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Haemophilus influenzae and Vibrio cholerae genes for mutH are able to fully complement a mutH defect in Escherichia coli
FEMS Microbiology Letters 208 (2002) 123^128
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Haemophilus in£uenzae and Vibrio cholerae genes for mutH are able to fully complement a mutH defect in Escherichia coli Peter Friedho¡ *, Babak Sheybani, Evangelos Thomas, Christian Merz, Alfred Pingoud Institut fu«r Biochemie (FB 08), Justus-Liebig-Universita«t, D-35392 Giessen, Germany Received 13 December 2001; accepted 18 December 2001 First published online 31 January 2002
Abstract MutH, MutL and MutS are essential components of the mismatch repair system in Escherichia coli. Whereas mutS and mutL genes are found in most organisms, the mutH gene is limited to some proteobacteria. We show here that the cloned genes of MutH from Vibrio cholerae and Haemophilus influenzae are able to fully complement a mutH defect in E. coli. Moreover, the purified proteins were shown to be dam methylation sensitive endonucleases, which can be activated by the E. coli MutL protein. These results allow to narrow down regions that are important for the interaction of MutH with MutL. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Mismatch repair ; Endonuclease ; Genetic complementation; Vibrio cholerae; Haemophilus in£uenzae
1. Introduction Mismatch repair is the primary mechanism for repair of replication errors in most living organisms, enhancing the ¢delity of DNA replication up to a factor of 1000 [1]. In Escherichia coli, mismatch repair is initiated by MutS binding to a mismatch followed by ATP hydrolysis and MutL binding, which leads to the activation of the strand discriminating endonuclease MutH to cleave the newly synthesized, unmethylated daughter strand at hemimethylated GATC sites. Proteins of the MutS and MutL family have been found in most sequenced genomes and, therefore, the principal mechanism of mismatch recognition and repair is believed to be evolutionarily conserved [2]. In contrast, homologs of the endonuclease MutH are limited to a small group of proteobacteria from the Q-subdivision. Therefore, the mechanism of strand discrimination mentioned above has to be di¡erent in most other prokaryotic and eukaryotic organisms, including humans.
* Corresponding author. Tel. : +49 (641) 99-35407; Fax: +49 (641) 99-35409. E-mail address : peter.friedho¡@chemie.bio.uni-giessen.de (P. Friedho¡).
In vitro, the activation of MutH can occur in a mismatch-dependent manner involving MutS and MutL [3] or in a mismatch-independent manner by MutL [4,5]. Physical interaction between MutH and MutL has been demonstrated by two hybrid assays [5,6], by the ability of MutH to bind to a MutL column [5] as well as by the MutL-mediated binding of MutH to a MutS column [7]. Although the structures of E. coli MutH [8] and the Nterminal domain of E. coli MutL [4] are known, the interaction sites of MutH and MutL have not yet been identi¢ed. It has been proposed that the less conserved C-terminal tail of MutH is important for the interaction with MutL [8]. Support for this was provided by the ¢nding that the deletion of the last 10 amino acid residues of MutH results in a loss of interaction with MutL [5]. More recently, it was shown that deletion of the last 15 amino acid residues results in a loss of in vivo function in a complementation assay, whereas the last ¢ve amino acid residues could be deleted without loss of function in vivo and in vitro [9]. Therefore, the pentapeptide (Ala-220^ Arg-224) was supposed to be the interaction region for MutL. However, the deletion of the last 15 amino acid residues resulted in a non-functional endonucleases, thereby making it di¤cult to investigate the e¡ect of the deletion on the activation of MutH by MutL. Hence, the role of the C-terminal tail of MutH in the process of activation by MutL remains elusive.
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 5 8 8 - 2
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In order get an idea which amino acid residues are involved in the interaction between MutH and MutL, we analyzed whether the related (60 and 56% identity) MutH proteins from two proteobacteria, Haemophilus in£uenzae and Vibrio cholerae, are able to substitute for the E. coli MutH protein in vitro and in vivo. Our ¢nding that the heterologous mutH genes can function in E. coli suggests that regions conserved in the MutH proteins from these three organisms are responsible for the MutL^MutH interaction, 2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions The bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains were grown in LB broth (Gibco BRL) in liquid culture or on agar plates at 37³C. The antibiotic concentrations used were: ampicillin 100 Wg ml31 , kanamycin 30 Wg ml31 and rifampicin 100 Wg ml31 . 2.2. Enzymes and chemicals Taq and Pfu DNA polymerase were purchased from Promega. T4 polynucleotide kinase, T4 DNA ligase and restriction endonucleases were from AGS. Dam methylase and V-exonuclease were purchased from NEB. [K-32 P]dATP was from NEN. Oligodeoxynucleotides were obtained form MWG-Biotech. 2.3. Site-directed mutagenesis Site-directed mutagenesis of E. coli MutH was carried out essentially as described earlier [10], with plasmid pTX417 as a template. Mutations were veri¢ed by sequencing the whole mutH gene.
2.4. PCR ampli¢cation and cloning of V. cholerae and H. in£uenzae mutH genes The 666-bp open reading frame of the V. cholerae mutH gene was ampli¢ed from V. cholerae O1 genomic DNA (clinical isolate 413854, University of Giessen, kindly provided by Prof. Dr. T. Chakraborty) by PCR with Pfu DNA polymerase using primers with a 5P-overhang, which introduce NdeI and XhoI sites. The sequences of the sense (5P-gcg gca gcc atA TGA AAC CAG CAC CGA CGA CTC AA-3P) and antisense primer (5P-gcc cca tcc tcg agC TAG GCG TAA TAG CGC TGC AGA ATT T-3P) were deduced from the genomic sequence of V. cholerae El Tor N16961 [11]. The cloning sites are underlined and the 5Poverhang indicated by lower case letters. The ampli¢cation product was trimmed with NdeI and XhoI and ligated into the T7 expression vector, pET-15b, that had been cleaved before with the corresponding restriction enzymes. DNA sequencing of the cloned V. cholerae mutH gene showed no di¡erences with the published genomic sequence. Similarly, the mutH gene of H. in£uenzae was cloned into the NdeI and BamHI sites of pET-15b using the sense (5P-cgg cag cca tAT GA TTC CAC AAA CCC TTG A-3P) and antisense primer (5P-gcc gga tcc caa gtT AAA GCG ACT TTG TTT CTA-3P) and genomic DNA from H. in£uenzae (strain ATCC 49247 kindly provided by Dr. R. Fislage). Primer sequences were deduced from the genomic sequence of H. in£uenzae Rd [12]. 2.5. Complementation of a mutH mutator phenotype The complementation assay is based on the mutator phenotype, which occurs when the bacteria lack the MutH protein. The mutation frequency is monitored by the frequency of mutations in the polB gene, causing resistance against rifampicin [9]. Single colonies of CC106 (wild-type) or TX2928 (mutH3 ) transformed with pET15b or derivatives thereof were grown in 3-ml LB cultures
Table 1 Strains and plasmids
E. coli strains CC106 TX2928 HMS174(VDE3) Plasmids pUC8 pET-15b pTX412 pTX417 pTX418 pTX417(D70A) pTX417(E77A) pTX417(K79A) pET-Hin-H pET-Vch-H
Relevant genotype, description
Reference, source
Wild-type mutHLS P90C [arav[lac-pro)XIII (FPlaciZ proB ] CC106 but mutH471 : :tn5;Kmr r F3 recA r3 K12 mK12 Rif
[21] [13] Novagen
Apr His-tag vector; Apr pET-15b (E. coli K-12 P (his6-tag-mutS )Hyb; Apr pET-15b (E. coli K-12 P (his6-tag-mutH )Hyb; Apr pET-15b (E. coli K-12 P (his6-tag-mutL )Hyb; Apr pTX417 with amino acid exchange D70A pTX417 with amino acid exchange E77A pTX417 with amino acid exchange K79A pET-15b (H. in£uenzae P (his6-tag-mutH )Hyb; Apr pET-15b (V. cholerae P (his6-tag-mutH )Hyb; Apr
[22] Novagen [13] [13] [13] this work this work this work this work this work
Apr , Kmr and Rifr indicate resistance to ampicillin, kanamycin and rifampicin, respectively.
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containing 100 Wg ml31 ampicillin, overnight at 37³C. Aliquots of 50 Wl from the undiluted or 1036 diluted culture were plated on LB agar containing 25 Wg ml31 ampicillin with or without 100 Wg ml31 rifampicin, respectively. Colonies were counted after overnight incubation at 37³C. 2.6. Puri¢cation of recombinant MutH, MutL and MutS proteins Recombinant His6 -tagged MutH, MutL and MutS proteins were expressed in HMS174(VDE3) and puri¢ed essentially by Ni^NTA chromatography as described earlier [13]. Protein concentrations were determined using the theoretical extinction coe¤cients [14]. 2.7. Circular dichroism (CD) CD spectra of MutH proteins (3 WM) in a 50 mM sodium phosphate bu¡er, pH 8.0, were recorded on a Jasco J-710 dichrograph at ambient temperature in a cylindrical cuvette of path length 0.05 cm. 2.8. Endonuclease activity test Endonuclease activity of MutH proteins was assayed with unmethylated or fully methylated pUC8 DNA. MutH (0.03^1 WM) was incubated with 20 ng Wl31 pUC8 in 10 mM Tris^HCl, 10 mM MgCl2 , 0.75 mM ATP and 0.05 mg ml31 BSA, pH 7.5, at 37³C, in the absence and presence of E. coli MutL protein (1^2 WM). Cleavage reactions were analyzed by 1.2% agarose gel electrophoresis. 2.9. Mismatch provoked DNA cleavage assay A 359-bp hemimethylated heteroduplex DNA containing a G/T mismatch at position 260 and a single hemimethylated GATC site at position 87, labeled in the unmethylated strand with 32 P, was generated by a
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modi¢cation of a method described earlier [15]. MutS (2 WM), MutL (1 WM) and MutH (0.1 WM) were incubated with 10 nM heteroduplex DNA in 10 mM Tris^HCl, 0.05 mg ml31 BSA, 100 mM KCl, 1.25 mM ATP, pH 7.9, for 10 min at 37³C. Reactions were stopped by addition of 1 volume formamide and subjected to gel electrophoresis on 6% polyacrylamide gels containing 6 M urea and then analyzed with an Instant Imager (Canberra Packard). 3. Results and discussion In this study the results of complementation experiments with the E. coli mismatch repair system (mutH3 , mutL , mutS ) and mutH genes from V. cholerae and H. in£uenzae are reported. It was not clear a priori whether a mutH gene from another proteobacteria functions in the context of the E. coli mismatch repair system, since the sequence alignment of the MutH proteins revealed that only Leu-222 of the above-mentioned C-terminal pentapeptide (cf. Section 1), considered to be essential for the MutH^MutL interaction, is conserved in all MutH proteins (see Fig. 1). Moreover, studies on complementation of mut-de¢cient E. coli strains with mut genes from other bacteria reported successful complementation in the case of the closely related mutL and mutS genes (85 and 88% identity) from Salmonella typhimurium [16], reduced complementation with the mutS gene of Pseudomonas putida (58% identity) [17] and no complementation with mutS or mutL genes from more distantly related organisms (Thermus aquaticus and Streptococcus pneumoniae) [18,19]. 3.1. Cloning, sequencing of MutH from V. cholerae and H. in£uenzae Cloning of the mutH genes from V. cholerae and H. in£uenzae was performed by PCR with primer sequences derived from the 5P- and 3P-end of the open reading frames
Table 2 In vivo complementation screen for MutH proteins Strain
Relevant genotype of plasmid
Relative mutation frequency (average)a
% Errorb
nc
CC106 CC106 CC106 TX2928 TX2928 TX2928 TX2928 TX2928 TX2928 TX2928
pET-15b; vector control Eco-H Eco-H (E77A) pET-15b, vector control Eco-H Hin-H Vch-H Eco-H (E77A) Eco-H (D70A) Eco-H (K79A)
1.0 0.3 5.0 257 1.1 1.8 1.2 145 165 123
96 106 46 65 124 93 88 42 73 58
11 7 5 33 38 9 11 4 5 4
a
The relative mutation frequency is given with respect to the mutation frequency of the wild-type strain CC106 with the plasmid pET-15b, which is the average number of rifampicin-resistant cells calculated per average number of viable cells, i.e. 4.6U1038 . b Errors are given as % standard deviation of the mutation frequency. c n is the number of independent experiments.
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Fig. 1. Sequence alignment and surface residue conservation of MutH proteins. A: Protein sequences were obtained via the NCBI Blast Interface and aligned using Clustal W [23]. Part of the sequences of MutH from Enterobacteriaceae (E. coli; Eco accession number NP_417308); Vibrionaceae (V. cholerae; Vch; NP_230317), Pasteurellaceae (H. in£uenzae, Hin; accession number NP_438565), Alteromonadaceae (Shewanella putrefaciens ; Spu) and Legionellaceae (Legionella pneumoniae; Lpn) as well as from Sau3AI (P16667), LlaKR2I (AAC77902) and Sth368I (CAC67532) are shown. Fully conserved residues are in black, and only partially conserved residues in gray. The active site residues D70, E77 and K79 are indicated by circles, the C-terminal pentapeptide containing L222 (indicated by a square) previously suggested to be involved in the interaction with MutL is boxed [9]. B: CK-carbon trace of MutH (pdb code 2azo chain B). The positions of the active site residues D70, E77 and K79 as well as L222 are indicated. C,D: Surface map of MutH. Residues conserved in both MutH and the related restriction endonucleases (C), or only in MutH but not in the restriction endonucleases (D) are colored black.
of the genomic DNA sequences [11,12] (see Section 2). One of these primers included the coding sequence for an N-terminal His6 -tag. DNA sequencing of the cloned genes revealed only one exchange of the non-conserved Arg168 with Leu in the case of the MutH protein from H. in£uenzae (in comparison with the published sequence). 3.2. In vivo complementation of a mutH-de¢cient E. coli strain The function of the MutH proteins from V. cholerae and H. in£uenzae was assessed by an in vivo complementation assay using a mutH-de¢cient E. coli strain. As a control for our in vivo complementation assay we used variants of the E. coli MutH protein, in which one of the three catalytic amino acid residues, Asp-70, Glu-77 or Lys-79, had been substituted by alanine. These variants are no longer able to complement a mutH3 -induced mutator phenotype in vivo but are interfering with the mismatch repair system in a wild-type E. coli strain (Table 2). Moreover, in vitro these variants were catalytically inactive though they were still able to bind to DNA (unpublished results, Merz, C. and Friedho¡, P). As can be seen in Table 2, the recombinant His6 -tagged MutH proteins from E. coli, V. cholerae and H. in£uenzae were all able to complement the mutH-de¢cient E. coli strain back to the wild-type level. Hence the amino acid di¡erences (60 and
56% identity with respect to the E. coli MutH protein) do not a¡ect the function of the endonucleases from the other organisms in the context of the E. coli mismatch repair system. 3.3. Expression and puri¢cation of MutH from V. cholerae and H. in£uenzae To demonstrate that the MutH proteins from V. cholerae and H. in£uenzae are functionally interacting with the E. coli mismatch repair proteins, especially MutL, we expressed the His6 -tagged proteins in E. coli. The proteins could be puri¢ed as soluble proteins to over 95% homogeneity as judged by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) (Fig. 2A). Moreover, the CD spectra of the three MutH proteins from E. coli, H. in£uenzae, and V. cholerae were identical, indicating that all three proteins adopt a similar overall fold as expected from the protein sequence similarity (Fig. 2B). 3.4. MutH from V. cholerae and H. in£uenzae functionally interact with the E. coli MutL and MutS proteins in vitro MutH endonuclease activity was monitored by the conversion of unmethylated plasmid from the supercoiled to the open circular form. At high enzyme concentrations
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V. cholerae and H. in£uenzae MutH showed cleavage of unmethylated plasmid DNA but no cleavage of fully methylated plasmid DNA, similarly to E. coli MutH (data not shown). At low protein concentration (30 nM), all three MutH proteins showed almost no DNA cleavage activity (Fig. 3). However, upon addition of E. coli MutL all three proteins were stimulated to cleave the supercoiled plasmid DNA to a comparable extent. Moreover, both heterologous MutH proteins were also activated by the E. coli MutS and MutL proteins in a mismatch-dependent manner under conditions where no mismatch-independent activation by MutL is observed (Fig. 4). Therefore, both heterologous MutH proteins are able to functionally interact with the E. coli MutL and MutS proteins in vitro and in vivo, suggesting that the di¡erences in the amino acid sequence between the MutH proteins, especially in the Cterminal tail, are not important for their interaction with
Fig. 3. In vitro activation of heterologous MutH proteins by MutL from E. coli. Nicking of 10 Wg ml3l unmethylated pUC8 DNA by 30 nM MutH from E. coli (Eco-H), H. in£uenzae (Hin-H) or V. cholerae (Vch-H) was measured in the absence or presence of 1 WM E. coli MutL. sc is the supercoiled and oc the open circular form of plasmid pUC8. The sc DNA is the substrate which is cleaved by MutH to give the open circular form of the plasmid. It is obvious that E. coli MutL can activate all three di¡erent MutH proteins to a comparable extent.
the other Mut proteins and, therefore, for the function of the protein. 3.5. Conclusions
Fig. 2. SDS^PAGE and CD analysis of recombinant MutH proteins. A: Increasing amounts (1, 2, 5 and 10 Wg) of recombinant E. coli MutH protein (Eco-H) and approximately 5 Wg of H. in£uenzae (HinH) and V. cholerae MutH protein (Vch-H) were subjected to SDS^ PAGE. The molecular masses (kg mol31 ) of marker proteins are indicated on the left. B: CD spectra of 3 WM MutH from E. coli (squares), H. in£uenzae (circles) and V. cholerae (triangles).
Our results do not support the claim that the C-terminal end of E. coli MutH is important for the interaction of MutH with MutL. The only fully conserved residue (Leu222) in the C-terminal tail of MutH is buried (less than 10% accessible surface area in the structure of MutH). Moreover, since at the position of Leu-222 there are also hydrophobic residues in three distantly related restriction endonucleases (Sau3AI, LlaKR2I and Sth368I) [20], this residue probably has a structural rather than a functional role in the interaction with MutL. This conclusion suggests that in the analysis of the interaction site of MutH with MutL one should focus on (surface) residues, which are conserved in the MutH protein family, and are not present in the distantly related restriction endonucleases (cf. Fig. 1).
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Fig. 4. Mismatch-dependent activation of heterologous MutH protein. Endonuclease activity of 100 nM MutH from E. coli (Eco-H), V. cholerae (Vch-H) and H. in£uenzae (Hin-H) was measured in the presence of 1 WM E. coli MutL (Eco-L) and in the absence or presence of 2 WM E. coli MutS protein (Eco-S) by analyzing nicking of a hemimethlyated DNA with a single G/T mismatch at position 260 and a GATC site at position 87 (cf. Section 2). It is obvious that a mismatch-dependent activation of all three di¡erent MutH proteins can occur.
Acknowledgements We thank Dr. Malcolm E. Winkler (University of Texas Medical School, Houston, TX, USA) for providing strains expressing E. coli His-tagged MutHLS proteins. Preliminary sequence data of MutH proteins were obtained from `The Institute for Genomic Research' website at http:// www.tigr.org and the `Columbia Genome Center'. The expert technical assistance of Ina Steindorf is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (Pi-122/12-3 and Pi-122/13-2), the Dr. Herbert Stolzenberg Stiftung and the Fonds der Chemischen Industrie. References [1] Modrich, P. and Lahue, R. (1996) Mismatch repair in replication ¢delity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65, 101^133. [2] Eisen, J.A. and Hanawalt, P.C. (1999) A phylogenomic study of DNA repair genes, proteins, and processes. Mutat. Res. 435, 171^ 213. [3] Au, K.G., Welsh, K. and Modrich, P. (1992) Initiation of methyldirected mismatch repair. J. Biol. Chem. 267, 12142^12148. [4] Ban, C. and Yang, W. (1998) Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis. Cell 95, 541^552. [5] Hall, M.C. and Matson, S.W. (1999) The Escherichia coli MutL protein physically interacts with MutH and stimulates the MutHassociated endonuclease activity. J. Biol. Chem. 274, 1306^1312.
[6] Mansour, C.A., Doiron, K.M.J. and Cupples, C.G. (2001) Characterization of functional interactions among the Escherichia coli mismatch repair proteins using a bacterial two-hybrid assay. Mutat. Res. 485, 331^338. [7] Spampinato, C. and Modrich, P. (2000) The MutL ATPase is required for mismatch repair. J. Biol. Chem. 275, 9861^9869. [8] Ban, C. and Yang, W. (1998) Structural basis for MutH activation in E.coli mismatch repair and relationship of MutH to restriction endonucleases. EMBO J. 17, 1526^1534. [9] Loh, T., Murphy, K.C. and Marinus, M.G. (2001) Mutational analysis of the MutH protein from Escherichia coli. J. Biol. Chem. 276, 12113^12119. [10] Kirsch, R.D. and Joly, E. (1998) An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res. 26, 1848^1850. [11] Heidelberg, J.F. et al. (2000) DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406, 477^483. [12] Fleischmann, R.D. et al. (1995) Whole-genome random sequencing and assembly of Haemophilus in£uenzae Rd. Science 269, 496^512. [13] Feng, G. and Winkler, M.E. (1995) Single-step puri¢cations of His6 MutH, His6 -MutL and His6 -MutS repair proteins of Escherichia coli K-12. BioTechniques 19, 956^965. [14] Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995) How to measure and predict the molar absorption coe¤cient of a protein. Protein Sci. 4, 2411^2423. [15] Prolla, T.A., Pang, Q., Alani, E., Kolodner, R.D. and Liskay, R.M. (1994) MLH1, PMS1, and MSH2 interactions during the initiation of DNA mismatch repair in yeast. Science 265, 1091^1093. [16] Haber, L.T., Pang, P.P., Sobell, D.I., Mankovich, J.A. and Walker, G.C. (1988) Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae. J. Bacteriol. 170, 197^202. [17] Kurusu, Y., Narita, T., Suzuki, M. and Watanabe, T. (2000) Genetic analysis of an incomplete mutS gene from Pseudomonas putida. J. Bacteriol. 182, 5278^5279. [18] Prudhomme, M., Martin, B., Mejean, V. and Claverys, J.P. (1989) Nucleotide sequence of the Streptococcus pneumoniae hexB mismatch repair gene: homology of HexB to MutL of Salmonella typhimurium and to PMS1 of Saccharomyces cerevisiae. J. Bacteriol. 171, 5332^ 5338. [19] Biswas, I. and Hsieh, P. (1996) Identi¢cation and characterization of a thermostable MutS homolog from Thermus aquaticus. J. Biol. Chem. 271, 5040^5048. [20] Friedho¡, P., Lurz, R., Luder, G. and Pingoud, A. (2001) Sau3AI, a monomeric type II restriction endonuclease that dimerizes on the DNA and thereby induces DNA loops. J. Biol. Chem. 276, 23581^ 23588. [21] Cupples, C.G. and Miller, J.H. (1989) A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc. Natl. Acad. Sci. USA 86, 5345^5349. [22] Vieira, J. and Messing, J. (1982) The pUC plasmids, an M13mp7derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259^268. [23] Higgins, D.G., Thompson, J.D. and Gibson, T.J. (1996) Using CLUSTAL for multiple sequence alignments. Methods Enzymol. 266, 383^402.
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Haemophilus in£uenzae and Vibrio cholerae genes for mutH are able to fully complement a mutH defect in Escherichia coli Peter Friedho¡ *, Babak Sheybani, Evangelos Thomas, Christian Merz, Alfred Pingoud Institut fu«r Biochemie (FB 08), Justus-Liebig-Universita«t, D-35392 Giessen, Germany Received 13 December 2001; accepted 18 December 2001 First published online 31 January 2002
Abstract MutH, MutL and MutS are essential components of the mismatch repair system in Escherichia coli. Whereas mutS and mutL genes are found in most organisms, the mutH gene is limited to some proteobacteria. We show here that the cloned genes of MutH from Vibrio cholerae and Haemophilus influenzae are able to fully complement a mutH defect in E. coli. Moreover, the purified proteins were shown to be dam methylation sensitive endonucleases, which can be activated by the E. coli MutL protein. These results allow to narrow down regions that are important for the interaction of MutH with MutL. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Mismatch repair ; Endonuclease ; Genetic complementation; Vibrio cholerae; Haemophilus in£uenzae
1. Introduction Mismatch repair is the primary mechanism for repair of replication errors in most living organisms, enhancing the ¢delity of DNA replication up to a factor of 1000 [1]. In Escherichia coli, mismatch repair is initiated by MutS binding to a mismatch followed by ATP hydrolysis and MutL binding, which leads to the activation of the strand discriminating endonuclease MutH to cleave the newly synthesized, unmethylated daughter strand at hemimethylated GATC sites. Proteins of the MutS and MutL family have been found in most sequenced genomes and, therefore, the principal mechanism of mismatch recognition and repair is believed to be evolutionarily conserved [2]. In contrast, homologs of the endonuclease MutH are limited to a small group of proteobacteria from the Q-subdivision. Therefore, the mechanism of strand discrimination mentioned above has to be di¡erent in most other prokaryotic and eukaryotic organisms, including humans.
* Corresponding author. Tel. : +49 (641) 99-35407; Fax: +49 (641) 99-35409. E-mail address : peter.friedho¡@chemie.bio.uni-giessen.de (P. Friedho¡).
In vitro, the activation of MutH can occur in a mismatch-dependent manner involving MutS and MutL [3] or in a mismatch-independent manner by MutL [4,5]. Physical interaction between MutH and MutL has been demonstrated by two hybrid assays [5,6], by the ability of MutH to bind to a MutL column [5] as well as by the MutL-mediated binding of MutH to a MutS column [7]. Although the structures of E. coli MutH [8] and the Nterminal domain of E. coli MutL [4] are known, the interaction sites of MutH and MutL have not yet been identi¢ed. It has been proposed that the less conserved C-terminal tail of MutH is important for the interaction with MutL [8]. Support for this was provided by the ¢nding that the deletion of the last 10 amino acid residues of MutH results in a loss of interaction with MutL [5]. More recently, it was shown that deletion of the last 15 amino acid residues results in a loss of in vivo function in a complementation assay, whereas the last ¢ve amino acid residues could be deleted without loss of function in vivo and in vitro [9]. Therefore, the pentapeptide (Ala-220^ Arg-224) was supposed to be the interaction region for MutL. However, the deletion of the last 15 amino acid residues resulted in a non-functional endonucleases, thereby making it di¤cult to investigate the e¡ect of the deletion on the activation of MutH by MutL. Hence, the role of the C-terminal tail of MutH in the process of activation by MutL remains elusive.
0378-1097 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 5 8 8 - 2
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In order get an idea which amino acid residues are involved in the interaction between MutH and MutL, we analyzed whether the related (60 and 56% identity) MutH proteins from two proteobacteria, Haemophilus in£uenzae and Vibrio cholerae, are able to substitute for the E. coli MutH protein in vitro and in vivo. Our ¢nding that the heterologous mutH genes can function in E. coli suggests that regions conserved in the MutH proteins from these three organisms are responsible for the MutL^MutH interaction, 2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions The bacterial strains and plasmids used in this study are listed in Table 1. E. coli strains were grown in LB broth (Gibco BRL) in liquid culture or on agar plates at 37³C. The antibiotic concentrations used were: ampicillin 100 Wg ml31 , kanamycin 30 Wg ml31 and rifampicin 100 Wg ml31 . 2.2. Enzymes and chemicals Taq and Pfu DNA polymerase were purchased from Promega. T4 polynucleotide kinase, T4 DNA ligase and restriction endonucleases were from AGS. Dam methylase and V-exonuclease were purchased from NEB. [K-32 P]dATP was from NEN. Oligodeoxynucleotides were obtained form MWG-Biotech. 2.3. Site-directed mutagenesis Site-directed mutagenesis of E. coli MutH was carried out essentially as described earlier [10], with plasmid pTX417 as a template. Mutations were veri¢ed by sequencing the whole mutH gene.
2.4. PCR ampli¢cation and cloning of V. cholerae and H. in£uenzae mutH genes The 666-bp open reading frame of the V. cholerae mutH gene was ampli¢ed from V. cholerae O1 genomic DNA (clinical isolate 413854, University of Giessen, kindly provided by Prof. Dr. T. Chakraborty) by PCR with Pfu DNA polymerase using primers with a 5P-overhang, which introduce NdeI and XhoI sites. The sequences of the sense (5P-gcg gca gcc atA TGA AAC CAG CAC CGA CGA CTC AA-3P) and antisense primer (5P-gcc cca tcc tcg agC TAG GCG TAA TAG CGC TGC AGA ATT T-3P) were deduced from the genomic sequence of V. cholerae El Tor N16961 [11]. The cloning sites are underlined and the 5Poverhang indicated by lower case letters. The ampli¢cation product was trimmed with NdeI and XhoI and ligated into the T7 expression vector, pET-15b, that had been cleaved before with the corresponding restriction enzymes. DNA sequencing of the cloned V. cholerae mutH gene showed no di¡erences with the published genomic sequence. Similarly, the mutH gene of H. in£uenzae was cloned into the NdeI and BamHI sites of pET-15b using the sense (5P-cgg cag cca tAT GA TTC CAC AAA CCC TTG A-3P) and antisense primer (5P-gcc gga tcc caa gtT AAA GCG ACT TTG TTT CTA-3P) and genomic DNA from H. in£uenzae (strain ATCC 49247 kindly provided by Dr. R. Fislage). Primer sequences were deduced from the genomic sequence of H. in£uenzae Rd [12]. 2.5. Complementation of a mutH mutator phenotype The complementation assay is based on the mutator phenotype, which occurs when the bacteria lack the MutH protein. The mutation frequency is monitored by the frequency of mutations in the polB gene, causing resistance against rifampicin [9]. Single colonies of CC106 (wild-type) or TX2928 (mutH3 ) transformed with pET15b or derivatives thereof were grown in 3-ml LB cultures
Table 1 Strains and plasmids
E. coli strains CC106 TX2928 HMS174(VDE3) Plasmids pUC8 pET-15b pTX412 pTX417 pTX418 pTX417(D70A) pTX417(E77A) pTX417(K79A) pET-Hin-H pET-Vch-H
Relevant genotype, description
Reference, source
Wild-type mutHLS P90C [arav[lac-pro)XIII (FPlaciZ proB ] CC106 but mutH471 : :tn5;Kmr r F3 recA r3 K12 mK12 Rif
[21] [13] Novagen
Apr His-tag vector; Apr pET-15b (E. coli K-12 P (his6-tag-mutS )Hyb; Apr pET-15b (E. coli K-12 P (his6-tag-mutH )Hyb; Apr pET-15b (E. coli K-12 P (his6-tag-mutL )Hyb; Apr pTX417 with amino acid exchange D70A pTX417 with amino acid exchange E77A pTX417 with amino acid exchange K79A pET-15b (H. in£uenzae P (his6-tag-mutH )Hyb; Apr pET-15b (V. cholerae P (his6-tag-mutH )Hyb; Apr
[22] Novagen [13] [13] [13] this work this work this work this work this work
Apr , Kmr and Rifr indicate resistance to ampicillin, kanamycin and rifampicin, respectively.
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containing 100 Wg ml31 ampicillin, overnight at 37³C. Aliquots of 50 Wl from the undiluted or 1036 diluted culture were plated on LB agar containing 25 Wg ml31 ampicillin with or without 100 Wg ml31 rifampicin, respectively. Colonies were counted after overnight incubation at 37³C. 2.6. Puri¢cation of recombinant MutH, MutL and MutS proteins Recombinant His6 -tagged MutH, MutL and MutS proteins were expressed in HMS174(VDE3) and puri¢ed essentially by Ni^NTA chromatography as described earlier [13]. Protein concentrations were determined using the theoretical extinction coe¤cients [14]. 2.7. Circular dichroism (CD) CD spectra of MutH proteins (3 WM) in a 50 mM sodium phosphate bu¡er, pH 8.0, were recorded on a Jasco J-710 dichrograph at ambient temperature in a cylindrical cuvette of path length 0.05 cm. 2.8. Endonuclease activity test Endonuclease activity of MutH proteins was assayed with unmethylated or fully methylated pUC8 DNA. MutH (0.03^1 WM) was incubated with 20 ng Wl31 pUC8 in 10 mM Tris^HCl, 10 mM MgCl2 , 0.75 mM ATP and 0.05 mg ml31 BSA, pH 7.5, at 37³C, in the absence and presence of E. coli MutL protein (1^2 WM). Cleavage reactions were analyzed by 1.2% agarose gel electrophoresis. 2.9. Mismatch provoked DNA cleavage assay A 359-bp hemimethylated heteroduplex DNA containing a G/T mismatch at position 260 and a single hemimethylated GATC site at position 87, labeled in the unmethylated strand with 32 P, was generated by a
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modi¢cation of a method described earlier [15]. MutS (2 WM), MutL (1 WM) and MutH (0.1 WM) were incubated with 10 nM heteroduplex DNA in 10 mM Tris^HCl, 0.05 mg ml31 BSA, 100 mM KCl, 1.25 mM ATP, pH 7.9, for 10 min at 37³C. Reactions were stopped by addition of 1 volume formamide and subjected to gel electrophoresis on 6% polyacrylamide gels containing 6 M urea and then analyzed with an Instant Imager (Canberra Packard). 3. Results and discussion In this study the results of complementation experiments with the E. coli mismatch repair system (mutH3 , mutL , mutS ) and mutH genes from V. cholerae and H. in£uenzae are reported. It was not clear a priori whether a mutH gene from another proteobacteria functions in the context of the E. coli mismatch repair system, since the sequence alignment of the MutH proteins revealed that only Leu-222 of the above-mentioned C-terminal pentapeptide (cf. Section 1), considered to be essential for the MutH^MutL interaction, is conserved in all MutH proteins (see Fig. 1). Moreover, studies on complementation of mut-de¢cient E. coli strains with mut genes from other bacteria reported successful complementation in the case of the closely related mutL and mutS genes (85 and 88% identity) from Salmonella typhimurium [16], reduced complementation with the mutS gene of Pseudomonas putida (58% identity) [17] and no complementation with mutS or mutL genes from more distantly related organisms (Thermus aquaticus and Streptococcus pneumoniae) [18,19]. 3.1. Cloning, sequencing of MutH from V. cholerae and H. in£uenzae Cloning of the mutH genes from V. cholerae and H. in£uenzae was performed by PCR with primer sequences derived from the 5P- and 3P-end of the open reading frames
Table 2 In vivo complementation screen for MutH proteins Strain
Relevant genotype of plasmid
Relative mutation frequency (average)a
% Errorb
nc
CC106 CC106 CC106 TX2928 TX2928 TX2928 TX2928 TX2928 TX2928 TX2928
pET-15b; vector control Eco-H Eco-H (E77A) pET-15b, vector control Eco-H Hin-H Vch-H Eco-H (E77A) Eco-H (D70A) Eco-H (K79A)
1.0 0.3 5.0 257 1.1 1.8 1.2 145 165 123
96 106 46 65 124 93 88 42 73 58
11 7 5 33 38 9 11 4 5 4
a
The relative mutation frequency is given with respect to the mutation frequency of the wild-type strain CC106 with the plasmid pET-15b, which is the average number of rifampicin-resistant cells calculated per average number of viable cells, i.e. 4.6U1038 . b Errors are given as % standard deviation of the mutation frequency. c n is the number of independent experiments.
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Fig. 1. Sequence alignment and surface residue conservation of MutH proteins. A: Protein sequences were obtained via the NCBI Blast Interface and aligned using Clustal W [23]. Part of the sequences of MutH from Enterobacteriaceae (E. coli; Eco accession number NP_417308); Vibrionaceae (V. cholerae; Vch; NP_230317), Pasteurellaceae (H. in£uenzae, Hin; accession number NP_438565), Alteromonadaceae (Shewanella putrefaciens ; Spu) and Legionellaceae (Legionella pneumoniae; Lpn) as well as from Sau3AI (P16667), LlaKR2I (AAC77902) and Sth368I (CAC67532) are shown. Fully conserved residues are in black, and only partially conserved residues in gray. The active site residues D70, E77 and K79 are indicated by circles, the C-terminal pentapeptide containing L222 (indicated by a square) previously suggested to be involved in the interaction with MutL is boxed [9]. B: CK-carbon trace of MutH (pdb code 2azo chain B). The positions of the active site residues D70, E77 and K79 as well as L222 are indicated. C,D: Surface map of MutH. Residues conserved in both MutH and the related restriction endonucleases (C), or only in MutH but not in the restriction endonucleases (D) are colored black.
of the genomic DNA sequences [11,12] (see Section 2). One of these primers included the coding sequence for an N-terminal His6 -tag. DNA sequencing of the cloned genes revealed only one exchange of the non-conserved Arg168 with Leu in the case of the MutH protein from H. in£uenzae (in comparison with the published sequence). 3.2. In vivo complementation of a mutH-de¢cient E. coli strain The function of the MutH proteins from V. cholerae and H. in£uenzae was assessed by an in vivo complementation assay using a mutH-de¢cient E. coli strain. As a control for our in vivo complementation assay we used variants of the E. coli MutH protein, in which one of the three catalytic amino acid residues, Asp-70, Glu-77 or Lys-79, had been substituted by alanine. These variants are no longer able to complement a mutH3 -induced mutator phenotype in vivo but are interfering with the mismatch repair system in a wild-type E. coli strain (Table 2). Moreover, in vitro these variants were catalytically inactive though they were still able to bind to DNA (unpublished results, Merz, C. and Friedho¡, P). As can be seen in Table 2, the recombinant His6 -tagged MutH proteins from E. coli, V. cholerae and H. in£uenzae were all able to complement the mutH-de¢cient E. coli strain back to the wild-type level. Hence the amino acid di¡erences (60 and
56% identity with respect to the E. coli MutH protein) do not a¡ect the function of the endonucleases from the other organisms in the context of the E. coli mismatch repair system. 3.3. Expression and puri¢cation of MutH from V. cholerae and H. in£uenzae To demonstrate that the MutH proteins from V. cholerae and H. in£uenzae are functionally interacting with the E. coli mismatch repair proteins, especially MutL, we expressed the His6 -tagged proteins in E. coli. The proteins could be puri¢ed as soluble proteins to over 95% homogeneity as judged by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) (Fig. 2A). Moreover, the CD spectra of the three MutH proteins from E. coli, H. in£uenzae, and V. cholerae were identical, indicating that all three proteins adopt a similar overall fold as expected from the protein sequence similarity (Fig. 2B). 3.4. MutH from V. cholerae and H. in£uenzae functionally interact with the E. coli MutL and MutS proteins in vitro MutH endonuclease activity was monitored by the conversion of unmethylated plasmid from the supercoiled to the open circular form. At high enzyme concentrations
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V. cholerae and H. in£uenzae MutH showed cleavage of unmethylated plasmid DNA but no cleavage of fully methylated plasmid DNA, similarly to E. coli MutH (data not shown). At low protein concentration (30 nM), all three MutH proteins showed almost no DNA cleavage activity (Fig. 3). However, upon addition of E. coli MutL all three proteins were stimulated to cleave the supercoiled plasmid DNA to a comparable extent. Moreover, both heterologous MutH proteins were also activated by the E. coli MutS and MutL proteins in a mismatch-dependent manner under conditions where no mismatch-independent activation by MutL is observed (Fig. 4). Therefore, both heterologous MutH proteins are able to functionally interact with the E. coli MutL and MutS proteins in vitro and in vivo, suggesting that the di¡erences in the amino acid sequence between the MutH proteins, especially in the Cterminal tail, are not important for their interaction with
Fig. 3. In vitro activation of heterologous MutH proteins by MutL from E. coli. Nicking of 10 Wg ml3l unmethylated pUC8 DNA by 30 nM MutH from E. coli (Eco-H), H. in£uenzae (Hin-H) or V. cholerae (Vch-H) was measured in the absence or presence of 1 WM E. coli MutL. sc is the supercoiled and oc the open circular form of plasmid pUC8. The sc DNA is the substrate which is cleaved by MutH to give the open circular form of the plasmid. It is obvious that E. coli MutL can activate all three di¡erent MutH proteins to a comparable extent.
the other Mut proteins and, therefore, for the function of the protein. 3.5. Conclusions
Fig. 2. SDS^PAGE and CD analysis of recombinant MutH proteins. A: Increasing amounts (1, 2, 5 and 10 Wg) of recombinant E. coli MutH protein (Eco-H) and approximately 5 Wg of H. in£uenzae (HinH) and V. cholerae MutH protein (Vch-H) were subjected to SDS^ PAGE. The molecular masses (kg mol31 ) of marker proteins are indicated on the left. B: CD spectra of 3 WM MutH from E. coli (squares), H. in£uenzae (circles) and V. cholerae (triangles).
Our results do not support the claim that the C-terminal end of E. coli MutH is important for the interaction of MutH with MutL. The only fully conserved residue (Leu222) in the C-terminal tail of MutH is buried (less than 10% accessible surface area in the structure of MutH). Moreover, since at the position of Leu-222 there are also hydrophobic residues in three distantly related restriction endonucleases (Sau3AI, LlaKR2I and Sth368I) [20], this residue probably has a structural rather than a functional role in the interaction with MutL. This conclusion suggests that in the analysis of the interaction site of MutH with MutL one should focus on (surface) residues, which are conserved in the MutH protein family, and are not present in the distantly related restriction endonucleases (cf. Fig. 1).
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Fig. 4. Mismatch-dependent activation of heterologous MutH protein. Endonuclease activity of 100 nM MutH from E. coli (Eco-H), V. cholerae (Vch-H) and H. in£uenzae (Hin-H) was measured in the presence of 1 WM E. coli MutL (Eco-L) and in the absence or presence of 2 WM E. coli MutS protein (Eco-S) by analyzing nicking of a hemimethlyated DNA with a single G/T mismatch at position 260 and a GATC site at position 87 (cf. Section 2). It is obvious that a mismatch-dependent activation of all three di¡erent MutH proteins can occur.
Acknowledgements We thank Dr. Malcolm E. Winkler (University of Texas Medical School, Houston, TX, USA) for providing strains expressing E. coli His-tagged MutHLS proteins. Preliminary sequence data of MutH proteins were obtained from `The Institute for Genomic Research' website at http:// www.tigr.org and the `Columbia Genome Center'. The expert technical assistance of Ina Steindorf is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (Pi-122/12-3 and Pi-122/13-2), the Dr. Herbert Stolzenberg Stiftung and the Fonds der Chemischen Industrie. References [1] Modrich, P. and Lahue, R. (1996) Mismatch repair in replication ¢delity, genetic recombination, and cancer biology. Annu. Rev. Biochem. 65, 101^133. [2] Eisen, J.A. and Hanawalt, P.C. (1999) A phylogenomic study of DNA repair genes, proteins, and processes. Mutat. Res. 435, 171^ 213. [3] Au, K.G., Welsh, K. and Modrich, P. (1992) Initiation of methyldirected mismatch repair. J. Biol. Chem. 267, 12142^12148. [4] Ban, C. and Yang, W. (1998) Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis. Cell 95, 541^552. [5] Hall, M.C. and Matson, S.W. (1999) The Escherichia coli MutL protein physically interacts with MutH and stimulates the MutHassociated endonuclease activity. J. Biol. Chem. 274, 1306^1312.
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