TY - JOUR
T1 - Cloning of Nt.CviQII nicking endonuclease and its cognate methyltransferase
T2 - M.CviQII methylates AG sequences
AU - Chan, Siu hong
AU - Zhu, Zhenyu
AU - Dunigan, David D.
AU - Van Etten, James L.
AU - Xu, Shuang yong
N1 - Funding Information:
We thank Richard Roberts, Iain Murray, and James Samuelson for helpful discussions and critical reading of the manuscript, Tom Evans for advice on the construction of C-terminal deletion variants, Jim Gurnon for isolating NY-2A DNA, Don Comb for support and encouragement. This work was partly supported by NIH National Human Genome Research Institute phase II STTR Grant 9R42HG003976-02. The CviQII N-M DNA sequences have been deposited in GenBank with Accession No. DQ355510 .
PY - 2006/9
Y1 - 2006/9
N2 - Chlorella virus NY-2A has a large, highly methylated dsDNA genome (45% of the cytosines are 5-methylcytosine and 37% of the adenines are N6-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 ↓ AG (R = A or G, ↓ 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.
AB - Chlorella virus NY-2A has a large, highly methylated dsDNA genome (45% of the cytosines are 5-methylcytosine and 37% of the adenines are N6-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 ↓ AG (R = A or G, ↓ 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.
KW - Chlorella virus
KW - Intein-mediated peptide ligation
KW - Methyltransferases
KW - Nicking endonucleases
KW - Restriction-modification system
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U2 - 10.1016/j.pep.2006.04.002
DO - 10.1016/j.pep.2006.04.002
M3 - Article
C2 - 16737828
AN - SCOPUS:33746830910
SN - 1046-5928
VL - 49
SP - 138
EP - 150
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -