C-terminal deletion mutants of the FokI restriction endonuclease

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

Research output: Contribution to journalArticle

Abstract

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
JournalGene
Volume133
Issue number1
DOIs
StatePublished - Oct 29 1993

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DNA Restriction Enzymes
Mutant Proteins
DNA
endodeoxyribonuclease FokI
Hydroxyl Radical
Trypsin
High Pressure Liquid Chromatography
Escherichia coli

Keywords

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

ASJC Scopus subject areas

  • Genetics

Cite this

C-terminal deletion mutants of the FokI restriction endonuclease. / Li, Lin; Wu, Louisa P.; Clarke, Robert; Chandrasegaran, Srinivasan.

In: Gene, Vol. 133, No. 1, 29.10.1993, p. 79-84.

Research output: Contribution to journalArticle

Li, Lin ; Wu, Louisa P. ; Clarke, Robert ; Chandrasegaran, Srinivasan. / C-terminal deletion mutants of the FokI restriction endonuclease. In: Gene. 1993 ; Vol. 133, No. 1. pp. 79-84.
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