Hyperactive and hmC dominant TET enzymes
AssigneeThe Trustees of the University of Pennsylvania
InventorsRahul Kohli, Monica Yun Liu
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Abstract
The present invention includes mutant AID, APOBEC, and Tet enzymes with improved functions. In one aspect the invention provides APOBEC fusion proteins comprising hyperactive deamination activity. In another aspect, the invention provides AID mutant proteins comprising hyperactive deamination activity. In yet another aspect, the invention provides mutant Tet proteins capable of stalling oxidation at a 5-hydroxymethylcytosine (hmC).
Claims17
A mutant Ten-Eleven Translocation 2 (TET2) enzyme comprising the amino acid sequence of SEQ ID NO:
- 21 or 22 except having one or two mutations in a catalytic domain active site, said mutation comprising a substitution of a threonine residue at position number 258 or 248, respectively, which
- i) stalls oxidation at a 5-hydroxymethylcytosine (hmC) when said threonine is substituted for a glutamine, an asparagine, an aspartic acid, a glutamic acid, or a valine, or
- ii) accelerates production of 5-carboxylcytosine by at least 20% as compared to wild type enzyme activity when said threonine is substituted with a serine.
A mutant human TET1 enzyme comprising a catalytic domain having the amino acid sequence of SEQ ID NO:
- 130 wherein except a threonine at position 6 is substituted with an amino acid selected from a glutamine, an asparagine, an aspartic acid, a glutamic acid or a valine, said substitution altering stalling activity as compared to wild type enzyme activity or accelerates production of 5-carboxylcytosine when said threonine is substituted with a serine as compared to wild type TET1 enzymes lacking said substitution.
A mutant human TET3 enzyme comprising a catalytic domain of SEQ ID NO:
- 134 except having a substitution of a threonine at position 6, said mutation altering stalling activity as compared to wild type enzyme activity when said threonine is substituted with an amino acid selected from a glutamine, an asparagine, an aspartic acid, a glutamic acid or a valine or accelerating production of 5-carboxylcytosine as compared to wild type enzyme activity when said threonine is substituted with a serine.
A mutant TET2 enzyme of SEQ ID NO:
- 22 or 21, wherein the mutant TET enzyme consists of
- (1) T248S/Q/N/D/E/V amino acid substitution or T258S/Q/N/D/E/V amino acid substitution, respectively, and, or a
- (2) Y778F amino acid substitution or Y440F amino acid substitution respectively, and, or
- (3) T248A+Y778F amino acid substitutions or T258A+Y440F amino acid substitutions, respectively.
Dependencies are inferred from the claim text.