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Modificatory DNA incision enzyme and its application method

A cleavage enzyme, unmodified technology, applied to modified DNA cleavage enzymes and their application fields, can solve problems such as difficulty in identifying common structural features, dissociation enzyme toxicity, etc.

Active Publication Date: 2007-01-31
NEW ENGLAND BIOLABS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This broad substrate specificity makes it difficult to identify common structural features of substrates selectively recognized and cleaved by enzymes (White, M.F., et al. J. Mol. Biol 269:647-664 (1997))
This broad substrate specificity also leads to toxicity of the resolvase in host cells

Method used

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  • Modificatory DNA incision enzyme and its application method
  • Modificatory DNA incision enzyme and its application method
  • Modificatory DNA incision enzyme and its application method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Example 1: Mutations at bridge sites and overexpression of the modified enzyme

[0121] Materials and methods

[0122] Restriction enzyme, nickase N.BstNB I, DNA polymerase, T4 ligase, T4 DNA kinase, β-agarase, λ exonuclease, maltose binding protein including plasmid pMAL-c2x (MBP) protein fusion expression and purification system, host Escherichia coli strains TB1 and ER2566, factor Xa protease, plasmid pUC(AT) containing a cruciform structure, and plasmid LITMUS28 were obtained from New England Biolabs Inc., Beverly , MA). Synthetic oligonucleotides were synthesized using standard techniques. T7 phage DNA encoding T7Endo I has a sequence corresponding to SEQ ID NO:1.

[0123] Recombinant DNA and Mutagenesis Methods

[0124] DNA manipulation and site-directed mutagenesis (Kunkel, Proc.Natl.Acad.Sci.USA.82 (2): 488-92 (1985Jan)) or PCR method according to Sambrook et al. Molecular Cloning (Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: a Laboratory Man...

Embodiment 2

[0154] Example 2: Characterization of two deletion mutants ME(PA / A) and ME(ΔPA)

[0155] 1. Cutting of cruciform DNA

[0156] [104] The stable cruciform structure on negatively supercoiled plasmids is structurally similar to the four-way linker of DNA. T7 Endo I decomposes both DNA structures with high efficiency (Parkinson, M.J. and Lilley, D.M.J.J. Mol. Biol. 270:169-178 (1997)). To compare the enzymatic activity between the different T7 Endo I mutants, we used cleavage of the cruciform-containing plasmid pUC(AT) as substrate to define the specific activity. One unit of activity is defined as the conversion of 1 μg of supercoiled pUC(AT) to a linear form (two-strand cleavage) or a nicked form (single-strand cleavage) within 30 minutes at 37°C in a 20 ml reaction. ) the amount of enzyme required. We measure specific activity by enzyme titration assay. result in figure 2 given in.

[0157] [105] in Mg 2+ Buffer, ME and ME(PA / A) have roughly the same specific activity, ...

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Abstract

Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with T7 Endo I. The modifed enzyme includes two catalytic centers separated by a beta-bridge where the beta-bridge contains at least one mutation having an effect of altering enzyme cleavage activity compared to the unmodified enzyme. Activities associated with the modified DNA cleaving enzyme that can be modulated in different reaction conditions include at least one of: (a) non-sequence specific nicking activity; (b) cleaving the second strand of a duplex DNA at a preexisting nick site to produce a linear duplex with a single strand overhang; (c) non-sequence specific DNA cleavage; (d) cleaving DNA flanking a mismatch; and (e) cleavage at a cruciform structure in a DNA duplex.

Description

Background of the invention [0001] [001] Recombination between sequences on two different dimers of DNA, or between sequences on the same DNA dimer separated by an intervening sequence, occurs in all self-replicating cells . Four-way Holliday junctions in DNA are a feature of recombination reactions and are produced in homologous as well as site-specific recombination reactions (Lilley, D.M.J. PNAS 94, 9513-9515( 1997)). The penultimate stage of recombination involves catalytic disassembly of the four-way linker. This catalysis is accomplished by structure-specific nucleases called resolvases (Aravind, L., et al. Nuc. Acid Res. 28:3417-3432 (2000)). Resolvase is ubiquitous in cells and is also expressed by viruses. Resolvases have various properties. Crystal structures of many resolvase enzymes have been reported, including that of T7 endonuclease I (T7Endo I) (Hadden, J.M., et al. Nat. Struct. Biol. 8:62-67 (2001)). [0002] [002] T7 Endo I has two catalytic centers tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12NC12N9/22C12Q1/68
CPCC12N9/22C12Q1/6827C12Q2521/301
Inventor C·关S·库马尔R·库塞拉
Owner NEW ENGLAND BIOLABS
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