The in Vitro Assembly of the EcoKI Type I DNA Restriction/Modification Enzyme and Its in Vivo Implications

Abstract

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.

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

“…Convincing evidence that endonuclease activity is associated only with the R 2 M 2 S 1 complex was obtained much later (45). For a time it appeared that EcoR124I and -II, members of the IC family, have one rather than two R subunits and that they retain endonuclease activity in the absence of AdoMet (75).…”

ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems

“…HsdS confers sequence specificity to both the modification and restriction activities. For EcoKI, the R-M system found in E. coli K-12, the stoichiometry of the subunits is 2HsdR, 2HsdM and 1HsdS (Dryden et al, 1997), commonly abbreviated to R 2 M 2 S 1 , and this complex coexists in vivo with a smaller one, M 2 S 1 , endowed with only modification activity (Dryden et al, 1993). A similar relationship is expected for EcoAI.…”

“…Other experiments have shown a lag of many generations before transconjugants become restriction proficient, despite transcription of the hsd genes . Post-translational control, possibly at the level of subunit assembly, could be a critical factor in causing a delay in the production of the restriction enzyme, and Dryden et al (1997) have used their data from an in vitro assembly pathway as the basis of such a mechanism. In the assembly pathway they propose, both inactive intermediates in the assembly pathway and the HsdR polypeptide could be susceptible to proteases.…”

“…In the assembly pathway they propose, both inactive intermediates in the assembly pathway and the HsdR polypeptide could be susceptible to proteases. A mutation isolated in E. coli C, which results in cell death after the acquisition of hsd genes (PrakashCheng et al, 1993), has identified a gene, hsdC, the product of which is a candidate for influencing the assembly pathway of EcoKI (Dryden et al, 1997).…”

“…It has been suggested that when hsd genes enter a recipient cell proteases could contribute to a post-transcriptional mechanism for ensuring the sequential production of the modification methylase before the restriction endonuclease (Kulik and Bickle, 1996;Dryden et al, 1997;J. Ryu, personal communication).…”