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 m ethylation 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 m odification phenotype apparently appears before the restriction phenot ype, but no evidence for transcriptional or translational control of t he genes and the resultant phenotypes has been found. Type I enzymes c ontain three types of subunit, S for sequence recognition, M for DNA m odification (methylation), and R for DNA restriction(cleavage), and ca n function solely as a M(2)S(1) methylase or as a R(2)M(2)S(1) bifunct ional methylase/nuclease. We show that the methylase is not stable at the concentrations expected to exist in vivo, dissociating into free M subunit and M(1)S(1), whereas the complete nuclease is a stable struc ture. The M(2)S(1) form can bind the R subunit as effectively as the M (2)S(1) methylase but possesses no activity; therefore, upon establish ment of the system in a new host, we propose that most of the R subuni t will initially be trapped in an inactive complex until the methylase has been able to modify and protect the host chromosome. We believe t hat the in vitro assembly pathway will reflect the in vivo situation, thus allowing the assembly process to at least partially explain the o bservations that the modification phenotype appears before the restric tion phenotype upon establishment of a type I system in a new host cel l.