Mutation in the specificity polypeptide of the type I restriction endonuclease R · EcoK that affects subunit assembly

Zinkevich, Vitaly; Heslop, Pauline; Glover, S. W.; Weiserová, Marie; Hubáček, J.; Firman, Keith

doi:10.1016/0022-2836(92)90210-b

Cited by 13 publications

(16 citation statements)

References 17 publications

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“…Unexpectedly common, but not obviously localized, were substitutions in HsdS that only impaired restriction but not modification. Such substitutions could affect the interaction of HsdS with HsdR (Zinkevich et al ., 1992; Weiserova and Firman, 1998) or they could influence the interaction of the complex with its DNA target.…”

Section: Discussionmentioning

confidence: 99%

Localization of a protein–DNA interface by random mutagenesis

O’Neill

Dryden

Murray

1998

EMBO J

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The type I restriction and modification enzymes do not possess obvious DNA-binding motifs within their target recognition domains (TRDs) of 150-180 amino acids. To identify residues involved in DNA recognition, changes were made in the amino-TRD of EcoKI by random mutagenesis. Most of the 101 substitutions affecting 79 residues had no effect on the phenotype. Changes at only seven positions caused the loss of restriction and modification activities. The seven residues identified by mutation are not randomly distributed throughout the primary sequence of the TRD: five are within the interval between residues 80 and 110. Sequence analyses have led to the suggestion that the TRDs of type I restriction enzymes include a tertiary structure similar to the TRD of the HhaI methyltransferase, and to a model for a DNA-protein interface in EcoKI. In this model, the residues within the interval identified by the five mutations are close to the protein-DNA interface. Three additional residues close to the DNA in the model were changed; each substitution impaired both activities. Proteins from twelve mutants were purified: six from mutants with partial or wildtype activity and six from mutants lacking activity. There is a strong correlation between phenotype and DNA-binding affinity, as determined by fluorescence anisotropy.

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Section: Discussionmentioning

confidence: 99%

Localization of a protein–DNA interface by random mutagenesis

O’Neill

Dryden

Murray

1998

EMBO J

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“…Mutations conferring an r Ϫ m ϩ phenotype have been identified in the hsdS genes of type IA and IC systems. In EcoR124I (189) these mutations are at the junction between a conserved region and a TRD, while in EcoKI they can be close to the junction (197) or at various positions within the TRD (131). A cautious interpretation of r Ϫ phenotypes for EcoKI, however, is required by the recent discovery that mutations in hsdM that result in inadequate modification induce host-mediated alleviation of restriction (111) (see section on modulation of restriction activity).…”

Section: R-m Complexmentioning

confidence: 99%

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

Murray

2000

Microbiol Mol Biol Rev

406

481

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SUMMARY Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.

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“…Plasmid pWU128 was constructed by ligating the Hin-dIII fragment of pBg3 [15] carrying the hsdR and hsdM genes and the proximal part of the hsdS gene into the HindIII site of the hsdSts2 gene in a derivative of plasmid pZH9 [11]. The HindIII site and the hsdM gene in plasmid pZH9 were removed by EcoRI-HpaI and SalI-BamHI deletions respectively.…”

Section: Plasmid Constructionsmentioning

confidence: 99%

“…We have investigated two point mutations in the hsdS gene of EcoKI showing a temperature-sensitive phenotype in vivo [11,12]. One of the mutations (Sts1) changes serine 340 to phenylalanine and displays a r ts m ts phenotype [13].…”

Section: Introductionmentioning

confidence: 99%

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Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties

Janscak

2000

FEMS Microbiology Letters

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Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser 340 Phe) and Sts2 (Ala 204 Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30³C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42³C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA^protein and proteinp rotein interactions, respectively. ß

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Mutation in the specificity polypeptide of the type I restriction endonuclease R · EcoK that affects subunit assembly

Cited by 13 publications

References 17 publications

Localization of a protein–DNA interface by random mutagenesis

Localization of a protein–DNA interface by random mutagenesis

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties

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