Enzymes and systems for synthesizing DNA
The disclosure provides novel topoisomerases which exhibit weakened ability to form a covalent bond to the 5′-(C/T)CCTT-3′recognition site in the presence of increased NaCl concentrations relative to wild-type, together with novel methods for synthesizing DNA in the 3′ to 5′ direction using the novel topoisomerase; and other compounds, compositions, methods and devices comprising or utilizing topoisomerases which exhibit reduced covalent bond formation to the 5′-(C/T)CCTT-3′ recognition site in the presence of increased NaCl concentrations, relative to wild-type.
1. A topoisomerase comprising the following sequence:
SEQ ID NO: 3
RALFYKDGKLFTDNNFLNPVSDDNPAYEVLQHVKIPTHLTDVVVYEQTWE
EALTRLIFVGSDSKGX 1 RX 2 X 3 FYGKMHVQNX 4 NAKRDRIFVRVYNVMKR
INCFINKNIKKSSTDSNYQLAVFMLMETMFFIRFGKMKYLKENETVGLLT
LKNKHIEISPDEIVIKFVGKDKVSHEFVVHKSNRLYKPLLKLTDDSSPEE
FLFNKLSERKVYECIKQFGIRIKDLRTYGVNYTFLYNFWTNVKSISPLPS
PKKLIALTIKQTAEVVGHTPSISKRAYMATTILEMVKDKNFLDVVSKTTF
DEFLSIVVDHVKSSTDG
wherein at least two of X 1 , X 2 , X 3 , and X 4 , are mutated from the residues in the wild type sequence [wherein in the wild-type sequence, X 1 is arginine (R), X 2 is glutamine (Q), X 3 is tyrosine (Y), and X 4 is arginine (R)] to glycine (G), alanine (A) or valine (V);
provided that where only two residues are mutated, X 1 and X 2 are not both mutated to A.
2. The topoisomerase of claim 1 wherein at least three or all of four of X 1 , X 2 , X 3 , and X 4 are mutated to A.
3. The topoisomerase of claim 1 wherein
X 2 and X 4 are A; or
X 1 , X 2 , and X 4 are A; or
X 1 , X 3 , and X 4 are A.
4. The topoisomerase of claim 1 which comprises one or more isolation sequences.
5. The topoisomerase of claim 1 which comprises SEQ ID NO: 4.