Skip to main content
Download PDF
Download ePub

Split MutT prevents the mutator phenotype of mutT-deficient Escherichia coli

Genes and Environment volume 46, Article number: 19 (2024) Cite this article

Abstract

Background

The Escherichia coli MutT (NudA) protein catalyzes the hydrolysis of an oxidized form of dGTP, 8-oxo-7,8-dihydro-dGTP (8-hydroxy-dGTP), and the spontaneous mutation frequency is elevated in E. coli cells deficient in the mutT gene.

Results

A split MutT, comprising the N-terminal (residues 1–95) and C-terminal (residues 96–129) peptides, was designed based on the known tertiary structure and linker insertion mutagenesis experiments. The mutator phenotype was complemented when the two peptides were separately expressed in mutT E. coli cells.

Conclusions

These results indicated that this split MutT functions as a nucleotide pool sanitization enzyme in vivo.

Introduction

Nucleoside 5’-triphosphates, DNA and RNA precursors, suffer various types of chemical damage and the lesioned nucleotides cause replicational and transcriptional mutagenesis. DNA repair is expected to minimize the effects of DNA damage on genetic information [1]. Meanwhile, nucleotide pool sanitization, the specific elimination of damaged nucleotides from the nucleotide pool, is universally recognized as a defense against the mutations induced by the damaged nucleotides [2, 3].

The Escherichia coli MutT (NudA) protein is a nucleotide pool sanitization enzyme specific for an oxidized form of dGTP, 8-oxo-7,8-dihydro-dGTP (dGOTP, 8-hydroxy-dGTP) [4]. The other substrates are the diphosphate derivative 8-oxo-7,8-dihydro-dGDP and the ribo tri-/di-phosphates [5]. The importance of nucleotide pool sanitization is supported by the presence of proteins with similar functions in bacterial and mammalian cells [6,7,8,9,10]. Moreover, the mutT deficiency in E. coli cells induces a mutator phenotype, and the frequency of A: T->C: G transversions, the signature of dGOTP, is elevated in the cells [11]. Thus, the MutT protein is the major system that prevents the mutagenesis induced by oxidized dGTP in bacterial cells [11, 12].

Some proteins can be split into two or more fragments while retaining similar activities to those of the native, uncleaved proteins, and this is called the fragment complementation system. For instance, RNase A digestion by subtilisin cleaves the peptide bond between Ala-20 and Ser-21 to produce the S peptide (residues 1–20) and S protein (residues 21–124). The two peptides reconstitute the noncovalently bound, active complex, RNase S [13]. Another example is E. coli β-galactosidase (LacZ). This protein can be separated into two peptides, LacZα and LacZΩ [14]. The β-galactosidase activity becomes functional (positive) when both peptides are present and this phenomenon, α complementation, has been used for cloning genes or gene fragments. Various proteins such as luciferase and enhanced green fluorescent protein have also been split into two parts [15,16,17,18] and applied to identify and quantify protein-protein interactions.

In this study, the author examined whether MutT can be split and retain its in vivo activity. First, linker insertion mutagenesis was performed to confirm a possible fragmentation site. Based on the result, the author divided the protein into the N-terminal (residues 1–95, Mu95) and C-terminal (residues 96–129, 96tT) peptides. Plasmid DNAs expressing Mu95 and 96tT, as proteins fused to glutathione-S-transferase (GST) and maltose binding protein (MBP), respectively, were introduced into mutT E. coli cells. The dual expressions of GST-Mu95 and 96tT-MBP in E. coli cells complemented the mutT deficiency, as expected. These results indicated that split MutT is functional in vivo.

Materials and methods

Materials

The oligodeoxyribonucleotides used for plasmid construction were purchased from Integrated DNA Technologies (Coralville, IA, USA) and Hokkaido System Science (Sapporo, Japan) in purified forms. The pGEX-6P-3 plasmid DNA was from Cytiva (Marlborough, MA, USA). The pET-MBP16b plasmid DNA containing the E. coli MBP gene, and the pBAD-MCS plasmid DNA containing the p15A ori, the chloramphenicol-resistance gene, the araBAD promoter, and the araC gene were constructed in our laboratory and will be reported elsewhere.

The pGST_MutT plasmid DNA was constructed in our laboratory by inserting the E. coli mutT gene into the pGEX-6P-3 plasmid at the BamHI and SalI sites, to construct the gene encoding the GST-MutT fusion protein. The gene fragments encoding the 1–95 and 96–129 peptides of MutT were separately amplified by PCR, using this plasmid as the template. The two fragments were joined by the linker 5’-dATGCATTGATAATAGATCT-3’, where the NsiI and BglII sites are italicized, in the second PCR. This PCR product was inserted into the pGEX-6P-3 vector at the BamHI and SalI sites. The sequence encoding a 30-residue glycine-serine linker, (GGGGS)6, 5’-dGGTGGAGGCGGTTCAGGCGGTGGAGGCTCCGGAGGTGGCGGAAGTGGCGGTGGCGGATCAGGTGGAGGTGGCAGCGGAGGCGGAGGTTCC-3’, was inserted at the NsiI and BglII sites, yielding pGST_MutT_95–96_GlySerLinker. The pGST_MutT_95–96_GlySerLinker(Ala53) was constructed by changing the 53rd codon GAA to GCA. The PCR fragment encoding the N-terminal 95 residues of MutT was inserted into the pGEX-6P-3 plasmid at the BamHI and SalI sites to yield pGST_MutT_1–95. The pGST_MutT_1–95(Ala53) plasmid was constructed by replacing the mutT(1–95) gene of pGST_MutT_1–95 with the mutT(1–95,Ala53) gene, prepared from pGST_MutT_95–96_GlySerLinker(Ala53).

The pBAD-MCS plasmid was digested with AccB1I and BstZ17I, and the chloramphenicol-resistance gene was replaced by the kanamycin-resistance gene, yielding pBAD-MCS-kanR. The genes encoding MBP plus the C-terminal His10-tag and the terminator sequence were amplified by PCR from pET-MBP16b and inserted into pBAD-MCS-kanR at the PstI site to yield pBAD-MBP. The region corresponding to the 96–129 peptide was amplified by PCR from pGST_MutT_95–96_GlySerLinker and ligated to pBAD-MBP at the XbaI and PstI sites, and the Gly-Ser linker was then inserted between the 96tT and MBP genes to yield pBAD-96tT-GSL-MBP. The pTac-MBP_lacIq and pTac-96tT-GSL-MBP_lacIq plasmid DNAs were constructed by replacing the araC gene plus the araBAD promoter of pBAD-MBP and pBAD-96tT-GSL-MBP, respectively, with the lacIq-tac promoter region amplified from pGEX-6P-3.

rpoB assay

KAM0003 mutT E. coli cells [19] were transformed by the plasmids containing the wild-type (wt) or mutant mutT gene, with and without either the pTac-MBP_lacIq or pTac-96tT-GSL-MBP_lacIq plasmid. The spontaneous rpoB mutant frequency was calculated according to the numbers of colonies on the titer and selection (100 µg/mL rifampicin) agar plates, as described previously [19, 20].

Results and discussion

Maintained activity of linker-inserted MutT mutant

The E. coli MutT protein is the product of the mutator mutT gene and catalyzes the hydrolysis of the mutagenic nucleotide dGOTP [4]. This protein is a member of the nucleotide hydrolyzing enzymes containing the “Nudix motif”, “MutT signature”, or “phosphohydrolase module” [21, 22]. The motif is GX5EX7REUXEEXGU (U = hydrophobic amino acid; X = any amino acid) and corresponds to residues 38–60 in MutT. It constitutes part of the phosphate binding portion for the substrate nucleotide. The protein catalyzed the hydrolysis of dGOTP/8-oxo-7,8-dihydro-dGDP and their corresponding ribonucleotides, and thus contains the amino acid residues that recognize the base moiety. Residues 23, 28, 35, 77, 78, and 119 reportedly participate in the recognition of the 8-oxo-7,8-dihydroguanine base [23, 24].

First, the author hypothesized that the positions for successful fragmentation would tolerate linker insertion mutagenesis. Based on the tertiary structure of MutT, the loop containing residues 95 and 96 was selected [23, 24]. The DNA fragment corresponding to a 30-residue glycine-serine linker, (GGGGS)6, was inserted into the mutT gene between codons 95 (Trp) and 96 (Gly) (Fig. 1). The mutant was designed to have the amino acid sequence W(95)-M-Q-(GGGGS)6-G-S-G(96). This mutant and the wt mutT genes were expressed as GST fusion proteins in E. coli cells deficient in the mutT gene. Spontaneous rpoB mutant frequencies were measured by rifampicin resistance.

Fig. 1
figure 1

The linker-inserted MutT mutant protein and the control proteins expressed in E. coli. The 30-residue glycine-serine linker (GGGGS)6 is indicated by the pale blue bar

Full size image

As shown in Fig. 2, the truncated mutant (1–95) did not complement the mutT deficiency. Meanwhile, the rpoB mutant frequency was lower when the linker-inserted mutant 95–96 L was expressed in the mutT cells. These results indicated that the inserted glycine-serine linker did not disrupt the catalytic activity of the MutT protein. Moreover, the author substituted Glu-53, an essential amino acid for catalysis, to Ala-53 in the 95–96 L MutT protein [24,25,26], and observed that the suppressive activity of 95–96 L MutT was lost by this substitution (Fig. 2). Thus, the author decided to divide MutT between Trp-95 and Gly-96.

Fig. 2
figure 2

Suppression of spontaneous mutation by expression of the linker-inserted mutant in mutT cells. Each dot represents the rpoB mutant frequency obtained from a single colony. The median values are shown in the upper part. Their relative values are also shown in parentheses. The 95% confidence interval values are 1.1 × 10–7 – 9.3 × 10–7, 1.5 × 10–7 – 2.6 × 10–7, 3.1 × 10–8 – 6.4 × 10–8, 2.5 × 10–7 – 4.1 × 10–7, and 1.1 × 10–8 – 4.0 × 10–8 for GST, 1–95, 95–96 L, 95–96 L (A53), and 1–129 (wt), respectively

Full size image

Mutation suppression by expression of split MutT

Next, the anti-mutator function of split MutT was examined. The N-95 residue (1–95) fragment was expressed as a GST fusion protein, GST-Mu95, as described above. The C-34 residue (96–129) fragment was expressed as an MBP fusion protein, 96tT-MBP (Fig. 3).

Fig. 3
figure 3

The proteins expressed in E. coli to evaluate the activity of split MutT. Mu95, GST-Mu95; 96tT, 96tT-MBP

Full size image

The author co-introduced two plasmid DNAs expressing GST-Mu95 and 96tT-MBP into mutT-deficient E. coli cells, and measured the spontaneous rpoB mutant frequencies. As shown in Fig. 4, the co-expression of GST-Mu95 and 96tT-MBP successfully suppressed the appearance of rpoB mutants. When the GST-Mu95 protein with the Ala-53 residue was expressed, the suppressive effect was lost. Thus, the separate expressions of Mu95 and 96tT result in the active complex formation and the hydrolysis of the substrates in E. coli cells.

Fig. 4
figure 4

Suppression of spontaneous mutation by split MutT expression in mutT cells. Each dot represents the rpoB mutant frequency obtained from a single colony. The median values are shown in the upper part. Their relative values, calculated using the mean of rpoB mutant frequencies of Mu95 + MBP and GST + 96tT as 1.0, are also shown in parentheses. The 95% confidence interval values are 1.4 × 10–7 – 2.1 × 10–6, 1.6 × 10–7 – 4.6 × 10–7, 1.1 × 10–8 – 1.2 × 10–7, and 1.8 × 10–7 – 3.1 × 10–7 for Mu95 + MBP, GST + 96tT, Mu95 + 96tT, and Mu95 (A53) + 96tT, respectively. Mu95, GST-Mu95; 96tT, 96tT-MBP

Full size image

Association of split fragments

In this study, MutT was split into Mu95 and 96tT, and the two fragments were separately expressed as proteins fused to GST and MBP, respectively, in E. coli. The dual expression complemented the mutT deficiency (Fig. 4), suggesting that the two parts associated and degraded dGOTP in the cells. When Glu-53, a catalytically important amino acid residue in Mu95, was mutated, the complementarity was lost. Thus, the two fragments seemed to have the ability to catalyze the hydrolysis of dGOTP in the complex.

The FK506 binding protein (FKBP) and the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR) form a heterodimer in the presence of rapamycin, an antifungal antibiotic [27, 28]. Likewise, the Mu95 and 96tT peptides might interact depending on dGOTP. Analyses of the binary complex of MutT plus the nucleotide indicated that amino acids 23, 28, 35, 77, 78, and 119 are important for the recognition of the 8-oxo-7,8-dihydroguanine base [23, 24]. The Mu95 and 96tT fragments contain residues 23–78 and 119, respectively, and O6 and N7-H of the base moiety are recognized by Asn-119. Since N7-H is present in 8-oxo-7,8-dihydroguanine but not in guanine, this residue could be crucial for dGOTP binding. Thus, the association of split fragments is suggested to be promoted by the oxidized nucleotide. This speculation is supported by our recent observation of split MutT [29]. The interaction of Ash (assembly helper tag)-Mu95 with the Ala-53 residue and hAG (Azami Green)-96tT allowed the visualization of intracellular dGOTP by fluorescent foci formation: the fluorescent foci are highly increased when dGOTP, but not dGTP, is introduced into human cells. Taken together, the association of Mu95 and 96tT probably occurs in the presence of dGOTP or other substrates.

Data availability

Data will be made available on request.

Abbreviations

dGOTP:

8-oxo-7,8-dihydro-dGTP

GST:

glutathione-S-transferase

MBP:

maltose binding protein

wt:

wild-type

References

  1. Barnes D, Lindahl T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annu Rev Genet. 2004;38:445–76.

    Article  PubMed  CAS  Google Scholar 

  2. Sekiguchi M, Tsuzuki T. Oxidative nucleotide damage: consequences and prevention. Oncogene. 2002;21:8895–904.

    Article  PubMed  CAS  Google Scholar 

  3. Kamiya H. Mutagenicity of oxidized DNA precursors in living cells: roles of nucleotide pool sanitization and DNA repair enzymes, and translesion synthesis DNA polymerases. Mutat Res. 2010;703:32–6.

    Article  PubMed  CAS  Google Scholar 

  4. Maki H, Sekiguchi M. MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis. Nature. 1992;355:273–5.

    Article  PubMed  CAS  Google Scholar 

  5. Ito R, Hayakawa H, Sekiguchi M, Ishibashi T. Multiple enzyme activities of Escherichia coli MutT protein for sanitization of DNA and RNA precursor pools. Biochemistry. 2005;44:6670–4.

    Article  PubMed  CAS  Google Scholar 

  6. Kamiya H, Murata-Kamiya N, Iida E, Harashima H. Hydrolysis of oxidized nucleotides by the Escherichia coli Orf135 protein. Biochem Biophys Res Commun. 2001;288:499–502.

    Article  PubMed  CAS  Google Scholar 

  7. Hori M, Fujikawa K, Kasai H, Harashima H, Kamiya H. Dual hydrolysis of diphosphate and triphosphate derivatives of oxidized deoxyadenosine by Orf17 (NtpA), a MutT-type enzyme. DNA Repair. 2005;4:33–9.

    Article  PubMed  CAS  Google Scholar 

  8. Mo J-Y, Maki H, Sekiguchi M. Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool. Proc Natl Acad Sci USA. 1992;89:11021–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Cai JP, Ishibashi T, Takagi Y, Hayakawa H, Sekiguchi M. Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides. Biochem Biophys Res Commun. 2003;305:1073–7.

    Article  PubMed  CAS  Google Scholar 

  10. Ishibashi T, Hayakawa H, Sekiguchi M. A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides. EMBO Rep. 2003;4:479–83.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Yanofsky C, Cox EC, Horn V. The unusual mutagenic specificity of an E. coli mutator gene. Proc Natl Acad Sci USA. 1966;55:274–81.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Kamiya H, Ishiguro C, Harashima H, Increased. A:T◊C:G mutations in the mutT strain upon 8-hydroxy-dGTP treatment: direct evidence for MutT involvement in the prevention of mutations by oxidized dGTP. J Biochem. 2004;136:359–62.

    Article  PubMed  CAS  Google Scholar 

  13. Richards FM, Vithayathil PJ. The preparation of subtilisin-modified ribonuclease and the separation of the peptide and protein components. J Biol Chem. 1959;234:1459–65.

    Article  PubMed  CAS  Google Scholar 

  14. Welply JK, Fowler AV, Zabin I. β-Galactosidase alpha-complementation. Overlapping sequences. J Biol Chem. 1981;256:6804–10.

    Article  PubMed  CAS  Google Scholar 

  15. Fujikawa Y, Nakanishi T, Kawakami H, Yamasaki K, Sato MH, Tsuji H, Matsuoka M, Kato N. Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format. Rice. 2014;7:11.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Barnard E, Timson DJ. Split-EGFP screens for the detection and localisation of protein-protein interactions in living yeast cells. Methods Mol Biol. 2010;638:303–17.

    Article  PubMed  CAS  Google Scholar 

  17. Blakeley BD, Chapman AM, McNaughton BR. Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo. Mol BioSyst. 2012;8:2036–40.

    Article  PubMed  CAS  Google Scholar 

  18. Cabantous S, Nguyen HB, Pedelacq JD, Koraïchi F, Chaudhary A, Ganguly K, Lockard MA, Favre G, Terwilliger TC, Waldo GS. A new protein-protein interaction sensor based on tripartite split-GFP association. Sci Rep. 2013;3:2854.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Hori M, Ishiguro C, Suzuki T, Nakagawa N, Nunoshiba T, Kuramitsu S, Yamamoto K, Kasai H, Harashima H, Kamiya H. UvrA and UvrB enhance mutations induced by oxidized deoxyribonucleotides. DNA Repair. 2007;6:1786–93.

    Article  PubMed  CAS  Google Scholar 

  20. Kamiya H, Iida E, Murata-Kamiya N, Yamamoto Y, Miki T, Harashima H. Suppression of spontaneous and hydrogen peroxide-induced mutations by a MutT-type nucleotide pool sanitization enzyme, the Escherichia coli Orf135 protein. Genes Cells. 2003;8:941–50.

    Article  PubMed  CAS  Google Scholar 

  21. Bessman MJ, Frick DN, O’Handley SF. The MutT proteins or Nudix hydrolases, a family of versatile, widely distributed, housecleaning enzymes. J Biol Chem. 1996;271:25059–62.

    Article  PubMed  CAS  Google Scholar 

  22. Nakabeppu Y. Molecular genetics and structural biology of human MutT homolog, MTH1. Mutat Res. 2001;477:59–70.

    Article  PubMed  CAS  Google Scholar 

  23. Saraswat V, Azurmendi HF, Mildvan AS. Mutational, NMR, and NH exchange studies of the tight and selective binding of 8-oxo-dGMP by the MutT pyrophosphohydrolase. Biochemistry. 2004;43:3404–14.

    Article  PubMed  CAS  Google Scholar 

  24. Nakamura T, Meshitsuka S, Kitagawa S, Abe N, Yamada J, Ishino T, Nakano H, Tsuzuki T, Doi T, Kobayashi Y, Fujii S, Sekiguchi M, Yamagata Y. Structural and dynamic features of the MutT protein in the recognition of nucleotides with the mutagenic 8-oxoguanine base. J Biol Chem. 2010;285:444–52.

    Article  PubMed  CAS  Google Scholar 

  25. Shimokawa H, Fujii Y, Furuichi M, Sekiguchi M, Nakabeppu Y. Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein. Nucleic Acids Res. 2000;28:3240–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Xia Z, Azurmendi HF, Mildvan AS. Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction. Biochemistry. 2005;44:15334–44.

    Article  PubMed  CAS  Google Scholar 

  27. Chen J, Zheng XF, Brown EJ, Schreiber SL. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA. 1995;92:4947–51.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Banaszynski LA, Liu CW, Wandless TJ. Characterization of the FKBP·rapamycin·FRB ternary complex. J Am Chem Soc. 2005;127:4715–21.

    Article  PubMed  CAS  Google Scholar 

  29. Fujikawa Y, Kawai H, Suzuki T, Kamiya H. Visualization of oxidized guanine nucleotides accumulation in living cells with split MutT. Nucleic Acids Res. 2024;52:6532–42.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The author thanks Dr. Tetsuya Suzuki for his advice and the pGST-MutT, pET-MBP16b, and pBAD-MCS plasmid DNAs.

Funding

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number JP 22K19381) to HK.

Author information

Authors and Affiliations

  1. Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan

    Hiroyuki Kamiya

Authors
  1. Hiroyuki Kamiya
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HK designed and conducted the experiments and wrote the manuscript.

Corresponding author

Correspondence to Hiroyuki Kamiya.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kamiya, H. Split MutT prevents the mutator phenotype of mutT-deficient Escherichia coli. Genes and Environ 46, 19 (2024). https://doi.org/10.1186/s41021-024-00314-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s41021-024-00314-8

Keywords

Genes and Environment

ISSN: 1880-7062

Contact us