Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is a genetically heterogeneous disorder caused by germ-line mutations in one of several DNA mismatch repair (MMR) genes, most commonly in hMSH2 and hMLH1. Human exonuclease 1 (hExo1) possesses both 5′exonuclease and flap endonuclease activities and plays a role in DNA repair, recombination, and replication. The enzyme interacts with MMR proteins, hMsh2, hMlh1, and hMsh3. Recently, eight missense mutations in hEXO1 were identified in atypical HNPCC patients, who have been screened to be negative for hMSH2, hMLH1, and hMSH6 mutations. To address the question of whether these mutations cause susceptibility to HNPCC, in vitro nuclease activity and protein-protein interaction assays were performed in this study. We found that two mutants, E109K and L410R, lost their exonuclease activities while retaining their capacity to bind to the DNA substrate. Three other mutants, P640S, G759E, and P770L, displayed a reduced capacity to interact with hMsh2. The combination of these three point mutations leads to the binding capacity with hMsh2 to nearly zero. Evidence made available in this study sheds light on the pathogenesis of HNPCC, perhaps initiated by an additional MMR gene, hEXO1.

HNPCC4 is an inherited autosomal dominant disorder that is characterized primarily by the development of early-onset colorectal cancer. Defects in five MMR genes are associated with HNPCC: hMSH2, hMLH1, hMSH6, hPMS1, and hPMS2(1). Together, hMSH2 and hMLH1 account for 50–60% of all mutations identified in HNPCC patients whose disease fulfills the Amsterdam criteria (2) and 30% of atypical HNPCCs (3, 4). The many HNPCC cases that are not associated with detectable mutations in hMSH2 or hMLH1 suggests that this disease may result from mutations in non-MMR genes or mutations in genes whose products act downstream of the MMR recognition step. Among them, hEXO1 has recently been the focus of much attention. Eukaryotic Exo1 was first identified in Schizosaccharomyces pombe(5). It has 5′ to 3′ exonuclease and 5′ flap endonuclease activities (5, 6). Sequence alignments indicate that Exo1 is homologous to Rad2 family nucleases (6, 7, 8, 9). The protein possesses a conserved NH4-terminal core nuclease domain and extensive COOH-terminal regions that interact with different MMR proteins. Abundant in vitro and in vivo evidence indicates that it is an excision nuclease candidate in MMR. Exo1-deficient yeast strains have a mutator phenotype and exhibit dinucleotide repeat instability (9, 10). Mutations in yeast EXO1 are epistatic with mutations in MSH2, MLH1, and PMS1(10, 11). Exo1 can interact physically with MMR proteins Msh2 and Mlh1 in both yeast and human cells and with Msh3 in human cells (10, 12, 13, 14, 15, 16). S. cerevisiae Exo1 plays a catalytic role in Msh2-mediated MMR (17), and hExo1 is required for both 5′ and 3′ excision reactions in MMR in an in vitro reconstitution system (18). This evidence may also suggest that HNPCC be result from its functional deficiency, similar to other components of the MMR pathway.

Recently, Wu et al.(19) found several hExo1 mutants in HNPCC patients who were shown to be negative for germ-line mutations in hMLH1, hMSH2, and hMSH6. Among a total of 30 alterations in hEXO1 exons, 16 were thought to be polymorphisms because they occurred at similar frequencies in both the patients and controls. Of the other 14 alterations unique to HNPCC patients, one is a splice-site mutation identified in a typical HNPCC family, and the other 13 included eight different missense mutations, all of which were found in atypical HNPCC patients. On the basis of sequence alignment and functional domain analysis (12, 13, 14, 15, 16, 20), we were able to assign nuclease activity to the first 391 amino acid residues and to identify regions responsible for protein-protein interactions with MMR proteins (Fig. 1). The location of each of the eight point mutations is indicated in Fig. 1. E109K is located in the NH4-terminal domain. L410R is located at the boundary between the internal nuclease domain and the region required for hMlh1 interaction. Both NH2-terminal and internal domains are necessary for exonuclease activity. Five other missense mutations, S610G, P640A, P640S, G759E, and P770L, are located COOH-terminal to the nuclease domain within a region required for interaction with the MMR proteins hMsh2 and hMlh1 (12, 13, 14, 15, 16). V27A is located NH4-terminal to the nuclease domain. These eight hExo1 missense mutants fulfill the following criteria supporting the assumption that the mutations are likely to be pathogenic: the mutations are located in critical regions for nuclease activity or interaction with Msh2 or Mlh1; they are not present in the healthy population; and hMSH2 and hMLH1 germ-line mutations are not present in individuals who contain these Exo1 mutations (19). To determine whether any of these mutations alter the ability of Exo1 to function in MMR, we carried out in vitro nuclease activity assays using purified proteins expressed in Escherichia coli and in vitro protein-protein interaction assays using wild type and mutant Exo1 proteins expressed in budding yeast.

Plasmids.

Plasmid pET28a (Novagen, Madison, WI) was used for expression of hExo1 and its mutants. A DNA fragment encoding hExo1 was amplified by PCR using hExo1b cDNA (6) as template and two synthetic oligonucleotides, 5′-CTAGCTAGCGGGATACAGGGAAAGCTAC-3′ and 5′-GCGGGATCCTTACTGGAATATTGCTCTTTG-3′, as forward and reverse primers, which contain NheI and BamHI restriction sites at their 5′ ends, respectively. The PCR products were digested with NheI and BamHI restriction enzymes and ligated into pET28a to produce pET28a-hExo1. Plasmids pBI and pBII were constructed based on yeast co-overexpression plasmid pESC-trp (Stratagene, La Jolla, CA). pBI was used for coexpression of hExo1 and hMlh1, whereas pBII was for hExo1 and hMsh2. A PCR product containing the hEXO1b coding region was cloned downstream of promoter G10 of pESC-trp using NotI and ClaI sites. The cDNA fragments encoding hMlh1 or hMsh2 were then inserted downstream of promoter G1 using BamHI and ApaI sites or SalI and ApaI sites, respectively. The resulting hExo1 protein was tagged at its COOH terminus with Flag whereas hMlh1 or hMsh2 was tagged with c-Myc. Both of the tags were built in the vector. The constructs encoding hExo1 mutants in this study were created using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) based on pET28a-hExo1, pBI, and pBII, with the primers listed in Table 1. All of the plasmid constructs were verified by DNA sequencing (DNA Sequencing Facility, City of Hope Cancer Center). All restriction enzymes and T4 ligases used in this study were purchased from New England BioLabs, Inc. (Beverly, MA), and PCR amplification and purification kits were from Qiagen, Inc. (Valencia, CA).

Expression and Purification of hExo1 and Its Mutants.

The pET28a-derived hEXO1 constructs were transformed into E. coli strain BL21 (DE3) (Novagen, Madison, WI) for protein expression. The E. coli BL21 (DE3) cells that harbor plasmid encoding His-tagged hExo1 or its mutants were grown at 37°C to an A600 of 1.0 and induced by 1 mm isopropyl-thio-β-galactopyranoside (4) for 4 h. Cells were harvested and lysed by sonication. The His-tagged fusion proteins were solubilized by a buffer containing 50 mm Tris-HCl (pH 7.9), 5 mm imidazole, 500 mm NaCl, and 8 m urea, and purified by Ni2+-chelating chromatography according to a procedure provided by Novagen. The purified denatured hExo1 or its mutants were refolded by direct dilution of the protein solution into refolding buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 1 m NDSB-201 (Calbiochem, La Jolla, CA). The protein solution was then extensively dialyzed against 50 mm Tris-Cl (pH 7.5), and 150 mm NaCl to remove NDSB and urea. The refolded proteins were concentrated using Centriprep concentrators (Millipore, Bedford, MA). The proteins were stored at −20°C after addition of an equal volume of glycerol.

Exonuclease Activity Assay and Kinetic Study of hExo1.

32P-labeled nick-specific DNA substrate was prepared according to a procedure published previously (21). One hundred ng of purified hExo1 or mutants were mixed with 1 pmol of DNA substrate in 15 μl of reaction buffer [50 mm Tris-HCl (pH 8.0), and 5 mm Mg2+]. The reaction was incubated at 30°C for 30 min and terminated by an equal volume of stop solution containing 95% formamide, 20 mm EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol. The product and substrate were then separated by 15% denaturing PAGE and visualized by autoradiography. For kinetic study of hExo1, the nick-specific nuclease activity of various amounts of hExo1 or its mutants was assayed. The separated reaction substrate and products were then quantified using the IPLab Gel program (Signal Analytics Corp., Vienna, VA).

DNA Substrate Binding Assay of hExo1 and Its Mutants.

The His-tagged fusion proteins were purified as described above. The purified proteins were refolded on the Ni2+-agarose beads. Ni2+-agarose beads or agarose-bound hExo1 or mutants were incubated at 4°C for 1 h, with 1 pmol of 32P-labeled nick-specific DNA substrate suspended in a buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 0.5 mm EDTA. The beads were then washed with the same buffer twice. The DNA substrate bound to the protein was determined by Beckman LS5000TD scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Values are means of four independent experiments.

Western Blotting.

Plasmids pBI, pBII, and derivatives with different mutations on hEXO1 were transformed individually into the yeast strain F3C15D (MATa, his3, leu2, ade2, exo1::URA3). Cultures of 50 ml were grown at 30°C in tryptophan minus synthetic dextrose minimal (SD-trp) medium to an A600 of 1.4 and induced by 2% galactose for 5 h according to the manufacturer’s recommendation (Stratagene, La Jolla, CA). Crude extracts were prepared in the buffer A containing 25 mm Tris-Cl (pH 7.4), 15 mm EDTA, 15 mm MgCl2, 1 mm NaN3, 0.1% Triton X-100, 10% glycerol, and 2× Protease Arrest (GenoTech, St. Louis, MO). Protein concentrations were determined with a Bio-Rad protein assay reagent kit (Bio-Rad, Hercules, CA), and the extracts were aliquoted and stored at −80°C. Co-overexpression of fusion proteins in yeast transformants was confirmed by Western analysis. Fifteen mg of protein from each sample were separated on a 4–15% gradient polyacrylamide gel (Bio-Rad), followed by blotting onto a nitrocellulose membrane. After probing with anti-hExo1N2 antibody (a gift from David M. Wilson, III, National Institute on Aging, Baltimore, MD) and detecting with enhanced chemiluminescence reagents, the membrane was stripped and then reprobed with anti-hMlh1 or anti-hMsh2 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Coimmunoprecipitation Analyses of hExo1-hMlh1 and hExo1-hMsh2.

All immunoprecipitation experiments were performed at 4°C. Approximately 200 μg of crude extracts prepared as described above were incubated with 20 μl of anti-Flag M2 antibody resin (Sigma Chemical Co., St. Louis, MO) for 2 h in buffer B (buffer A without glycerol but supplemented with 150 mm NaCl). Reactions were centrifuged at 1500 rpm for 15 s, and the supernatants were removed. The agarose beads were then washed four times with 400 μl of buffer B. The coprecipitated products were eluted with the sample buffer containing 62.5 mm Tris-Cl (pH 6.8), 5% β-mercaptoethanol, 2% SDS, 10% glycerol, and 0.002% bromphenol blue and separated on 4–15% gradient polyacrylamide gel. Immunoblots were performed as described above using anti-hExo1N2 antibody and enhanced chemiluminescence, followed by stripping and reprobing using anti-hMlh1 or anti-hMsh2 antibody. The densities of the desired protein bands were quantified with the IPLab Gel program. The relative amount of hMlh1 or hMsh2, which bound to wild-type or mutant hExo1, was determined by normalizing the intensity of hMlh1 or hMsh2 band to the intensity of corresponding hExo1 band. The amount of hMlh1 or hMsh2 bound to wild-type hExo1 is arbitrarily set as 1.

In an in vitro MMR reconstitution system, the exonuclease activity of hExo1 was required for 5′ mismatch excision (18). To test whether the eight hExo1 mutations identified in HNPCC lost their MMR excision activity, we constructed E. coli expression vectors encoding wild-type hExo1 and its mutants and purified the recombinant proteins (Fig. 2,A). In vitro nuclease assays were carried out using the recombinant wild-type and mutant proteins and a 5′-labeled nicked double-stranded DNA substrate to detect exonuclease activity required for the excision step of MMR. As shown in Fig. 2,B, wild-type hExo1 possesses 5′ to 3′ exonuclease activity as reported previously (6, 20). Except for E109K and L410R, all other mutants manifest approximately the same activity as the wild type. No digested bands were visible when E109K and L410R mutant proteins were incubated with the DNA substrate. Dynamic cleavage curves versus protein concentrations shown in Fig. 2,C further confirm the results in Fig. 2,B. This result is consistent with our expectations, given that E109 is located in the region required for nuclease activity. L410 is located at the boundary between the internal domain and the region required for Msh2 interaction. According to a previously published sequence alignment (8), both E109 and L410 are conserved amino acid residues among higher eukaryotic cells. Loss of activity attributable to the E109R mutation suggests that the acidic residue may be also involved in the catalytic center besides the previously identified active center residues (Ref. 20 and Fig. 1). Exo1 proteins containing the E109K and L410R mutations retained their ability to bind to the DNA substrate (Fig. 2,D), suggesting that the deficiency in nuclease activity was attributable to a loss of catalytic function. No loss of nuclease activity was observed for the V27A mutation, although this amino acid is also conserved within members of the Exo1 nuclease family. However, V27 is located in the NH2 terminus of the nuclease domain of hExo1 (Fig. 1). We have found that the first 29 amino acid residues were not absolutely required for nuclease activity of this protein.5 This may explain the lack of any effect of a mutation at this site in our assay system. There are cleavage products (11, 12, and 13 nucleotides) immediately under those representing 14 nucleotide substrates in Fig. 2 B. These bands may be the products from a 3′ hydrolytic activity of hExo1. Similar observations were reported in an in vitro reconstitution system (18). Therefore, it is likely that hExo1 possesses 5′ and 3′ nuclease activities, both of which are required in mismatch-directed excision.

Exo1 physically and functionally interacts with major MMR components (10, 11, 12, 13, 14, 15, 16, 17, 18). The interaction between hExo1 and hMlh1 or hMsh2 may be important for targeting hExo1 to its proper location during the MMR excision process. In an attempt to determine whether the COOH-terminal mutants are defective in their interaction with MMR proteins, we co-overexpressed hExo1 and its mutants with hMlh1 or hMsh2 in yeast cells (Fig. 3,A). Cell lysates were used for in vitro immunoprecipitation (pull-down) assays. As shown in Fig. 3,B, the relative amount of hMlh1 that coprecipitated with hExo1 mutants was similar to that observed with wild-type hExo1. This was also true for L410R, which is located in the region for hMlh1 interaction. In contrast, the relative amount of hMsh2 that was pulled down with the hExo1 mutants varied considerably. Mutants P640S, G759E, and P770L pulled down approximately one-third of the amount of hMsh2 that was observed with wild-type hExo1 (Fig. 3,C). Interestingly, these mutations were located in the region required for hMsh2 interaction (Fig. 1). However, the P640A mutation arising at the same site as P640S was not as severely affected in its ability to interact with hMsh2. This is probably attributable to the replacement of a non-polar amino acid with a polar amino acid in the case of P640S. None of the eight single amino acid modifications was sufficient to completely abolish the interaction between hExo1 and hMsh2. To enhance the effect of the individual mutations, we constructed yeast expression vectors that encode combined double or triple mutations of P640S, G759E, and P770L. The double mutants PS/PL (P640S/P770L) and GE/PL (G759E/P770L) pulled down very little hMsh2, whereas the triple mutant PS/GE/PL (P640S/G759E/P770L) did not pull down any detectable hMsh2. Unexpectedly, we observed that mutant PS/GE (P640S/G759E) pulled down an even greater amount of hMsh2 protein than either of the single mutants did. Presumably, the simultaneous replacement of these two residues altered the conformation of hExo1, resulting in a stronger interaction with hMsh2.

As mentioned above, hExo1 is a multifunctional molecule. It participates in multiple DNA metabolic processes, including DNA recombination, replication, and repair. It is likely that other proteins interact with hExo1 to determine the location and process through which it acts. For its involvement in MMR, hMsh2 may direct hExo1 to the site of action. Other proteins then interact with hMsh2 and hExo1 to form a complex capable of completing the repair. Interestingly, in a genetic screen designed to identify proteins functionally interacting with yeast Exo1 (yExo1), mutations generated at Msh2 and Mlh1, which caused only weak MMR defects in cells with wild-type yExo1, resulted in a strong MMR defect when combined with the exo1 null mutation (22). These findings suggest that Exo1 facilitates interactions between Mlh1 and Msh2. Thus, Exo1 may play an important structural role in forming or maintaining protein complexes that function in MMR in addition to its role as a nuclease. In the current study, we identified three mutations that resulted in reduced binding between hExo1 and hMsh2. It is possible that amino acid residues 640, 759, and 770 of hExo1 play a critical role in maintaining this stabilizing interaction between these two proteins. Mutants V27A, S610G, and P640A, although not showing a phenotype in our assays, may perturb the stability of the MMR complex. Destabilization of interactions between MMR proteins as a result of mutations in hEXO1 could be one mechanism that promotes the development of HNPCC.

In contrast to our understanding of prokaryotic MMR, the mispair removal process in the corresponding eukaryotic pathway is as yet largely uncharacterized. The HNPCC syndrome has been primarily linked to defects in MMR, especially those resulting from mutations in hMSH2 and hMLH1. Findings that hEXO1 missense mutations exist in atypical HNPCC patients without germ-line mutations in major MMR proteins (19) and that functional alterations take place in some of these hExo1 mutants in our studies provide further evidence that Exo1 serves as an excision exonuclease in MMR. On the basis of the bacterial model of MMR, it is likely that hExo1 acts in concert with other nucleases to excise mismatched bp. Our study indicates that several of the HNPCC-associated hEXO1 mutations yielded only a partial defect in functional assays. This may be attributable to the evidence of the redundant excision activities. Mutations in another redundant MMR protein, hMsh6, are only associated with atypical HNPCC (23, 24, 25). In contrast, the unique MMR components hMsh2 and hMlh1 mutations are associated with typical HNPCC, resulting in a total loss of the MMR function (26). Furthermore, mutations in different functional classes of MMR proteins may promote HNPCC through different mechanisms. hMsh2, hMlh1, and hExo1 play roles both in MMR and in DNA recombination. Further systematic in vivo assays will be required to determine which pathways are affected by specific mutations in these proteins.

Genetic testing of individuals at a high risk of developing certain hereditary conditions is a powerful emerging strategy for the prevention of disease. However, a persistent ambiguity will arise regarding the functional significance of missense codons identified by gene sequencing. If sufficient biochemical, clinical, and population data are lacking, then it becomes impossible to state with confidence whether a sequence variation is pathogenic or simply a natural variation in the human population. Accumulation of the mapping data such as the work reported here will increase the effectiveness of genetic testing programs. In combination with biochemical reconstitution, human population studies monitoring the association of hEXO1 gene defects with cancer and/or the construction of an EXO1 knockout and knock-in of these point mutations in mice should further clarify whether these hExo1 mutants play important roles in tumorigenesis, especially in the generation of HNPCC.

Fig. 1.

Functional motifs of hExo1 and the locations of hExo1 missense mutations identified in atypical HNPCC. Designation of the nuclease domain including N and I motifs (both indicated as striped boxes) is based on previously published amino acid sequence alignments (6, 7, 8). Conserved acidic amino acid residues required for the nuclease active center of the Rad2 family nucleases and identified by site-directed mutagenesis are also indicated with hollow circles under the boxes, i.e., D30, D78, E150, D171, D173, and D225, from left to right(6, 7, 8). The protein-protein interaction region is designated based on the results of domain analysis (12, 13, 14, 15, 16). Reverted triangles above the boxes and lines indicate eight missense mutations identified in atypical HNPCC patients.

Fig. 1.

Functional motifs of hExo1 and the locations of hExo1 missense mutations identified in atypical HNPCC. Designation of the nuclease domain including N and I motifs (both indicated as striped boxes) is based on previously published amino acid sequence alignments (6, 7, 8). Conserved acidic amino acid residues required for the nuclease active center of the Rad2 family nucleases and identified by site-directed mutagenesis are also indicated with hollow circles under the boxes, i.e., D30, D78, E150, D171, D173, and D225, from left to right(6, 7, 8). The protein-protein interaction region is designated based on the results of domain analysis (12, 13, 14, 15, 16). Reverted triangles above the boxes and lines indicate eight missense mutations identified in atypical HNPCC patients.

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Fig. 2.

In vitro nuclease activity and substrate binding capability of hExo1 and mutants. A, purification of hExo1 proteins. His-tagged recombinant hExo1 and its mutants were overexpressed and purified as described in the “Materials and Methods.” B, nuclease activity of hExo1 and mutants. The cleavage reaction was carried out by mixing 1 pmol of 32P-labeled, nick-specific DNA substrate and 100 ng of purified wild-type hExo1 and its mutants as indicated. 14 nt oligo is the labeled DNA substrate, whereas 3 nt, 2 nt, and 1 nt are the cleaved products. C, kinetics of hExo1 and its mutants. Exonuclease activity was assayed with various amounts of wild-type hExo1, V27A, E109K, L410R, and S610G. The DNA substrate and cleaved products were analyzed by a 4–15% denaturing PAGE. Densities of the cleavage products and remaining substrates representing the relative nuclease activities were then quantified with the IPLab Gel program. D, DNA substrate binding capability of hExo1 and its nuclease domain-defective mutants. Ni2+-agarose bound wild-type hExo1, V27A, E109K, or L410R was mixed with 1 pmol of 32P-labeled, nick-specific DNA substrate at 4°C for 1 h. Radioactivity bound to the beads was quantified by scintillation counter. The relative amount of DNA substrate bound to each protein was calculated by normalizing the radioactivity associated with protein-beads to the corresponding protein concentrations. The relative amount of DNA substrate bound to wild-type hExo1 was arbitrarily set as 1. Values are means of three independent experiments. Bars, SD.

Fig. 2.

In vitro nuclease activity and substrate binding capability of hExo1 and mutants. A, purification of hExo1 proteins. His-tagged recombinant hExo1 and its mutants were overexpressed and purified as described in the “Materials and Methods.” B, nuclease activity of hExo1 and mutants. The cleavage reaction was carried out by mixing 1 pmol of 32P-labeled, nick-specific DNA substrate and 100 ng of purified wild-type hExo1 and its mutants as indicated. 14 nt oligo is the labeled DNA substrate, whereas 3 nt, 2 nt, and 1 nt are the cleaved products. C, kinetics of hExo1 and its mutants. Exonuclease activity was assayed with various amounts of wild-type hExo1, V27A, E109K, L410R, and S610G. The DNA substrate and cleaved products were analyzed by a 4–15% denaturing PAGE. Densities of the cleavage products and remaining substrates representing the relative nuclease activities were then quantified with the IPLab Gel program. D, DNA substrate binding capability of hExo1 and its nuclease domain-defective mutants. Ni2+-agarose bound wild-type hExo1, V27A, E109K, or L410R was mixed with 1 pmol of 32P-labeled, nick-specific DNA substrate at 4°C for 1 h. Radioactivity bound to the beads was quantified by scintillation counter. The relative amount of DNA substrate bound to each protein was calculated by normalizing the radioactivity associated with protein-beads to the corresponding protein concentrations. The relative amount of DNA substrate bound to wild-type hExo1 was arbitrarily set as 1. Values are means of three independent experiments. Bars, SD.

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Fig. 3.

Analysis of protein-protein interactions between hExo1 and hMlh1 or hMsh2. A, Western blotting to confirm the coexpression of hExo1 and hMlh1 or hMsh2 in yeast cells. Yeast cells harboring plasmids pESC or its derivatives pBI or pBII were grown and induced by 2% galactose for 2 or 5 h. Cell lysates were analyzed by Western blotting as described in “Materials and Methods.” Upper panel, blotting result with anti-hExo1N2 antibody. The membrane was then stripped and reprobed with anti-hMlh1 antibody or anti-hMsh2 antibody. The result was shown in the bottom panel. B, analysis of interaction between single hExo1 mutants and hMlh1 by immunoprecipitation (IP) and Western blotting. Cell lysates from yeast cells coexpressing c-Myc-tagged hMlh1 and Flag-tagged wild-type or mutant hExo1 were prepared and subjected to immunoprecipitation using agarose-conjugated anti-Flag M2 antibody. The coimmunoprecipitated products were analyzed by Western blotting. The upper panel shows Flag-tagged hExo1 and its mutants that were pulled down by agarose beads. The middle panel shows hMlh1, which was coimmunoprecipitated with wild-type hExo1 or its mutants. The lower panel shows the relative quantity of hMlh1 pulled down. C, interaction of hExo1 single mutants with hMsh2. Coimmunoprecipitation and Western blotting analysis were performed on cell lysates prepared from yeast cells coexpressing c-Myc-tagged hMsh2 and Flag-tagged wild-type hExo1 or its mutants. The amount of hExo1 or its mutants and the amount of hMsh2 that was coprecipitated with hExo1 are displayed in upper and middle panels, respectively. The lower panel shows the relative quantity of hMsh2 pulled down. D, interaction of hExo1 double and triple mutants with hMsh2. Mutants P640S/G759E (PS/GE), P640S/P770L (PS/PL), G759E/P770L (GE/PL), and P640S/G759E/P770L (PS/GE/PL) were created sequentially based on the constructs containing single hEXO1 mutation. Cell lysates of yeast transformants were prepared and were subjected to immunoprecipitation and Western blotting. Similarly, the upper panel shows different hExo1 proteins pulled down by agarose beads. The middle panel shows the hMsh2 coprecipitated with different hExo1 proteins. The lower panel shows the relative quantity of hMsh2 pulled down. All of the desired bands in B, C, and D were quantified with the IPLab Gel program. The relative amount of hMlh1 or hMsh2 that bound to wild-type or mutant hExo1 was determined by normalizing the intensity of hMlh1 or hMsh2 band to the intensity of the corresponding hExo1 band. The amount of hMlh1 or hMsh2 bound to wild-type hExo1 is arbitrarily set as 1. Bars, SD.

Fig. 3.

Analysis of protein-protein interactions between hExo1 and hMlh1 or hMsh2. A, Western blotting to confirm the coexpression of hExo1 and hMlh1 or hMsh2 in yeast cells. Yeast cells harboring plasmids pESC or its derivatives pBI or pBII were grown and induced by 2% galactose for 2 or 5 h. Cell lysates were analyzed by Western blotting as described in “Materials and Methods.” Upper panel, blotting result with anti-hExo1N2 antibody. The membrane was then stripped and reprobed with anti-hMlh1 antibody or anti-hMsh2 antibody. The result was shown in the bottom panel. B, analysis of interaction between single hExo1 mutants and hMlh1 by immunoprecipitation (IP) and Western blotting. Cell lysates from yeast cells coexpressing c-Myc-tagged hMlh1 and Flag-tagged wild-type or mutant hExo1 were prepared and subjected to immunoprecipitation using agarose-conjugated anti-Flag M2 antibody. The coimmunoprecipitated products were analyzed by Western blotting. The upper panel shows Flag-tagged hExo1 and its mutants that were pulled down by agarose beads. The middle panel shows hMlh1, which was coimmunoprecipitated with wild-type hExo1 or its mutants. The lower panel shows the relative quantity of hMlh1 pulled down. C, interaction of hExo1 single mutants with hMsh2. Coimmunoprecipitation and Western blotting analysis were performed on cell lysates prepared from yeast cells coexpressing c-Myc-tagged hMsh2 and Flag-tagged wild-type hExo1 or its mutants. The amount of hExo1 or its mutants and the amount of hMsh2 that was coprecipitated with hExo1 are displayed in upper and middle panels, respectively. The lower panel shows the relative quantity of hMsh2 pulled down. D, interaction of hExo1 double and triple mutants with hMsh2. Mutants P640S/G759E (PS/GE), P640S/P770L (PS/PL), G759E/P770L (GE/PL), and P640S/G759E/P770L (PS/GE/PL) were created sequentially based on the constructs containing single hEXO1 mutation. Cell lysates of yeast transformants were prepared and were subjected to immunoprecipitation and Western blotting. Similarly, the upper panel shows different hExo1 proteins pulled down by agarose beads. The middle panel shows the hMsh2 coprecipitated with different hExo1 proteins. The lower panel shows the relative quantity of hMsh2 pulled down. All of the desired bands in B, C, and D were quantified with the IPLab Gel program. The relative amount of hMlh1 or hMsh2 that bound to wild-type or mutant hExo1 was determined by normalizing the intensity of hMlh1 or hMsh2 band to the intensity of the corresponding hExo1 band. The amount of hMlh1 or hMsh2 bound to wild-type hExo1 is arbitrarily set as 1. Bars, SD.

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH Grant R01 CA85344 (to B. H. S.).

4

The abbreviations used are: HNPCC, hereditary nonpolyposis colorectal cancer; CRC, colorectal cancer; MMR, mismatch repair; Exo1, exonuclease 1; hExo1, human exonuclease 1.

5

B. Shen, unpublished data.

Table 1

Oligonucleotides used to create point mutations in the hEXO1 gene

Mutation and oligo nameOligo sequence (5′–3′)a
V27A  
 V27A-F GTATAAAGGGCAGGTAGcAGCTGTGGATACATATTG 
 V27A-R CAATATGTATCCACAGCTgCTACCTGCCCTTTATAC 
E109K  
 E109K-F GCAACTTCTTCGTaAGGGGAAAGTCTCGG 
 E109K-R CCGAGACTTTCCCCTtACGAAGAAGTTGC 
L410R  
 L410R-F CTAAAGGGTTAAATCgCCCAAGGAAATCATCC 
 L410R-R GGATGATTTCCTTGGGcGATTTAACCCTTTAG 
S610G  
 S610G-F CACTAAGAAGTTGTTTTgGTTGGTCTGGAGGTC 
 S610G-R GACCTCCAGACCAACcAAAACAACTTCTTAGTG 
P640S  
 P640S-F GAAAGAGCGATTCCtCCACCTCTTTGCCTGAG 
 P640S-R CTCAGGCAAAGAGGTGGaGGAATCGCTCTTTC 
P640A  
 P640A-F GAAAGAGCGATTCCgCCACCTCTTTGCCTGAG 
 P640A-R CTCAGGCAAAGAGGTGGcGGAATCGCTCTTTC 
G759E  
 G759E-F GATCAAACCTCTAGaACCTGCCAGAGCCAG 
 G759E-R CTGGCTCTGGCAGGTtCTAGAGGTTTGATC 
P770L  
 P770L-F GCTGAGCAAGAAGCtGGCAAGCATCCAGAAG 
 P770L-R CTTCTGGATGCTTGCCaGCTTCTTGCTCAGC 
Mutation and oligo nameOligo sequence (5′–3′)a
V27A  
 V27A-F GTATAAAGGGCAGGTAGcAGCTGTGGATACATATTG 
 V27A-R CAATATGTATCCACAGCTgCTACCTGCCCTTTATAC 
E109K  
 E109K-F GCAACTTCTTCGTaAGGGGAAAGTCTCGG 
 E109K-R CCGAGACTTTCCCCTtACGAAGAAGTTGC 
L410R  
 L410R-F CTAAAGGGTTAAATCgCCCAAGGAAATCATCC 
 L410R-R GGATGATTTCCTTGGGcGATTTAACCCTTTAG 
S610G  
 S610G-F CACTAAGAAGTTGTTTTgGTTGGTCTGGAGGTC 
 S610G-R GACCTCCAGACCAACcAAAACAACTTCTTAGTG 
P640S  
 P640S-F GAAAGAGCGATTCCtCCACCTCTTTGCCTGAG 
 P640S-R CTCAGGCAAAGAGGTGGaGGAATCGCTCTTTC 
P640A  
 P640A-F GAAAGAGCGATTCCgCCACCTCTTTGCCTGAG 
 P640A-R CTCAGGCAAAGAGGTGGcGGAATCGCTCTTTC 
G759E  
 G759E-F GATCAAACCTCTAGaACCTGCCAGAGCCAG 
 G759E-R CTGGCTCTGGCAGGTtCTAGAGGTTTGATC 
P770L  
 P770L-F GCTGAGCAAGAAGCtGGCAAGCATCCAGAAG 
 P770L-R CTTCTGGATGCTTGCCaGCTTCTTGCTCAGC 
a

Three bolded letters in the sequence represent the code of a mutated amino acid. The lowercase letters are the substituted nucleotide residues.

We thank David M. Wilson, III at the National Institute on Aging, Baltimore, MD for generously providing the anti-hExo1N2 antibody, Guo-Min Li at University of Kentucky Medical Center for hMSH2 cDNA, and Lene J. Rasmussen at Roskilde University, Denmark, for hMLH1 cDNA. We also thank Tim O’Connor, Steve Alas, and Douglas Thrower for critical reading of the manuscript.

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