN1 can remove mismatches from the α-segment in an in vitro reaction, referred to as the α-segment error editing (AEE) assay. This action is strongly stimulated by MutSα; FEN1 mutants are defective in the AEE reaction, and these mutations result in a mutator phenotype and increased cellular transformation in mouse cells and increased cancer susceptibility in mice. Results Pol α error editing during Okazaki fragment maturation To identify the protein machinery responsible for editing the α-segment during OFM, we designed five sets of DNA substrates that mimic the α-segment (Supplementary Fig S1). The first set (Supplementary Fig S1A) is a pair of gapped-flap substrates starting with an "A" in the three or four nucleotide (nt) single-stranded (ss) DNA region and with or without a "C/T" mismatch in the downstream duplex. C is unique in the entire downstream template sequence. Incorporation of radiolabeled T in the matched substrate is used to monitor normal flap removal, gap filling, and ligation, referred to here as RNA primer removal (RPR). Incorporation of radiolabeled G in the mismatched substrate is used to monitor removal of the flap containing the mismatch, gap filling, and ligation, referred to here as AEE. The second pair of substrates uses the same principle, but contains a T in the ssDNA region and a G/T mismatch in the downstream duplex, with the G also being unique in the entire downstream template sequence. We obtained similar results with both pairs of substrates and use them interchangeably in the experiments below, as indicated in the text, figures, and Figure legends. In the second set of substrates (Supplementary Fig S1B), we varied the distance between the first paired nt of the downstream duplex and mismatched nts (indicated as X nt), where X nt is the 3rd, 6th, 12th, or 18th nt. The third set of substrates was designed to test the nuclease activities of EXO1, DNA2 and wild-type (WT) and mutant FEN1. These substrates include one with a 3′ single nt flap and a 40-nt-long 5′ flap, two without a 3′ flap but with different lengths of 5′ flaps (long 5′ flap = 40 nt and short 5′ flap = 5 nt), and one with a nicked duplex DNA without a flap (Supplementary Fig S1C). Set 4 (D1, 2, 3, 4) contains gapped substrates with or without an upstream 3′ flap, and nicked substrates with or without an upstream 3′ flap, used to elucidate the best substrate for the FEN1/MSH2 complex in the removal of Pol α errors (Supplementary Fig S1D). Finally, set five contains substrates that are similar to A1 and A2 but contain an RNA flap (Supplementary Fig S1E). The oligonucleotides used to construct these substrates are listed in Supplementary Table S1. We performed the RPR assay using the first pair of substrates with or without a downstream mismatch, following a procedure that was previously described (Turchi et al, 1994; Zheng et al, 2007a, 2008). This procedure involves incubating nuclear extracts (NEs) with the indicated DNA substrates and nts. In the RPR assays, the gapped-flap substrate was designed in such a way that the first ss DNA nt is an A. In the reaction mixture, we have included radiolabeled dTTP and the other three non-labeled nts. Therefore, if the flap is cleaved, the gap is filled by polymerization and the nick is sealed to generate an 80-nt-long radiolabeled DNA product; then, we know that all of the functional components necessary for OFM are intact in NEs. Indeed, in the first experiment described in Fig 1, we observed a full length product from the 80 nt DNA fragment and 39–42 nt intermediate products incorporating [α-32P] dTTP, indicating that the NEs had the functional enzymatic components needed for flap removal, gap filling, and nick sealing. When incubated with WT NEs, substrates with or without Pol α errors produced similar amounts of non-ligated (39–42 nt) and ligated product (80 nt) (Fig 1A). However, when we incubated the NEs with the same substrates in the presence of [α-32P] dGTP instead of [α-32P] dTTP, the substrate with the C/T mismatch showed significantly more ligated product compared with the reaction product from the substrate without a mismatch (Fig 1B). Because the C is unique in the entire downstream template sequence, the 80-nt-long product is only generated when the first portion of the downstream nts, up to the mismatched nt, T, is removed and radiolabeled G is incorporated. This experiment was repeated with two similar substrates that contained RNA in the 5′ flap to mimic the RNA primer. We obtained very similar results using the RNA and DNA substrates, as we have previously described (Qiu et al, 1999b) (Supplementary Fig S2). Therefore, in the subsequent in vitro biochemical experiments, we used the DNA flap substrates. Figure 1. RNA primer removal (RPR) and α-segment error editing (AEE) assays RPR efficiency without (A1) or with (A2) a mismatch in the downstream duplex DNA region using NEs. One microgram of the NE was incubated with 500 fmol gap substrates containing a 15-nucleotide (nt) DNA flap at 37°C for 5, 10, 20, 40, 60, and 80 min. All reactions were carried out in a buffer containing 5 μCi [α-32P] dTTP and 50 μM dGTP, dCTP, and dTTP. Lanes 3–8 show substrate A1; lanes 9–14 show substrate A2; lane 1 shows molecular weight markers; lane 2 shows the reaction with A1 without enzyme. AEE efficiency without (A1) or with (A2) a mismatch in the downstream duplex DNA region using NEs. One microgram of the NE was incubated with 500 fmol of the gap substrates containing a 15-nt DNA flap at 37°C for 5, 10, 20, 40, 60, and 80 min. All reactions were carried out in a buffer containing 5 μCi [α-32P] dGTP and 50 μM dATP, dCTP, and dTTP. Lanes 3–8 show substrate A1; lanes 9–14 show substrate A2; lane 1 shows molecular weight markers; lane 2 shows the reaction with A1 without enzyme. AEE assay to show the extended gap-filling products beyond the mismatch. One microgram of the MEF NE was incubated with 500 fmol of the 5′ end upstream primer and 32P-labeled flap DNA substrates without (A1) or with (A2) a mismatch in the downstream DNA duplex. Reactions were carried out at 37°C for 10, 20, 40, 60, 80, and 100 min. The designed substrate is illustrated on the top of the panels. See also Supplementary Fig S1 and Supplementary Table S1. Lanes 4–9 show substrate A1; lanes 11–16 show substrate A2; lane 1 shows molecular weight markers; lane 2 is blank, lanes 3 and 10 show the reactions with A1 and A2, respectively, without enzyme. Data information: Numbers at the bottom of the panels are the lane numbers for the various reactions. Prod., product. Source data are available online for this figure. Source Data for Figure 1 [embj201489865-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint The result in Fig 1B indicates that there is an error editing mechanism triggered by a mismatch. To further test this hypothesis, we radiolabeled the two substrates at the 5′ end of the upstream primer. This allowed visualization of extended "nick translation" products beyond the mismatch. Similar amounts of the ligated product were generated with both of the substrates as long as the RPR reactions were completed (Fig 1C). However, extended gap-filling products beyond the ss DNA region (38–52 nt) appeared in a time-dependent manner for the mismatch substrate only (Fig 1C). This confirms the presence of a mechanism for mismatch editing during OFM. How far can editing proceed beyond the ssDNA region or the gap? We designed a series of substrates with a "T/C" mismatch at the 3rd, 6th, 12th, and 18th nt from the gap in the downstream duplex, simulating Pol α errors at different positions in the α-segment. After incubating WT NEs with the four DNA substrates shown in Supplementary Fig S1B in the presence of [α-32P] dATP, we found that overall RPR efficiency was similar (Fig 2A). However, when we incubated NEs with [α-32P] dCTP to assay 5′ end error editing efficiency, there were very large differences among these four substrates in the amount of product generated (Fig 2B). The substrate with a mismatch at the 3rd nt was edited out most efficiently (Fig 2B, lanes 2–5). As the mismatch was located farther from the gap, the editing efficiency decreased (Fig 2B, lanes 6–9) such that little signal was observed when the distance was 12 nts or longer (Fig 2B, lanes 10–13). These results indicated that the OFM machinery primarily edits mismatches within 1–12 nt of the gap. Figure 2. AEE is mismatch-location dependent RPR efficiency with a mismatch at different locations in the downstream duplex DNA. One microgram of the NE was incubated with 500 fmol of the gap substrates containing a 6-nt DNA flap and a variable-length spacer region (B1–4 corresponds to 3, 6, 12, and 18 nt, respectively) at 37°C for 0, 5, 10, 20, 40, and 60 min. All reactions were carried out in a buffer containing 5 μCi [α-32P] dATP and 50 μM dGTP, dCTP, and dTTP. Lanes 3–7 show substrate B1; lanes 9–13 show substrate B2; lanes 15–19 show substrate B3; lanes 21–25 show substrate B4; lane 1 shows molecular weight markers; lanes 2, 8, 14, and 20 show the reactions with B1, B2, B3, and B4, respectively, without enzyme. AEE efficiency with a base mismatch at different locations on the downstream duplex DNA. One microgram of the NE was incubated with 500 fmol of the gap substrates containing a 6-nt DNA flap and a variable-length spacer region (B1–4 corresponds to 3, 6, 12, and 18 nt, respectively) at 37°C for 10, 20, 40, and 60 min. All reactions were carried out in a buffer containing 5 μCi [α-32P] dCTP and 50 μM dATP, dCTP, and dTTP. Lanes 2–5 show substrate B1; lanes 6–9 show substrate B2; lanes 10–13 show substrate B3; lanes 14–17 show substrate B4; lane 1 shows molecular weight markers. Data information: The designed substrate is illustrated on the top of the panels. The numbers on the bottom are the lane numbers. Prod., product. Source data are available online for this figure. Source Data for Figure 2 [embj201489865-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint The concerted flap endonuclease and exonuclease actions of FEN1 are critical for α-segment error editing Several nucleases, including FEN1, DNA2, and EXO1, have been proposed to play a role in RPR during OFM in yeast and mammalian cells (Waga & Stillman, 1994; Qiu et al, 1999a; Bae et al, 2001a), but the nuclease responsible for AEE has not been identified. Using the established RPR and AEE assays with purified proteins (Supplementary Fig S3), we have determined the efficiency of each of the nucleases in the RPR and AEE reactions (Fig 3). We found that DNA2 was not able to complete the RPR reaction to produce the ligated products, as it was not able to completely remove the ss flap. With the residual flap left over, the ligase was not able to complete the ligation portion of the RPR reaction (Fig 3A lane 5 and Fig 3B lanes 7–10). In addition, DNA2 was not able to remove the embedded mismatches due to its lack of EXO activity on the double-stranded DNA duplex. It completely failed to perform the AEE reaction (Fig 3C lane 5 and Fig 3D lanes 7–10). On the other hand, the major function of EXO1 is to remove the mismatches from the DNA duplex, but it has little capacity to remove the flap. Therefore, as expected, EXO1 failed to produce any RPR or AEE products (Fig 3A and C, lane 6, Fig 3B and D, lanes 11–14). FEN1 has both flap endonuclease (FEN) and EXO activities and was able to efficiently generate both the RPR and AEE products (Fig 3A and C, lane 4, Fig 3B and D, lanes 3–6). We used nuclease activity assays to show that all three enzymes are active with their standard substrates (Supplementary Fig S1C and Supplementary Fig S4). Figure 3. FEN1 is the nuclease for AEE RPR in a reconstitution assay with the purified individual nucleases: FEN1, EXO1, or DNA2 and [α-32P] dTTP. 100 fmol of Pol δ, 300 fmol of PCNA, 300 fmol of RFC, and 240 fmol of Lig I were mixed with 500 fmol of hFEN1, hDNA2, or EXO1, as indicated. The mixture was then incubated with 50 fmol of substrate in the reaction buffer containing 5 µCi [α-32P] dTTP and 50 µM of each of the other three nucleotides. The reactions were carried out at 37°C for 60 min. The same reactions as (A) were carried out for 10 min (lanes 3, 7, and 11), 30 min (lanes 4, 8, and 12), 60 min (lanes 5, 9, and 13), and 90 min (lanes 6, 10, and 14). AEE in a reconstitution assay with the individual nucleases: FEN1, EXO1, or DNA2 and [α-32P] dGTP. The reactions were carried out at 37°C for 60 min. [α-32P] dGTP, instead of [α-32P] dTTP, was included in the reactions. All other conditions are the same as in (A, B). The same reactions as in (C) were carried out for 10 min (lanes 3, 7, and 11), 30 min (lanes 4, 8, and 12), 60 min (lanes 5, 9, and 13), and 90 min (lanes 6, 10, and 14). Data information: Substrate A2, containing a downstream C/T mismatch, was used in all of the reactions. Numbers at the bottom of the panels are the lane numbers for the various reactions. Prod., product. Source data are available online for this figure. Source Data for Figure 3 [embj201489865-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint To determine the role and molecular mechanism of FEN1 in AEE, we employed two FEN1 mutants previously identified in cancer cells, E160D and A159V (Zheng et al, 2007b). We characterized their nuclease activity profiles using four standard substrates designed based on a series of publications regarding the FEN1 substrates (Kao et al, 2002) (Supplementary Figs S1C and S5). These substrates included a duplex double-flap DNA with a 3′ single nt flap and a 40-nt-long 5′ flap, two substrates without the 3′ flap but with different lengths of 5′ flaps (long 5′ flap = 40 nt and short 5′ flap = 5 nt), and one nicked duplex DNA without any flap. The E160D mutant retained 100% of WT double-flap endonuclease activity (Supplementary Fig S5A). However, its activity was approximately 50% and 45%, respectively, for cleaving the single-flap substrate with a ss flap of 40 and 5 nts in length (Supplementary Fig S5B and C), and no activity was observed with a nicked DNA substrate (Supplementary Fig S5D). In contrast, the A159V mutant retained approximately 65% of WT activity with the double-flap substrate, but had no activity with the other three substrates (Supplementary Fig S5E–H). To test whether the different degrees of defect in the mutant activity profiles affect α-segment repair efficiency and disease susceptibility in vivo, we generated a mouse model carrying the FEN1 point mutation A159V. We constructed the FEN1 A159V mouse using a gene targeting approach (Supplementary Fig S6A), as previously described for creation of the FEN1 E160D mouse line (Zheng et al, 2007b). The genotype of the A159V mouse was confirmed by Southern blotting and DNA sequence analysis (Supplementary Fig S6B and C). No live homozygous FEN1 A159V mice were obtained. All homozygous FEN1 A159V embryos died before the E9.5 stage. Heterozygous FEN1 A159V mutant mice were viable. In the RPR assays, WT and WT/A159V NEs from mouse embryonic fibroblast (MEF) cells were incubated with the substrate with a downstream mismatch in the presence of [α-32P] dTTP for the RPR assay. The amount of ligated product for A159V was similar to that of WT or E160D, indicating that the A159V mutation did not affect the RPR efficiency (Fig 4A). However, when we incubated the WT and WT/A159V NEs with the substrate in the presence of [α-32P] dGTP (AEE), a condition where the radioactivity would only be incorporated when the mismatched nt is excised, the WT/A159V NE only produced about half of the ligated product compared with the WT or E160D NEs (Fig 4B). In addition, we reconstituted the RPR and AEE assays with the purified WT and mutant FEN1 enzymes and found that E160D was able to complete the RPR process by efficiently cleaving the 5′ flap, whereas A159V failed to do so (Fig 4C). On the other hand, neither A159V nor E160D was able to complete the AEE reactions (Fig 4D), indicating that it is the concerted actions of the flap endonuclease and EXO activities that complete the AEE functional pathway. Figure 4. The concerted FEN and EXO actions of FEN1 are critical in AEE RPR efficiency with the A2 substrate, [α-32P] dTTP, and NEs prepared from MEFs with the indicated genotypes. One microgram NE was incubated with 500 fmol of the substrate A2 and 5 μCi [α-32P] dTTP at 37°C for 10, 20, 40, 60, and 80 min. AEE efficiency with the substrate A2, [α-32P] dGTP, and NEs prepared from the MEFs of the indicated genotypes. One microgram of the NE was incubated with 500 fmol of the substrate A2 and 5 μCi [α-32P] dGTP at 37°C for 10, 20, 40, 60, and 80 min. RPR efficiency in the reconstitution assay with the substrates A1 and A2, WT and mutant FEN1 enzymes, and [α-32P] dTTP at 3°C for 80 min. AEE efficiency in the reconstitution assay with WT and mutant FEN1 enzymes and [α-32P] dGTP at 37°C for 80 min. All reactions were carried out in a buffer containing 5 μCi [α-32P] dTTP or [α-32P] dGTP and 50 μM of the other three nucleotides. Data information: In (A, B), lanes 3–7 show the reactions with the WT MEF NEs; lanes 8–12 show the reactions with the WT/A159V MEF NEs; lanes 13–17 show the reactions with the E160D MEF NEs; lane 1 shows the molecular weight markers; lane 2 shows the reaction without NEs. Numbers at the bottom of the panels are the lane numbers for the various reactions. Lig. Prod., ligated product. AV, the FEN1 mutant A159V.