The in vitro assembly of the EcoKI type I DNA restriction/modification enzyme and its in vivo implications

D T F Dryden, L P Cooper, P H Thorpe, O Byron

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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 M(2)S(1) methylase or as a R(2)M(2)S(1) 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 M(1)S(1), whereas the complete nuclease is a stable structure. 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 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.

Original languageEnglish
Pages (from-to)1065-1076
Number of pages12
Issue number5
Publication statusPublished - 4 Feb 1997


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