McrA variants and uses thereof
View Patent ↗Compositions and methods are provided in which the composition is a protein with at least 50% but less than 100% amino acid sequence identity with McrA or is a variant McrA protein with at least one amino acid sequence modification. The variant or protein has the property of cleaving DNA with methylated cytosine and not hydroxymethylated cytosine in a target DNA sequence, or substantially lacks catalytic activity while maintaining binding activity. Methods are provided in which the protein or McrA variant are used to identify methylation sites either by cleavage or by binding to the methylation site in the presence of a marker or by binding to an immobilized protein or McrA variant.
1. An isolated E. coli McrA variant able to cleave DNA comprising methylcytosine but not hydroxymethylcytosine, the variant comprising an amino acid sequence which differs from the amino acid sequence of its parent E. coli McrA polypeptide solely by:
(a) a modification at a position corresponding to amino acid 67 of SEQ ID NO: 2; or
(b) a modification at a position corresponding to amino acid 67 of SEQ ID NO: 2 and at one or more positions selected from the group consisting of those corresponding to amino acids 3, 15, 20, 58, 60, 74, 118, 128, 157, 158, 159, 167, 174, and 177 of SEQ ID NO: 2.
2. The E. coli McrA variant according to claim 1 , wherein the E. coli McrA variant is encoded by a gene which results from making one or more mutations in the polynucleotide of SEQ ID NO: 1.
3. A method for separating methylated DNA from unmethylated DNA, comprising:
(a) immobilizing one or more E. coli McrA variants according to claim 1 ;
(b) contacting the one or more E. coli McrA variants with a DNA sample;
(c) selectively binding DNA containing methylated cytosine residues; and
(d) separating methylated DNA from unmethylated DNA.
4. A method for identifying methylated DNA comprising:
(a) binding one or more E. coli McrA variants according to claim 1 to a DNA substrate; and
(b) identifying the extent and optionally the location of binding of the E. coli McrA variants by means of a reporter tag, thus identifying the DNA substrate as methylated.
5. The method according to claim 4 , wherein the reporter tag is a fluorescent, chemiluminescent or radioactive tag.
6. A method for selectively cleaving methylated DNA, comprising mixing DNA with a preparation comprising the E. coli McrA variant of claim 1 under conditions suitable for cleavage of the DNA at methylation sites in the DNA.
7. The method according to claim 6 , wherein the nucleotide sequences of the methylation sites are selected from the group consisting of C 5m CGG, C 5m CSGG, and 5m CG.