Molecular cloning of EcoRII endonuclease and methylase genes

Kosykh, V.G.; Buryanov, Yа. I.; Bayev, A.A.

doi:10.1007/bf00337884

Cited by 43 publications

(15 citation statements)

References 6 publications

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“…2), the molecular mass of the native His-tagged EcoRII ENase was determined to be 85 kDa, which is approximately twice that of the protein monomer (26). This would confirm that the His 6 -tagged enzyme is a dimer in solution as was previously shown by gel filtration through Sephadex G-100 for the native ENase (26).…”

Section: Resultssupporting

confidence: 78%

Cooperative Binding Properties of Restriction Endonuclease EcoRII with DNA Recognition Sites

Reuter

Kupper

Meisel³

et al. 1998

Journal of Biological Chemistry

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EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA. To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction was observed at dimer/site ratios of 0.25 and 0.5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites. A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch. Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance. At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.Concerning their substrate requirements, certain restriction endonucleases (ENases) 1 that interact at a distance with at least two DNA recognition sites resemble proteins involved in processes like transcription control, DNA recombination, and replication in both prokaryotes and eukaryotes (1-6). In addition to EcoRII, the first ENase for which this special characteristic was described (Ref. 7; for a review see 8), comprehensive studies were also performed on NaeI. Electron microscopy of DNA-NaeI complexes revealed loop structures connecting NaeI sites on the same DNA molecule (9). Another extraordinary type II ENase is SfiI, whose tetrameric enzyme structure bridges two specific recognition sites and cleaves them simultaneously (10, 11). A few other ENases are considered to belong to this particular ENase group (12, 13) termed IIe, characterized by requiring a second DNA (effector) site to perform cleavage (14).The complex mode of action of these particular ENases appears to limit their efficiency in DNA restriction and may reflect additional biological functions besides the destruction of invading DNA. Other type II ENases (non IIe) like EcoRI and EcoRV use the simpler and more powerful mechanism of linear diffusion along the helical pitch of the DNA that enables them to find every single recognition site and effectively restrict foreign DNA (15, 16). Some experimental results support the idea of an evolutionary relationship of type IIe ENases with other protein families; Jo and Topal (17) discovered a sequence motif shared by ENase NaeI and the active site of human DNA ligase where the essential lysine 43 of the ligase is replaced by leucine in NaeI. A single amino acid substitution, L43K, in NaeI converts the ENase to a DNA topoisomerase with pref...

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Section: Resultssupporting

confidence: 78%

Cooperative Binding Properties of Restriction Endonuclease EcoRII with DNA Recognition Sites

Reuter

Kupper

Meisel³

et al. 1998

Journal of Biological Chemistry

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“…EcoRII restriction endonuclease was isolated in an electrophoretically homogeneous state according to Kosykh et al [11]. T4 polynucleotide kinase, T4 DNA ligase, restriction endonuclease EcoRI and AluI, terminal deoxynucleotidyl transferase (DNTFase) were from NIKTI BAV (Novosibirsk, USSR).…”

Section: Methodsmentioning

confidence: 99%

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages

et al. 1985

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“…EcoRI (22), EcoRII (23), Hha II (15), and Pst I (24). It is uncertain why attempts to clone other systems have not succeeded.…”

Section: Properties Ofpaer7mentioning

confidence: 99%

Cloned restriction/modification system from Pseudomonas aeruginosa.

Gingeras¹,

Brooks²

1983

Proc. Natl. Acad. Sci. U.S.A.

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DNA fragments from Pseudomonas aeruginosa carrying the PaeR7 restriction/modification genes have been cloned in the plasmid vector pBR322 and propagated in Escherichia coli A subclone (pPAORM3.8) has been constructed that contains the complete restriction/modification system on a 3.8-kilobase DNA fragment. Digestion of the pPAORM3.8 plasmid with nuclease BAL-31 has yielded two types of clones. One type contains an active methylase gene but no active endonuclease gene; such clones will modify the DNA but not restrict the growth of incoming phage in vivo. The second type contains an active endonuclease gene but no active methylase gene, as judged both by in vivo tests and by the activity ofthe cell extracts in vitro. Although extracts of cells containing these plasmids display restriction endonuclease activity, these bacteria are unable to restrict the growth ofincoming phage. Furthermore, chromosomal and phage DNA isolated from these host cells are not protected against cleavage by PaeR7 in vitro. The properties of PaeR7 endonuclease and methylase enzymes have also been examined. The PaeR7 restriction endonuclease recognizes and cleaves the sequence C J T-C-G-A-G, as does Xho I. However, there exists a canonical Xho I site at 26.5% on the adenovirus 2 genome which is totally refractory to PaeR7 cleavage but is cut by Xho I. Under conditions of low salt, high glycerol, and high enzyme concentrations, a "PaeR7*" activity is found that is similar to that observed for EcoRI. Finally, evidence is presented that the PaeR7 methylase modifies the adenine residue within the recognition sequence.In many bacteria, restriction/modification systems produce strain-specific enzymes that allow host cells to recognize and destroy foreign DNA. This is accomplished by a restriction endonuclease that makes double-strand scissions at a limited number of specific sites on the DNA. In addition to restriction activity, such bacterial strains possess a corresponding DNA methylase that modifies specific adenine or cytosine residues within the sequence recognized by the nuclease. The methylation protects the cell's DNA against the action of its own restriction enzymes (1-4).The genes for restriction/modification systems may be encoded on chromosomal, phage, or plasmid DNAs (1). The PaeR7 system belongs to the third category. It is encoded by a 42-kilobase (kb) plasmid, pMG7, carried in Pseudomonas aeruginosa (5). We were able to use the system's plasmid location to facilitate its cloning.We have used molecular cloning to transfer the PaeR7 restriction/modification system into Escherichia coli. Furthermore, we have been able to divide the system and generate two types of subclones: those that express only the methylase gene, and those that express only the endonuclease gene. The viability of the latter class of clones is surprising because the chromosomal DNA from these clones is susceptible to PaeR7 cleavage. Restriction Enzymes and 5' Mono-and Dinucleotide Analysis. The restriction endonucleases Sal I, HincII, BstNI, EcoRI, BamHI...

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Molecular cloning of EcoRII endonuclease and methylase genes

Cited by 43 publications

References 6 publications

Cooperative Binding Properties of Restriction Endonuclease EcoRII with DNA Recognition Sites

Cooperative Binding Properties of Restriction Endonuclease EcoRII with DNA Recognition Sites

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages

Cloned restriction/modification system from Pseudomonas aeruginosa.

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