Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.
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PMID:Cloning the KpnI restriction-modification system in Escherichia coli. 199 32

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.
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PMID:Cloning and characterization of the MboII restriction-modification system. 202 May 40

The restriction-modification system HgiDI from Herpetosiphon giganteus strain Hpa2 has been cloned in E. coli in a two-step procedure. Selection of the methyltransferase (M.HgiDI) gene in vitro was performed using the heterologous restriction endonuclease AhaII, an isoschizomer of Acyl and HgiDI (GRCGYC). Cloning of the complete HgiDI endonuclease (R.HgiDI) gene could only be achieved in recipient cells harbouring a recombinant plasmid, which was expressing the corresponding methyltransferase and could thereby prevent the host from self-destruction of its genetic material. The HgiDI restriction-modification system was sequenced and functionally correlated with two open reading frames of 309 (M) and 359 (R) codons. In homology studies M.HgiDI showed significant similarities to 20 other m5C-methyltransferases and turned out to be the most compact enzyme of this group described so far. Initial attempts for overexpression of M.HgiDI and partial purification of R.HgiDI have been successful.
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PMID:Stepwise cloning and molecular characterization of the HgiDI restriction-modification system from Herpetosiphon giganteus Hpa2. 202 May 44

The complete type II restriction-modification system HgiBI of Herpetosiphon giganteus strain Hpg5 recognizing the AvaII specific DNA sequence GGWCC has been cloned and expressed functionally active in Escherichia coli. A considerable acceleration in cloning could be achieved by preparing a size restricted library after application of a related hybridization probe. Both methyltransferase (437 codons) and restriction endonuclease gene (274 codons) were found to be encoded on a 3.6 kilobases ClaI/HincII fragment in the same transcriptional orientation separated by one triplett only. Protein sequence comparisons revealed a close resemblance of M.HgiBI to the group of m5C-methyltransferases, especially to M.BanI from Bacillus aneurinolyticus with the related recognition sequence GGYRCC. In contrast, no significant similarities have been observed for the associated endonuclease R.HgiBI with any other restriction enzyme described so far, even not with the isoschizomeric R.SinI from Salmonella infantis, or with R.BanI.
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PMID:Isolation and genetic structure of the AvaII isoschizomeric restriction-modification system HgiBI from Herpetosiphon giganteus Hpg5: M.HgiBI reveals high homology to M.BanI. 206 38

We have developed an assay that allows analysis of the activity of EcoRI restriction endonuclease (ENase) and its mutants in vivo. This assay is based on the fact that wild type (wt) EcoRI ENase is toxic for Escherichia coli cells not expressing the EcoRI methyltransferase (MTase). The viability factor defined by the ratio of the viable counts of E. coli cultures having or not having expressed the ecoRIR gene for a defined time is 10(-6) for wt EcoRI ENase and close to one for a totally inactive EcoRI ENase mutant. While the EcoRI MTase (M.EcoRI) provides substantial protection against the toxic effects of the wt EcoRI ENase and several of the mutants, some mutants become more toxic in the presence of M.EcoRI. Twenty-four different DNA-binding-site mutants of EcoRI ENase were characterized in their activity in vivo with this assay. The results obtained allow us to conclude that the structural integrity of the region at and around aa 200 seems to be very critical for the enzymatic function of EcoRI ENase: nonconservative replacements there lead to viability factors of 1-10(-2). While our results indicate that the region around aa 144 and 145 is also involved in the EcoRI ENase-catalyzed reaction, it is also evident that the effects of mutation there are not as large: viability factors of approx. 10(-3) are obtained even for drastic replacements. These results are discussed in the light of the x-ray structure analysis of an EcoRI ENase-DNA recognition complex.
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PMID:Probing the function of individual amino acid residues in the DNA binding site of the EcoRI restriction endonuclease by analysing the toxicity of genetically engineered mutants. 214 67

The EcoRV restriction/modification system consists of two enzymes that recognize the DNA sequence GATATC. The EcoRV restriction endonuclease cleaves DNA at this site, but the DNA of Escherichia coli carrying the EcoRV system is protected from this reaction by the EcoRV methyltransferase. However, in vitro, the EcoRV nuclease also cleaves DNA at most sites that differ from the recognition sequence by one base pair. Though the reaction of the nuclease at these sites is much slower than that at the cognate site, it still appears to be fast enough to cleave the chromosome of the cell into many fragments. The possibility that the EcoRV methyltransferase also protects the noncognate sites on the chromosome was examined. The modification enzyme methylated alternate sites in vivo, but these were not the same as the alternate sites for the nuclease. The excess methylation was found at GATC sequences, which are also the targets for the dam methyltransferase of E. coli, a protein that is homologous to the EcoRV methyltransferase. Methylation at these sites gave virtually no protection against the EcoRV nuclease: even when the EcoRV methyltransferase had been overproduced, the cellular DNA remained sensitive to the EcoRV nuclease at its noncognate sites. The viability of E. coli carrying the EcoRV restriction/modification system was found instead to depend on the activity of DNA ligase. Ligase appears to proofread the EcoRV R/M system in vivo: DNA, cut initially in one strand at a noncognate site for the nuclease, is presumably repaired by ligase before the scission of the second strand.
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PMID:Fidelity of DNA recognition by the EcoRV restriction/modification system in vivo. 217 80

The genes encoding SsoI and SsoII restriction endonuclease (ENase) and methyltransferase (MTase) are located on the small plasmids P6 and P4, respectively, of Shigella sonnei strain 47. Functions provided by plasmids P5, P7 and P9, which include colicinogenicity and immunity to colicin E1, resistance to streptomycin (Sm), and conjugative DNA transfer, respectively, have also been identified. The genes of the SsoII restriction-modification (R-M) system have been cloned into Escherichia coli expressing the 35-kDa (ENase) and 43-kDa (MTase) products. A restriction map of the P4 plasmid DNA was determined, and the approximate location of the genes encoding SsoII ENase and MTase (ssoIIR and ssoIIM) on that have been established. SsoI is an isoschisomer of EcoRI and SsoII cleaves the 5'-/CCNGG/recognition sequence producing 5'-protruding 5-nt long cohesive ends.
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PMID:Characterization of the genetic determinants of SsoII-restriction endonuclease and modification methyltransferase. 218 34

The genes coding for the GGNCC specific Sau96I restriction and modification enzymes were cloned and expressed in E. coli. The DNA sequence predicts a 430 amino acid protein (Mr: 49,252) for the methyltransferase and a 261 amino acid protein (Mr: 30,486) for the endonuclease. No protein sequence similarity was detected between the Sau96I methyltransferase and endonuclease. The methyltransferase contains the sequence elements characteristic for m5C-methyltransferases. In addition to this, M.Sau96I shows similarity, also in the variable region, with one m5C-methyltransferase (M.SinI) which has closely related recognition specificity (GGA/TCC). M.Sau96I methylates the internal cytosine within the GGNCC recognition sequence. The Sau96I endonuclease appears to act as a monomer.
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PMID:Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes. 220 26

The CHO-UV-1 mutant, a Chinese hamster ovary cell with defective postreplication recovery of DNA, is 2- to 4-fold more sensitive than its wild-type counterpart (CHO-77256) to the lethal effects of ethylating agents and UV radiation; it is also hypersensitive (10- to 20-fold) to some DNA-methylating and -cross-linking agents. We studied the CHO-UV-1 mutant further to define its phenotype in terms of DNA damage induction and repair, methyltransferase activity, and effects of caffeine on mutational and lethal responses. Both wild-type and CHO-UV-1 cells incurred similar levels and types of damage when exposed to UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, or N-methyl-N-nitrosourea. The rate and extent of repair of Micrococcus luteus endonuclease-sensitive sites after UV irradiation or treatment with N-methyl-N'-nitro-N-nitrosoguanidine were also equivalent in these two cell types. Twenty % of the initial endonuclease-sensitive sites induced in either cell line remained at 18 h after UV irradiation; approximately 8% of the sites after N-methyl-N'-nitro-N-nitrosoguanidine exposure were present in both parental and CHO-UV-1 cells after a 17-h repair period. Moreover, the ability of CHO-UV-1 to resynthesize and ligate DNA during excision repair was similar to that of its parent. Neither CHO-UV-1 nor CHO-77256 had appreciable levels of O6-methylguanine-DNA methyltransferase activity which ameliorates the cytotoxicity of alkylating agents. Caffeine, a known inhibitor of postreplication repair, decreased the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyltransferase locus by 40-55% in CHO-77256 but not in CHO-UV-1. These results rule out defective excision repair as a factor in the hypersensitivity of the CHO-UV-1 mutant to DNA-damaging agents. Hence, this cell line appears to derive from a mutation affecting nonexcision repair processes and should be useful in clarifying the mechanism(s) of postreplication recovery of DNA in mammalian cells.
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PMID:Genetic and biochemical characterization of the CHO-UV-1 mutant defective in postreplication recovery of DNA. 231 21

A new site-specific class-II restriction endonuclease, MamI, has been discovered in the nonsporulating Gram+ Microbacterium ammoniaphilum. MamI recognition sequence and cleavage positions were deduced using experimental and computer-assisted mapping and sequencing approaches. MamI cleavage specificity corresponds to: [formula: see text] The novel 43-kD enzyme recognizes a palindromic hexanucleotide interrupted by four ambiguous nucleotides. MamI cleavage positions are located in the center of the recognition sequence resulting in blunt-ended fragments after cleavage in the presence of Mg2+ ions. MamI is inhibited by N6-methyladenine residues. In case of overlapping sequences of MamI and Escherichia coli-coded DNA modification methyltransferase M.EcodamI (5'-[formula: see text]-3'), cleavage of DNA isolated from E. coli wild-type cells will be inhibited. By applying incubation conditions forcing star activity, relaxing of MamI sequence specificity is observed (MamI*).
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PMID:MamI, a novel class-II restriction endonuclease from Microbacterium ammoniaphilum recognizing 5'-GATNN decreases NNATC-3'. 240 11


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