On the structure and operation of type I DNA restriction enzymes.
Type I DNA restriction enzymes are large, molecular machines possessing DNA methyltransferase, ATPase, DNA translocase and endonuclease activities. The ATPase, DNA translocase and endonuclease activities are specified by the restriction (R) subunit of the enzyme. We demonstrate that the R subunit of the Eco KI type I restriction enzyme comprises several different functional domains. An N-terminal domain contains an amino acid motif identical with that forming the catalytic site in simple restriction endonucleases, and changes within this motif lead to a loss of nuclease activity and abolish the restriction reaction. The central part of the R subunit contains amino acid sequences characteristic of DNA helicases. We demonstrate, using limited proteolysis of this subunit, that the helicase motifs are contained in two domains. Secondary structure prediction of these domains suggests a structure that is the same as the catalytic domains of DNA helicases of known structure. The C-terminal region of the R subunit can be removed by elastase treatment leaving a large fragment, stable in the presence of ATP, which can no longer bind to the other subunits of Eco KI suggesting that this domain is required for protein assembly. Considering these results and previous models of the methyltransferase part of these enzymes, a structural and operational model of a type I DNA restriction enzyme is presented.
Davies
G P
GP
Institute of Cell and Molecular Biology, University of Edinburgh, The King's Buildings, Edinburgh, EH9 3JR, UK.
Martin
I
I
Sturrock
S S
SS
Cronshaw
A
A
Murray
N E
NE
Dryden
D T
DT
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D014364:Tryptophan / Q000378:metabolism
D010446:Peptide Fragments; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI; D015253:Deoxyribonucleases, Type I Site-Specific; D010196:Pancreatic Elastase; D004265:DNA Helicases