Process for producing recombinant McrBC endonuclease and cleavage of methylated DNA
First Claim
1. A recombinant modified cytosine restriction endonuclease, McrBC, obtainable from Escherichia coli comprising two active components McrBL and McrC, wherein the endonuclease cleaves a methylated DNA fragment at the Rm5C (N40-100) Rm5C recognition site.
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Abstract
The present invention relates to a recombinant McrBC endonuclease obtainable from Escherichia coli, two components of which, McrBL and McrC, have been purified in active form. McrBC is active in the presence of GTP and at a low pH. The McrBC endonuclease is also substantially free of a third component, McrBS, which is believed to inhibit or otherwise interfere with the activity of the enzyme. McrBC has various desirable properties, including the ability to recognize a methylated DNA sequence and also its ability to cleave such a sequence in the presence of GTP. Also provided is a process for the production of recombinant McrBC endonuclease, a process for the determination of the modification state of DNA a process for the determination of an epigenetic alteration or defect (including "imprinting"), as well as a process for identifying and isolating additional enzymes that cleave modified DNA.
47 Citations
17 Claims
- 1. A recombinant modified cytosine restriction endonuclease, McrBC, obtainable from Escherichia coli comprising two active components McrBL and McrC, wherein the endonuclease cleaves a methylated DNA fragment at the Rm5C (N40-100) Rm5C recognition site.
- 3. A construct comprising a nucleic acid sequence encoding the McrBL component of the McrBC endonuclease, wherein said construct substantially does not express McrBS.
- 4. A construct which is pER276.
- 9. A construct comprising a nucleic acid sequence encoding the McrBL and McrC component of the McrBC endonuclease, wherein said construct substantially does not express McrBS.
- 13. A method for producing recombinant McrBC having two active components McrBL and McrC, comprising isolating a nucleic acid sequence encoding McrBL and nucleic acid sequence encoding McrC, inserting the isolated DNA into the same or separate vectors to form one or more recombinant vectors, transforming a host cell with the one or more recombinant vectors and culturing the transformed host cells under conditions suitable for expression of the active components of the McrBC nuclease.
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16. A method of treating cytosine-modified DNA with recombinant endonuclease, McrBC, which has two active components McrBL and McrC, comprising determining a recognition sequence of the McrBC endonuclease for a cytosine-modified DNA, mapping locations for McrBC cleavage, and digesting the cytosine-modified DNA substrate with McrBC endonuclease.
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17. A method of cleaving cytosine-modified DNA having the sequence Rm5C (N40-100) 5m5c with recombinant endonuclease McrBC which has two active components, McrBL and McrC, comprising contacting the cytosine-modified DNA with the McrBC endonuclease under suitable conditions.
Specification