The in vitro assembly of the EcoKI type I DNA restriction/modification enzyme and its in vivo implications.
Type I DNA restriction/modification enzymes protect the bacterial cell from viral infection by cleaving foreign DNA which lacks N6-adenine methylation within a target sequence and maintaining the methylation of the targets on the host chromosome. It has been noted that the genes specifying type I systems can be transferred to a new host lacking the appropriate, protective methylation without any adverse effect. The modification phenotype apparently appears before the restriction phenotype, but no evidence for transcriptional or translational control of the genes and the resultant phenotypes has been found. Type I enzymes contain three types of subunit, S for sequence recognition, M for DNA modification (methylation), and R for DNA restriction(cleavage), and can function solely as a M2S1 methylase or as a R2M2S1 bifunctional methylase/nuclease. We show that the methylase is not stable at the concentrations expected to exist in vivo, dissociating into free M subunit and M1S1, whereas the complete nuclease is a stable structure. The M1S1 form can bind the R subunit as effectively as the M2S1 methylase but possesses no activity; therefore, upon establishment of the system in a new host, we propose that most of the R subunit will initially be trapped in an inactive complex until the methylase has been able to modify and protect the host chromosome. We believe that the in vitro assembly pathway will reflect the in vivo situation, thus allowing the assembly process to at least partially explain the observations that the modification phenotype appears before the restriction phenotype upon establishment of a type I system in a new host cell.
Dryden
D T
DT
Institute of Cell & Molecular Biology, University of Edinburgh, U.K. David.Dryden@ed.ac.uk
Cooper
L P
LP
Thorpe
P H
PH
Byron
O
O
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI