Cloning of Nt.CviQII nicking endonuclease and its cognate methyltransferase: M.CviQII methylates AG sequences

Siu hong Chan, Zhenyu Zhu, David D. Dunigan, James L. Van Etten, Shuang yong Xu

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)138-150
Number of pages13
JournalProtein Expression and Purification
Volume49
Issue number1
DOIs
StatePublished - Sep 2006

Keywords

  • Chlorella virus
  • Intein-mediated peptide ligation
  • Methyltransferases
  • Nicking endonucleases
  • Restriction-modification system

ASJC Scopus subject areas

  • Biotechnology

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