C-terminal deletion mutants of the FokI restriction endonuclease

Lin Li, Louisa P. Wu, Robert Clarke, Srinivasan Chandrasegaran

Research output: Contribution to journalArticle


We have constructed two C-terminal deletion mutants of the FokI restriction endonuclease by using the polymerasechain-reaction technique and expressed them in Escherichia coli. The two mutant proteins (MP) of 41 and 30 kDa, were purified to homogeneity and their DNA-binding properties were characterized. The 41-kDa MP specifically binds the DNA sequence, 5′-GGATG 3′-CCTAC, like the wild-type (wt) FokI, but does not cleave DNA. The 30-kDa MP does not bind DNA. The affinity of the 41-kDa MP for the DNA substrate is comparable to that of wt FokI. The 41-kDa MP interacts with its substrate like the wt FokI, as revealed by hydroxyl radical footprinting experiments. In the presence of a DNA substrate, the 41-kDa MP is cleaved by trypsin into a 30-kDa N-terminal fragment and an 11-kDa C-terminal fragment. Addition of the HPLC-purified 11-kDa C-terminal fragment to the 30-kDa MP restores its sequence-specific DNA-binding property. These results confirm that the N-terminal 41-kDa fragment of the FokI ENase constitutes the DNA recognition domain of the ENase.

Original languageEnglish (US)
Pages (from-to)79-84
Number of pages6
Issue number1
StatePublished - Oct 29 1993


  • Catalytic domain
  • Flavobacterium okeanokoites
  • hydroxyl radical footprinting
  • protein-DNA interaction
  • recognition domain

ASJC Scopus subject areas

  • Genetics

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