Linked Life Data

11273713

Source:http://linkedlifedata.com/resource/pubmed/id/11273713

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
2001-3-29
pubmed:abstractText
We report a genetic and biochemical analysis of a target recognition domain (TRD) of EcoKI, a type I restriction and modification enzyme. The TRDs of type I R-M systems are within the specificity subunit (HsdS) and HsdS confers sequence specificity to a complex endowed with both restriction and modification activities. Random mutagenesis has revealed that most substitutions within the amino TRD of EcoKI, a region comprising 157 amino acid residues, have no detectable effect on the phenotype of the bacterium, even when the substitutions are non- conservative. The structure of the TRD appears to be robust. All but one of the six substitutions that confer a restriction-deficient, modification-deficient (r(-)m(-)) phenotype were found to be in the interval between residues 80 and 110, a region predicted by sequence comparisons to form part of the protein-DNA interface. Additional site-directed mutations affecting this interval commonly impair both restriction and modification. However, we show that an r(-) phenotype cannot be taken as evidence that the EcoKI complex lacks endonuclease activity; in response to even a slightly impaired modification efficiency, the endonuclease activity of EcoKI is destroyed by a process dependent upon the ClpXP protease. Enzymes from mutants with an r(-)m(-) phenotype commonly retain some sequence-specific activity; methylase activity can be detected on hemimethylated DNA substrates and residual endonuclease activity is implied whenever the viability of the r(-)m(-) bacterium is dependent on ClpXP. Conversely, the viability of ClpX(-) r(-)m(-) bacteria can be used as evidence for little, or no, endonuclease activity. Of 14 mutants with an r(-)m(-) phenotype, only six are viable in the absence of ClpXP. The significance of four of the six residues (G91, G105, F107 and G141) is enhanced by the finding that even conservative substitutions for these residues impair modification, thereby conferring an r(-)m(-) phenotype.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0022-2836
pubmed:author
pubmed:copyrightInfo
Copyright 2001 Academic Press.
pubmed:issnType
Print
pubmed:day
30
pubmed:volume
307
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
951-63
pubmed:dateRevised
2009-11-3
pubmed:meshHeading
pubmed-meshheading:11273713-Adenosine Triphosphatases, pubmed-meshheading:11273713-Amino Acid Sequence, pubmed-meshheading:11273713-Amino Acid Substitution, pubmed-meshheading:11273713-Binding Sites, pubmed-meshheading:11273713-Chromosomes, Bacterial, pubmed-meshheading:11273713-DNA Restriction Enzymes, pubmed-meshheading:11273713-DNA-Binding Proteins, pubmed-meshheading:11273713-Endopeptidase Clp, pubmed-meshheading:11273713-Enzyme Activation, pubmed-meshheading:11273713-Escherichia coli, pubmed-meshheading:11273713-Escherichia coli Proteins, pubmed-meshheading:11273713-Fluorescence Polarization, pubmed-meshheading:11273713-Molecular Sequence Data, pubmed-meshheading:11273713-Mutation, pubmed-meshheading:11273713-Phenotype, pubmed-meshheading:11273713-Plasmids, pubmed-meshheading:11273713-Protein Structure, Tertiary, pubmed-meshheading:11273713-Serine Endopeptidases, pubmed-meshheading:11273713-Site-Specific DNA-Methyltransferase (Adenine-Specific), pubmed-meshheading:11273713-Substrate Specificity, pubmed-meshheading:11273713-Transduction, Genetic
pubmed:year
2001
pubmed:articleTitle
Target recognition by EcoKI: the recognition domain is robust and restriction-deficiency commonly results from the proteolytic control of enzyme activity.
pubmed:affiliation
Institute of Cell and Molecular Biology, University of Edinburgh, Darwin Building, Mayfield Road, King's Buildings, Edinburgh, EH9 3JR, UK.
pubmed:publicationType
Journal Article