TY - JOUR
T1 - C-terminal deletion mutants of the FokI restriction endonuclease
AU - Li, Lin
AU - Wu, Louisa P.
AU - Clarke, Robert
AU - Chandrasegaran, Srinivasan
N1 - Copyright:
Copyright 2014 Elsevier B.V., All rights reserved.
PY - 1993/10/29
Y1 - 1993/10/29
N2 - 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.
AB - 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.
KW - Catalytic domain
KW - Flavobacterium okeanokoites
KW - hydroxyl radical footprinting
KW - protein-DNA interaction
KW - recognition domain
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U2 - 10.1016/0378-1119(93)90227-T
DO - 10.1016/0378-1119(93)90227-T
M3 - Article
C2 - 8224897
AN - SCOPUS:0027518230
SN - 0378-1119
VL - 133
SP - 79
EP - 84
JO - Gene
JF - Gene
IS - 1
ER -