Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

IncP alpha plasmids, exemplified by RP4, are remarkable for their broad host range. They contain strikingly few cleavage sites for many commonly used type II restriction enzymes but an overabundance of sites for certain enzymes that target G + C-rich sequences. To identify factors responsible for these distributions, the recently compiled nucleotide sequence of RP4 was analysed to determine the frequency of tetra- and hexanucleotide motifs in the 49 kb plasmid backbone. This is defined as the sectors encoding basic plasmid functions. The overabundant restriction targets in RP4 are concentrated in the backbone and contain overlapping copies of CGGC/GCCG, identified as the most abundant tetranucleotide motif in the plasmid. Motif frequencies in the RP4 backbone are shown to be similar to those in Pseudomonas aeruginosa, a natural host of RP4, with the notable exception that a number of 6-bp palindromes are underrepresented in the plasmid. It is proposed that 6-bp palindromes were counterselected as type II restriction enzyme recognition sequences. Conjugative transfer of RP4 and R751 (IncP beta) is unusually sensitive to restriction compared to enterobacterial plasmids of the IncFII and IncI1 groups, implying that IncP plasmids experienced particularly strong selection for loss of restriction targets. Pseudomonas spp. of rRNA homology group I specify many type II restriction enzymes that target 6-bp palindromes and are candidates for the evolutionary hosts of IncP alpha plasmids.
J Mol Biol 1996 May 10
PMID:Distribution of restriction enzyme recognition sequences on broad host range plasmid RP4: molecular and evolutionary implications. 864 2

BsoBI is a type II restriction enzyme found in Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5'-CYCGRG-3' (Y=C or T; R=A or G) and cleaves between the first and second base to generate a four-base 5' extension. The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695-699]. Here we report the overexpression of BsoBI restriction endonuclease gene in E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive expression of BsoBI from pRRS is lethal to E. coli in the absence of the cognate methylase. The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.
 ...
PMID:Overexpression of BsoBI restriction endonuclease in E. coli, purification of the recombinant BsoBI, and identification of catalytic residues of BsoBI by random mutagenesis. 909 56

It is thought that most of the type II restriction endonucleases interact with DNA as homodimers. Cfr10I is a typical type II restriction enzyme that recognises the 5'-Pu decreases CCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in isolation. The only SfiI restriction enzyme that recognises the long interrupted recognition sequence 5'-GGCCNNNNNGGCC has been previously reported to operate as a tetramer however, its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine, and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that, in contrast to the wild-type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I, since the DNA cleavage activity of the W220A mutant is < 0.1% of that of the wild-type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I.
J Mol Biol 1999 Sep 03
PMID:The Cfr10I restriction enzyme is functional as a tetramer. 1051 46

This chapter outlines seven synthetic and molecular biology techniques that allow the controlled synthesis of nucleic acid libraries. Specifically: (1) The high-diversity chemical synthesis of point mutations; (2) the high-diversity chemical synthesis of point deletions; (3) the split-bead approach for constructing point mutation or deletion libraries with limited sequence diversity; (4) pool deprotection, gel purification, and quality-control techniques; (5) large-scale polymerase chain reaction amplification for the generation of high-diversity double-stranded deoxyribonucleic acid libraries; (6) type II restriction enzyme digestion techniques for the construction of long-sequence libraries containing minimal fixed sequence; and (7) extension techniques for the rapid synthesis of long, low-diversity oligonucleotide sequences.
Methods Mol Biol 2005
PMID:Nucleic acid library construction using synthetic DNA constructs. 1533 15