[Substrate specificity of restriction endonuclease Eco781].
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Abstract:
The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.Keywords:
Isoschizomer
Cleave
Recognition sequence
Cleavage (geology)
Homing endonuclease
Sequence (biology)
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Homing endonuclease
Isoschizomer
Recognition sequence
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Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.
Homing endonuclease
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Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4-8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.
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FokI
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Abstract Type II restriction endonuclease activities detected in various Neisseria species were characterized for sequence specificity and precise site of cleavage. Nsi CI isolated from N. sicca C351 cleaves the sequence 5′-GAT↓ATC-3′ ( Eco RV isoschizomer); Nme CI from N. meningitidis C114 and Nph I from N. pharyngis C245 cleave 5′-N↓GATCN-3′ ( Mbo I isoschizomers); Ngo PII and Ngo PIII from N. gonorrhoeae P9-2 cleave at 5′-CC↓GCGG-3′ ( Sac II isoschizomer) and 5′-GG↓CC-3′ ( Hae III isoschizomer), respectively. Chromosomal DNA isolated from these strains and two other N. meningitidis strains (which lacked detectable endonuclease activities), was found to be refractive to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases.
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The recombinant plasmid pGEM4Z-ras DNA which was methylated on dam and dcm sites outside the PvuII recognition sequence was digested with restriction endonuclease PvuII, and one of the three PvuII sites was about 16-fold less efficient to cleave than either of the other two. On the contrary, the three PvuII sites were cleaved at about the same rate on the unmethylated DNA molecule. The results show that the cleavage inhibition of the methylated DNA on the certain PvuII site was caused by methylation outside the PvuII recognition sequence. Maybe a adjacent methylated dam site *A was responsible for the less efficient cleavage. This observation suggests that methylation outside the recognition sequence may be considered a new factor in the kinetic experiment of restriction endonuclease.
Cleavage (geology)
Recognition sequence
Cleave
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The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.
Isoschizomer
Cleave
Recognition sequence
Cleavage (geology)
Homing endonuclease
Sequence (biology)
Cite
Citations (1)
New restriction endonucleases, Bsp153AI and BspM39I, were isolated from Bacillus species strains 153A and M39, respectively. The enzymes recognize and cleave the nucleotide sequence [sequence: see text] and are true isoschizomers of restriction endonuclease PvuII.
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Homing endonuclease
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Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.
Homing endonuclease
Nuclease
FokI
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Cleavage (geology)
Linker
Zinc finger nuclease
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Isoschizomer
Recognition sequence
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