Study of a suppressor of mut T in Escherichia coli

Ray, Usharanjan

doi:10.1007/bf00270007

Cited by 3 publications

(4 citation statements)

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“…Since we noticed that the MutT catalytic core was widely distributed throughout nature (2), we have begun to characterize other proteins that share this common motif in order to uncover its biochemical mechanism and to catalogue the physiological function of proteins harboring the signature sequence. These MutT-like proteins may be of special interest, because there have been reports of suppressors of the mutT Ϫ phenotype in E. coli (36,37). Such genes could code for proteins with similar functions to MutT that could also be involved in the maintenance of the fidelity of DNA replication.…”

Section: Discussionmentioning

confidence: 99%

A Novel GDP-Mannose Mannosyl Hydrolase Shares Homology with the MutT Family of Enzymes

Frick

Townsend

Bessman

1995

Journal of Biological Chemistry

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The product of the Escherichia coli orf1.9, or yefc, gene (GenBank accession number L11721) has been expressed under the control of a T7 promoter, purified to apparent homogeneity, and identified as a novel enzyme that hydrolyzes GDP-mannose or GDP-glucose to GDP and the respective hexose. The enzyme has little or no activity on other nucleotides, dinucleotides, nucleotide sugars, or sugar phosphates. It has a pH optimum between 9.0 and 9.5, a K m of 0.3 mM, and a V max of 1.6 mol min

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Section: Discussionmentioning

confidence: 99%

A Novel GDP-Mannose Mannosyl Hydrolase Shares Homology with the MutT Family of Enzymes

Frick

Townsend

Bessman

1995

Journal of Biological Chemistry

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“…Although the transformed strain produced large quantities of the enzyme (data not shown), the results in Table III clearly demonstrate that orf17 does not complement mutT. Thus, it is most likely not the putative suppressor of mutT referred to by Ray (27) or Desiraju (28). Furthermore, a strain of E. coli, lacking the orf17 gene, has no significant enhancement in mutation frequency when compared to its wild type parent (Table III).…”

Section: Table IImentioning

confidence: 91%

Escherichia coli orf17 Codes for a Nucleoside Triphosphate Pyrophosphohydrolase Member of the MutT Family of Proteins

O'Handley

Frick

Bullions

et al. 1996

Journal of Biological Chemistry

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The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the ␤-phosphorus to produce dAMP and inorganic pyrophosphate. It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg 2؉, a K m of 0.8 mM and a k cat of 5.2 s ؊1 at 37°C for dATP. dAMP is a weak competitive inhibitor with a K i of approximately 4 mM, while PP i is a much stronger inhibitor with an apparent K i of approximately 20 M.The enzyme contains the highly conserved signature sequence GXVEX 2 ETX 6 REVXEEX 2 I designating the MutT family of proteins. However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT ؊ mutator phenotype, and thus must serve a different biological role in the cell.Members of the MutT family of proteins are categorized by a conserved amino acid motif originally identified as an important functional region in the Escherichia coli MutT and the Streptococcus pneumoniae MutX antimutator proteins (1). This conserved amino acid signature sequence was found, by computer search, to be present in a number of open reading frames broadly distributed throughout nature, from viruses to humans (1, 2), and one of these, orf17 (GenBank D10165, 1992), is the subject of this paper. The orf17 gene is located at 41 min on the E. coli chromosome (3) just upstream of the ruvC gene (4, 5), which codes for a Holliday junction-specific endonuclease (4).Both the MutT and MutX proteins are nucleoside triphosphatases (6, 7), as are the corresponding proteins from Proteus vulgaris (8) and from humans (9), and all four are implicated in preventing mutations. On the other hand, two other proteins containing the MutT signature sequence are not nucleoside triphosphatases. One of them, a nucleoside pyrophosphatase, prefers NADH as its substrate (10), while the other, GDPmannose mannosyl hydrolase, prefers GDP-mannose (11). Neither of these latter two enzymes has been linked to a mutagenic pathway. A feature common to all six enzymes containing the signature sequence is the hydrolysis of a substrate containing a nucleoside diphosphate linkage.In this paper, we describe the cloning and expression of the orf17 gene, and the purification and characterization of the Orf17 protein. Like the first four enzymes described above, it is a nucleoside triphosphatase. However, unlike those four enzymes, which prefer dGTP as a substrate over the other canonical (deoxy)nucleoside triphosphates, Orf17 has a unique preference for dATP, and it does not appear to be involved in antimutagenesis. EXERIMENTAL PROCEDURES MaterialsEnzymes-Thermus aquaticus DNA polymerase was from PerkinElmer, and other enzymes used in standard cloning procedures were from Life Technologies, Inc., Stratagene, or U. S. Biochemical Corp. Yeast inorganic pyrophosphata...

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“…First, identification of an aberration in the nucleotide sequence of the mutant gene would establish that the wild-type gene cloned by ourselves (4) and Akiyama et al (1) was indeed the mutT gene and not some other factor which suppresses the mutTI phenotype (9). Second, a comparison of the defective gene product with our purified enzyme produced from the wild-type gene (4) might provide important insights into the biochemical basis of the mutator effect.…”

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confidence: 97%

“…As such, it could be argued that the cloned gene actually represents some suppressor of the mutTl phenotype (9) and not the gene itself. However, the results reported here which show that an IS] insertion into the wild-type gene can be recovered from the mutTI mutator strain strongly support the notion that the complementary factor is, indeed, the wild-type gene itself, and the absence of discernible protein or nucleoside triphosphatase activity most likely accounts for the mutator activity of mutT) cells.…”

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confidence: 99%

Characterization of the defect in the Escherichia coli mutT1 mutator gene

et al. 1990

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Study of a suppressor of mut T in Escherichia coli

Abstract: A suppressor mutation of E. coli mut T has been isolated and mapped in the lue--ace(E/F) region.

Cited by 3 publications

References 14 publications

A Novel GDP-Mannose Mannosyl Hydrolase Shares Homology with the MutT Family of Enzymes

A Novel GDP-Mannose Mannosyl Hydrolase Shares Homology with the MutT Family of Enzymes

Escherichia coli orf17 Codes for a Nucleoside Triphosphate Pyrophosphohydrolase Member of the MutT Family of Proteins

Characterization of the defect in the Escherichia coli mutT1 mutator gene

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