The Escherichia coliMutL Protein Physically Interacts with MutH and Stimulates the MutH-associated Endonuclease Activity
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1306
ISSN1083-351X
AutoresMark C. Hall, Steven W. Matson,
Tópico(s)RNA modifications and cancer
ResumoAll possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins. A two-hybrid screen previously identified a physical interaction between MutL and UvrD. Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected. A series of truncations from the NH2 and COOH termini of MutL demonstrated that the COOH-terminal 218 amino acids were sufficient for the two-hybrid interaction with MutH. Removal of a small number of residues from either the NH2 or COOH termini of MutH eliminated the two-hybrid interaction with MutL. Protein affinity chromatography experiments confirmed that MutL, but not MutS, physically associates with MutH. Furthermore, MutL greatly stimulated the d(GATC)-specific endonuclease activity of MutH in the absence of MutS and a mispaired base. Stimulation of the MutH-associated endonuclease activity by MutL was dependent on ATP binding but not ATP hydrolysis. Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP. These results suggest that MutL activates the MutH-associated endonuclease activity through a physical interaction during methyl-directed mismatch repair in Escherichia coli. All possible pairwise combinations of UvrD, MutL, MutS, and MutH were tested using the yeast two-hybrid system to identify potential interactions involving mismatch repair proteins. A two-hybrid screen previously identified a physical interaction between MutL and UvrD. Although several other known interactions were not observed, a novel interaction between MutL and MutH was detected. A series of truncations from the NH2 and COOH termini of MutL demonstrated that the COOH-terminal 218 amino acids were sufficient for the two-hybrid interaction with MutH. Removal of a small number of residues from either the NH2 or COOH termini of MutH eliminated the two-hybrid interaction with MutL. Protein affinity chromatography experiments confirmed that MutL, but not MutS, physically associates with MutH. Furthermore, MutL greatly stimulated the d(GATC)-specific endonuclease activity of MutH in the absence of MutS and a mispaired base. Stimulation of the MutH-associated endonuclease activity by MutL was dependent on ATP binding but not ATP hydrolysis. Further stimulation of this reaction by MutS required the presence of a DNA mismatch and a hydrolyzable form of ATP. These results suggest that MutL activates the MutH-associated endonuclease activity through a physical interaction during methyl-directed mismatch repair in Escherichia coli. The methyl-directed mismatch repair pathway in Escherichia coli functions to correct DNA biosynthetic errors that arise during chromosomal replication and to discourage recombination between substantially diverged DNA sequences (1Modrich P. Lahue R. Annu. Rev. Biochem. 1996; 65: 101-133Crossref PubMed Scopus (1337) Google Scholar). Inactivation of the mismatch repair system results in elevated spontaneous mutation rates (2Nevers P. Spatz H.C. Mol. Gen. Genet. 1975; 139: 233-243Crossref PubMed Scopus (99) Google Scholar). The pathway has been reconstituted in vitro and involves the action of eight proteins (3Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (446) Google Scholar). Initiation of mismatch repair requires MutS, MutL, and MutH in addition to a DNA mismatch, ATP, and Mg2+, and results in the generation of a nick in the unmethylated (nascent) strand of a nearby hemimethylated d(GATC) sequence (4Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar). The transient hemimethylated state of d(GATC) sequences after replication serves as a signal to direct repair to the nascent DNA strand (5Lu A.-L. Clark S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4639-4643Crossref PubMed Scopus (212) Google Scholar, 6Pukkila P.J. Peterson J. Herman G. Modrich P. Meselson M. Genetics. 1983; 104: 571-582Crossref PubMed Google Scholar). MutS recognizes and binds the mismatched base (7Su S.-S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (262) Google Scholar, 8Su S.-S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar). MutL binds the MutS-mismatch complex (9Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar), and MutH is stimulated to catalyze the endonucleolytic cleavage at the d(GATC) site in the presence of MutL and MutS (4Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar). After the initiation stage of mismatch repair, DNA unwinding is initiated at the nick by DNA helicase II (UvrD) and proceeds to a point beyond the error (10Dao V. Modrich P. J. Biol. Chem. 1998; 273: 9202-9207Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Grilley M. Griffith J. Modrich P. J. Biol. Chem. 1993; 268: 11830-11837Abstract Full Text PDF PubMed Google Scholar). Excision of the error-containing DNA strand is facilitated by the action of one of several exonucleases (depending on the polarity of the reaction) which serve to degrade the single-stranded DNA (ssDNA) 1The abbreviations used are: ssDNA, single-stranded DNA; PCR, polymerase chain reaction; BSA, bovine serum albumin; ATPγS, adenosine 5′-O-(3-thiotriphosphate); AMP-PNP, adenosine 5′-(β,γ-imino) triphosphate; MES, 4-morpholineethanesulfonic acid. as it is unwound by UvrD (11Grilley M. Griffith J. Modrich P. J. Biol. Chem. 1993; 268: 11830-11837Abstract Full Text PDF PubMed Google Scholar, 12Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar). In the presence of ssDNA-binding protein, DNA polymerase III holoenzyme catalyzes repair synthesis on the resulting gapped DNA molecule to restore the correct sequence, and DNA ligase seals the final nick (3Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (446) Google Scholar). The E. coli MutH protein possesses a weak endonuclease activity that is specific for unmethylated d(GATC) sequences (13Welsh K.M. Lu A.-L. Clark S. Modrich P. J. Biol. Chem. 1987; 262: 15624-15629Abstract Full Text PDF PubMed Google Scholar). In the presence of ATP, MutS, MutL, and a hemimethylated DNA substrate containing a mismatched base pair, the MutH-associated endonuclease activity is greatly stimulated (4Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar). However, the mechanism by which the MutH endonuclease activity is activated by the MutS-MutL complex is not known. Recently, we identified a physical interaction between the MutL and UvrD proteins using a yeast two-hybrid screen with UvrD as bait (14Hall M.C. Jordan J.R. Matson S.W. EMBO J. 1998; 17: 1535-1541Crossref PubMed Scopus (128) Google Scholar). Simultaneously, a biochemical interaction was reported between MutL and UvrD (15Yamaguchi M. Dao V. Modrich P. J. Biol. Chem. 1998; 273: 9197-9201Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). To identify other potential interactions involving E. coli mismatch repair proteins, all possible pairwise combinations of MutS, MutL, MutH, and UvrD were tested for interactions using the yeast two-hybrid system. An interaction was identified between MutL and MutH which was subsequently confirmed by affinity chromatography. The weak endonuclease activity of MutH on unmethylated d(GATC) sequences was greatly stimulated by MutL. Surprisingly, this stimulation of the activity of MutH occurred in the absence of MutS and a mismatched base pair, suggesting that MutL is the component of the MutS-MutL complex responsible for activating MutH during mismatch repair in vivo and that the activation occurs via a direct physical interaction. In addition, the stimulation of MutH by MutL was dependent on ATP but not ATP hydrolysis. These results suggest an additional role for the MutL protein in coordinating activities during mismatch repair in E. coli. pGAD424 and pGBT9, and yeast HF7c and SFY526 were from the Matchmaker two-hybrid system (CLONTECH). pCYB1, pCYB2, and all components of the Impact I protein purification system were from New England Biolabs. E. coliGE1752ΔuvrD (16Mendonca V.M. Kaiser-Rogers K. Matson S.W. J. Bacteriol. 1993; 175: 4641-4651Crossref PubMed Google Scholar) and GE1752mutS::Tn5 (17.Kaiser-Rogers, K. A., Escherichia coli DNA Helicase II: Construction and Characterization of ATPase "A" Site Mutants and Deletion Mutants Involving Helicase II, Helicase IV, and Rep Protein. Ph.D. dissertation, 1991, 66, 70, Department of Biology, University of North Carolina, Chapel Hill, NC.Google Scholar) were constructed previously in this laboratory. HMS174 (recA1 hsdR (rK12-mK12+) RifR) was from Novagen. BL21(DE3)mutS::Tn5 was constructed by P1 transduction (18Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 201-205Google Scholar) using GE1752mutS::Tn5 as the donor strain and BL21(DE3) as the recipient. Several Kanrtransductants were selected, colony purified, and screened for a mutator phenotype. To ensure that the mutator phenotype was caused by the mutant mutS allele, complementation experiments were performed using a high copy number plasmid that expressed MutS. To prepare M13mp18 ssDNA, phage infection of E. coli XL-1 Blue (Stratagene) and collection of phage particles were performed as described (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 4.26-4.32Google Scholar). Phage particles were purified on a CsCl gradient (0.438 g of CsCl/ml; 83,000 × g for 24 h at 25 °C). After isolation from the gradient and dialysis against 10 mm Tris-HCl (pH 8.0) to remove CsCl, the phage particles were treated with 200 μg/ml proteinase K and 0.1% SDS for 1 h at 50 °C. M13mp18 ssDNA was purified from phage particles by sequential extractions with buffered phenol, 25:24:1 phenol:chloroform:isoamyl alcohol, and 24:1 chloroform:isoamyl alcohol followed by ethanol precipitation. M13mp18 RF DNA was prepared from phage-infected XL-1 Blue cells as described (20$$Google Scholar). T7 DNA polymerase was purified previously according to a published procedure (21Fischer H. Hinkle D.C. J. Biol. Chem. 1980; 255: 7956-7964Abstract Full Text PDF PubMed Google Scholar). All enzymes used for cloning and PCR were from New England Biolabs with the exception of T4 DNA ligase, which was from Boehringer Mannheim. Nucleotides were from Amersham Pharmacia Biotech. Construction of pGAD424-UvrD, pGAD424-MutL, pGBT9-UvrD, and pGBT9-MutL was described previously (14Hall M.C. Jordan J.R. Matson S.W. EMBO J. 1998; 17: 1535-1541Crossref PubMed Scopus (128) Google Scholar). The coding regions of mutS and mutH were amplified by PCR from E. coli K-12 genomic DNA using Vent DNA polymerase. Oligonucleotide primers for amplifying themutS gene contained restriction enzyme sites that allowed cloning of the mutS coding sequence into theEcoRI and BamHI sites of pGAD424 and pGBT9, creating a translational fusion with the Gal4 transcriptional activation domain and DNA binding domain, respectively, for use in the yeast two-hybrid system. In addition, these primers contained restriction enzyme sites that allowed cloning of mutS into the NdeI and SmaI sites of pCYB2 for overexpression and purification of MutS using the Impact I protein purification system. Likewise, primers for amplifying themutH coding sequence contained restriction enzyme sites that allowed cloning of mutH into the EcoRI andBamHI sites of pGAD424 and pGBT9 and into theNdeI and SapI sites of pCYB1. pET3c-MutL was constructed by subcloning the NdeI-BamHI fragment containing the mutL coding sequence from pGAD424 into theNdeI and BamHI sites of pET3c (Novagen). Deletions from each end of themutL gene in pGAD424 were constructed previously (14Hall M.C. Jordan J.R. Matson S.W. EMBO J. 1998; 17: 1535-1541Crossref PubMed Scopus (128) Google Scholar). To construct mutHΔ38N and mutHΔ10C, the appropriate portion of the mutH gene was amplified by PCR using Vent DNA polymerase. The oligonucleotide primers used in these reactions contained restriction enzyme sites that allowed cloning of each PCR product into the EcoRI and BamHI sites of pGBT9, creating a translational fusion with the Gal4 DNA binding domain. Potential interactions between mismatch repair genes were tested in yeast HF7c by cotransformation of all possible combinations of pGAD424 and pGBT9 harboring theuvrD, mutL, mutS, and mutHgenes. After selection of cotransformants on complete synthetic media lacking tryptophan and leucine, cells were transferred to complete synthetic media lacking tryptophan, leucine, and histidine and supplemented with 1 mm 3-amino-1,2,4-triazole. HF7c contains a HIS3 reporter gene that requires a two-hybrid interaction for expression. Yeast SFY526 containing a lacZreporter gene was used to confirm any interactions by monitoring β-galactosidase activity in the presence of the color-producing substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. β-Galactosidase activity was quantified (where indicated) as described by the supplier using the substrate o-nitrophenyl β-d-galactopyranoside, and results were expressed as Miller units (18Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 201-205Google Scholar). Purification of MutL from GE1752ΔuvrD was described previously (14Hall M.C. Jordan J.R. Matson S.W. EMBO J. 1998; 17: 1535-1541Crossref PubMed Scopus (128) Google Scholar). To overexpress MutL in an E. coli strain lacking a functionalmutS gene product, a 10.5-liter culture of BL21(DE3)mutS::Tn5 containing pET3c-MutL was grown at 30 °C in 2 × YT medium. Protein expression was induced with 0.5 mm isopropyl β-d-thiogalactopyranoside at an A 600 nm of 2.0, and incubation at 30 °C was continued for 5 h. Cells (118 g) were harvested by centrifugation and washed with M9 minimal medium salts. Cell lysis and protein purification were performed essentially as described previously (9Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar) with a few exceptions. First, 10% glycerol was included in all column buffers. Second, a Bio-Rex 70 column was used as the initial chromatographic step. Subsequently, the first hydroxylapatite chromatographic step was used, and the second hydroxylapatite column was eliminated. Third, a Superose 12 HR 10/30 high performance liquid chromatography sizing column (Amersham Pharmacia Biotech) was used for the final purification step instead of a Sephadex G-150 column. Storage buffer for MutL was 25 mm Tris-HCl (pH 7.5), 200 mm NaCl, 1 mm 2-mercaptoethanol, 0.1 mm EDTA, and 50% glycerol. To overexpress MutH, three 1-liter cultures of HMS174 containing pCYB1-MutH were grown at 37 °C to an A 600 nmof 1.2 in 2 ×YT medium. Protein expression was induced by the addition of isopropyl β-d-thiogalactopyranoside to 0.5 mm, and growth was continued at 30 °C for 5 h. Cells (12 g) were collected by centrifugation and resuspended in column buffer (20 mm Tris-HCl (pH 8.0), 750 mm NaCl, 0.1 mm EDTA, 0.1% Triton X-100, 10% glycerol, and 0.1 mm phenylmethylsulfonyl fluoride). Cells were lysed by sonication, and protein purification was performed using a 20-ml chitin column (4.1 cm × 4.9 cm2) equilibrated in column buffer essentially as recommended for the Impact I system. To overexpress MutH in an E. coli strain lacking a functional mutS gene product, a 10.5-liter culture of GE1752mutS::Tn5 containing pCYB1-MutH was grown at 30 °C to an optical density of 3.0 in 2 × YT medium. Protein expression was induced by the addition of isopropyl β-d-thiogalactopyranoside to 0.5 mm, and growth was continued for 5 h at 30 °C. Cells were harvested by centrifugation, washed once with M9 minimal medium salts, and resuspended in column buffer (20 mm Tris-HCl (pH 8.0), 500 mm NaCl, 0.2% Triton X-100, 0.5 mm EDTA, 10% glycerol, and 0.1 mm phenylmethylsulfonyl fluoride). Cells (70 g) were lysed by sonication and purified on a 20-ml chitin column (4.1 cm × 4.9 cm2) according to the Impact I purification protocol. The chitin column was equilibrated and washed with column buffer, and intein-induced self-cleavage was initiated with cleavage buffer (20 mm Tris-HCl (pH 8.0), 200 mm NaCl, 0.1 mm EDTA, 10% glycerol, and 100 mm 2-mercaptoethanol). The cleavage reaction was allowed to proceed for 72 h before elution of MutH from the chitin column. Pooled MutH (18 mg in 27.5 ml) was dialyzed extensively against 20 mm Tris-HCl (pH 8.0), 200 mm NaCl, 0.1 mm EDTA, and 10% glycerol to remove the 2-mercaptoethanol. MutH was precipitated with 60% ammonium sulfate and resuspended in 4 ml of 20 mm Tris-HCl (pH 8.0), 200 mm NaCl, 0.1 mm EDTA, and 10% glycerol and dialyzed against two 500-ml volumes of this buffer to remove ammonium sulfate. MutH was loaded in 1-ml aliquots onto a Superose 12 HR 10/30 sizing column at a flow rate of 0.2 ml/min to separate MutH from a prominent contaminating protein of approximately 75 kDa. Both preparations of MutH were stored in 25 mm Tris-HCl (pH 8.0), 200 mm NaCl, 0.1 mm EDTA, and 50% glycerol. To overexpress MutS, four 750-ml cultures of HMS174 containing pCYB2-MutS were grown at 37 °C to an A 600 nmof 0.7 in 2 × YT medium. Protein expression was induced by the addition of isopropyl β-d-thiogalactopyranoside to 0.5 mm, and growth was continued for an additional 5 h at 30 °C. Cells were harvested by centrifugation and resuspended in column buffer (20 mm Tris-HCl (pH 8.0), 750 mmNaCl, 0.1 mm EDTA, 0.1% Triton X-100, 10% glycerol, and 0.1 mm phenylmethylsulfonyl fluoride). Cells (9 g) were lysed by sonication and purified using a 20-ml chitin column (4.1 cm × 4.9 cm2) essentially as recommended for the Impact I system. Purified MutS was stored in 25 mm Tris-HCl (pH 8.0), 200 mm NaCl, 0.1 mm EDTA, and 50% glycerol. All protein concentrations were determined using the Bio-Rad protein assay. Because of the nature of the intein cleavage reaction in the Impact I system, purified MutS contained two extra amino acids on the COOH terminus (proline and glycine). The amino acid sequence of MutH purified using the Impact I system was identical to native MutH. The oligonucleotides 5′-GGTACCGAGTTCGAATTCG-3′ and 5′-GGTACCGAGCTCGAATTCG-3′ were used to generate covalently closed duplex M13mp18 DNA containing a single G-T mismatch (heteroduplex) and no mismatch (homoduplex), respectively. The G-T mismatch in the heteroduplex substrate disrupted a SacI site in the polylinker of M13mp18. The presence of the mismatch was confirmed by digestion of the heteroduplex substrate withSacI. Both oligonucleotides anneal to identical positions in the M13mp18 polylinker, and all manipulations used to generate the heteroduplex and homoduplex substrates were identical. Before annealing, oligonucleotides were phosphorylated using T4 polynucleotide kinase. Annealing mixtures (65 μl) contained 100 mm Tris-HCl (pH 8.0), 20 mm MgCl2, 140 pmol of oligonucleotide, and 4.6 pmol of M13mp18 ssDNA molecules. These mixtures were heated to 94 °C for 3 min and cooled 1 °C/min to 30 °C in a Perkin-Elmer 2400 thermal cycler. Components of the extension reaction were added to the annealing mixtures such that a final volume of 130 μl was achieved containing 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 8 μg/ml bovine serum albumin (BSA), 500 μm each dNTP, and 8.3 mm dithiothreitol. Extension reactions were incubated at 30 °C for 30 min with enough T7 DNA polymerase to achieve complete conversion of M13mp18 ssDNA to duplex molecules. To achieve covalently closed molecules, 750 μm ATP and 1 unit of T4 DNA ligase (Boehringer Mannheim) were added to each extension reaction. Extension reactions were pooled and covalently closed, and nicked circular DNA was separated on a CsCl/EtBr gradient as described (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 4.26-4.32Google Scholar). MutH-catalyzed endonuclease reactions (16 μl) contained 20 mm Tris-HCl (pH 7.6), 4 mm MgCl2, 20 mm NaCl, 50 μg/ml BSA, and 50 ng of the appropriate DNA substrate. When present, ATP, ATPγS, and AMP-PNP were 1.25 mm. When present, MutL and MutS were added immediately before initiation of the reactions with the indicated concentration of MutH. All protein dilutions were made in 10 mm Tris-HCl (pH 8.0). All reactions were incubated at 37 °C for 15 min and quenched with 4 μl of 5 × dye solution (25% glycerol, 100 mm EDTA, and 0.025% bromphenol blue). Reaction products were subjected to electrophoresis on 0.8% agarose gels in the presence of 0.5 μg/ml EtBr to separate covalently closed and nicked circular DNA species. Agarose gels were subsequently irradiated with a hand-held UV (254 nm) lamp for 30 min, restained for 30 min with 0.5 μg/ml EtBr, and destained with deionized and distilled water. Gels were illuminated with UV light and photographed using an Eagle Eye II still video imaging system (Stratagene). 4.5 mg of purified MutH was covalently coupled to approximately 750 μl of Affi-Gel 10 resin as described by the supplier (Bio-Rad) in 25 mm MES (pH 6.4), 200 mm NaCl, and 20% glycerol for 12 h at 4 °C. The coupling reaction was quenched with 25 mm ethanolamine (pH 8.0) for 1 h, and the resin was transferred to a chromatography column (inner diameter = 0.75 cm). The coupling efficiency was greater than 50% based on quantitation of protein in the initial column flow-through using the Bio-Rad protein assay. The column was equilibrated with affinity buffer (25 mmTris-HCl (pH 7.5), 10% glycerol, 2.5 mm 2-mercaptoethanol, and 3 mm MgCl2) containing 50 mmNaCl. Approximately 100 μg of the indicated protein, diluted to a 1-ml volume in affinity buffer plus 50 mm NaCl, was applied to the MutH affinity column at a flow rate of 10 ml/h. The column was washed four times with 500 μl of affinity buffer plus 50 mm NaCl, collecting each wash as an individual fraction. The column was eluted with four 500-μl volumes of affinity buffer plus 1 m NaCl, collecting each as an individual fraction. Fractions were analyzed for protein content by electrophoresis on a 10% polyacrylamide gel in the presence of SDS followed by staining with Coomassie Brilliant Blue. A control column containing chicken egg white lysozyme covalently coupled to Affi-Gel 10 resin was constructed previously (14Hall M.C. Jordan J.R. Matson S.W. EMBO J. 1998; 17: 1535-1541Crossref PubMed Scopus (128) Google Scholar). Experiments using this column were performed exactly as described for the MutH affinity column. Previously, we identified a physical interaction between the methyl-directed mismatch repair proteins MutL and UvrD using a yeast two-hybrid screen of an E. coli genomic library with UvrD as bait (14Hall M.C. Jordan J.R. Matson S.W. EMBO J. 1998; 17: 1535-1541Crossref PubMed Scopus (128) Google Scholar). To identify other potential interactions between components of the mismatch repair system, the mutS andmutH genes were amplified by PCR from E. coliK-12 genomic DNA and cloned into the two-hybrid system vectors pGAD424 and pGBT9 as described under "Experimental Procedures." All possible pairings of uvrD, mutL, mutS, and mutH were tested for interactions in yeast HF7c cells containing a HIS3 two-hybrid reporter gene. Although the previously described dimerization of MutL (9Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar), oligomerization of MutS (7Su S.-S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (262) Google Scholar), and interaction between MutL and MutS (9Grilley M. Welsh K.M. Su S.-S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar) were not detected using the yeast two-hybrid system (data not shown), a potential interaction between MutL and MutH was observed (Fig.1). Interestingly, the interaction between MutL and MutH was only observed when MutL was fused to the Gal4 transcriptional activation domain and MutH was fused to the Gal4 DNA binding domain. To confirm that the pGAD424-MutH construct expressing MutH as a fusion with the Gal4 transcriptional activation domain did not contain a mutation resulting in the loss of an interaction with the MutL-Gal4 DNA binding domain fusion, mutH was subcloned from pGBT9-MutH into pGAD424. Again, an interaction was not observed with the MutL-Gal4 DNA binding domain fusion. These results were confirmed using yeast SFY526, containing a lacZ reporter gene under the control of a promoter other than the HIS3 reporter gene in HF7c. The reason for the observed "polarity" in the two-hybrid interaction between MutL and MutH is not known. Purified MutH protein (Fig.2) was covalently coupled to an activated agarose resin (Affi-Gel 10) as described under "Experimental Procedures." To confirm a physical interaction between MutL and MutHin vitro, 100 μg of purified MutL was applied to the MutH affinity column. The column was washed with buffer containing 50 mm NaCl, and bound protein was eluted with buffer containing 1 m NaCl. A large fraction of the applied MutL was retained on the MutH column after the 50 mm NaCl wash steps and was eluted with 1 m NaCl (Fig.3 A). In contrast, when an identical experiment was performed using an Affi-Gel 10 column covalently coupled to chicken egg white lysozyme, the applied MutL was found exclusively in the flow-through and 50 mm NaCl wash fractions (Fig. 3 B). Therefore, MutL was specifically retained on the MutH affinity column because of a physical interaction with MutH.Figure 3MutL is specifically retained on a MutH affinity column. Approximately 100 μg of MutL (panel A), BSA (panel C), or MutS (panel D) was applied to a 750-μl Affi-Gel 10 column to which purified MutH had been covalently coupled as described under "Experimental Procedures." Likewise, 100 μg of MutL was applied to an Affi-Gel 10 column containing covalently coupled chicken egg white lysozyme (panel B). In all panels: lane 1, flow-through (FT); lanes 2–5, 50 mmNaCl wash fractions; lanes 6–9, 1 m NaCl elution fractions. Each lane contains 36 μl of the corresponding fraction. All fractions were 500 μl with the exception of the flow-through, which was 1 ml. Molecular mass markers were: rabbit muscle phosphorylase b, 97.4 kDa; BSA, 66.2 kDa.View Large Image Figure ViewerDownload (PPT) To ensure further that the interaction between MutL and MutH observed using affinity chromatography was specific, 100 μg each of BSA and MutS were applied to the MutH affinity column. Using the same experimental protocol used for MutL, neither BSA nor MutS was retained to a significant extent on the column (Fig. 3, C andD). These results support the yeast two-hybrid results and suggest that a physical interaction exists between MutL and MutH. To identify the regions of MutL and MutH responsible for the two-hybrid interaction, a series of truncations was made from the NH2 and COOH termini of both proteins. TruncatedmutL alleles were generated in pGAD424 and tested for an interaction in the presence of pGBT9-MutH in yeast SFY526 (Fig.4). Likewise, truncated mutHalleles were generated in pGBT9 and tested for an interaction in the presence of pGAD424-MutL. SFY526 contains a lacZ reporter gene encoding β-galactosidase. The relative strengths of interactions were measured using a spectrophotometric assay that monitors the cleavage of o-nitrophenyl β-d-galactopyranoside by β-galactosidase. Results are reported as Miller units (18Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 201-205Google Scholar). Removal of 293, 344, or 397 amino acids from the NH2terminus of MutL (MutLΔ293N, MutLΔ344N, and MutLΔ397N) did not eliminate the two-hybrid interaction with MutH. In contrast, removal of 438 amino acids from the NH2 terminus (MutLΔ438N) or 59 amino acids from the COOH terminus (MutLΔ59C) of MutL completely eliminated the two-hybrid interaction with MutH. These results indicate that the COOH-terminal 218 amino acids of MutL are necessary and sufficient to maintain this interaction and therefore contain the MutH interaction interface. Removal of 38 amino acids from the NH2 terminus (MutHΔ38N) or 10 amino acids from the COOH terminus (MutHΔ10C) of MutH eliminated the two-hybrid interaction with MutL. Thus, we were unable to define the interaction interface of MutH in more detail. It is possible that both ends of MutH contribute to the interaction domain. Alternatively, one or both of these truncation mutants may not be expressed or maintained as stable proteins in the yeast cells. These results are strikingly similar to those observed for the MutL-UvrD interaction (14Hall M.C. Jordan J.R. Matson S.W. EMBO J. 1998; 17: 1535-1541Crossref PubMed Scopus (128) Google Scholar). The COOH-terminal 218 amino acids of MutL were also sufficient to maintain the two-hybrid interaction with UvrD, whereas both the NH2 and COOH termini of UvrD were required for the interaction with MutL. The d(GATC)-specific endonuclease activity of MutH is relatively weak in the absence of other components of the mismatch repair system (13Welsh K.M. Lu A.-L. Clark S. Modrich P. J. Biol. Chem. 1987; 262: 15624-15629Abstract Full Text PDF PubMed Google Scholar). However, this activity is markedly stimulated in
Referência(s)