Cloning of Nt.CviQII nicking endonuclease and its cognate methyltransferase: M.CviQII methylates AG sequences.
Chlorella virus NY-2A has a large, highly methylated dsDNA genome (45% of the cytosines are 5-methylcytosine and 37% of the adenines are N(6)-methyladenine). Here, we report the cloning, expression, and characterization of the NY-2A-encoded CviQII nicking-modification (N-M) system. The nicking endonuclease, Nt.CviQII, recognizes R downward arrowAG (R=A or G, downward arrow indicating cleavage site) sequences and cleaves the phosphodiester bond 5' to the adenosine. Because of the difficulty in cloning and expressing the wild-type Nt.CviQII, C-terminal truncation mutants were generated and full-length Nt.CviQII was reconstructed by intein-mediated peptide ligation. The truncation mutants and the reconstructed full-length Nt.CviQII have the same recognition and cleavage specificity as the native enzyme. Full-length and truncated Nt.CviQII produced by a cell-free transcription/translation system have similar reaction rates. The methyltransferase, M.CviQII, was also cloned and expressed. It modifies the adenine in AG doublets of DNA in vitro and in vivo in Escherichia coli. To our knowledge, M.CviQII is the first adenine methyltransferase that recognizes a dinucleotide. Therefore, M.CviQII may be a useful reagent for blocking endonuclease cleavage when restriction sites overlap with AG sequences.
Chan
Siu-hong
SH
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Zhu
Zhenyu
Z
Dunigan
David D
DD
Van Etten
James L
JL
Xu
Shuang-yong
SY
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D019175:DNA Methylation; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism
D014764:Viral Proteins; D008780:Methyltransferases; D004720:Endonucleases