Induction of EcoRII methyltransferase: evidence for autogenous control

Abstract

The cytosine analog 5-azacytidine kills Escherichia coli cells that carry plasmids expressing EcoRII DNA (cytosine 5)methyltransferase under control of its own promoter. We previously showed that this enzyme binds tightly to azacytidine-containing DNA in vitro and proposed that such binding is lethal in vivo. In support of this proposal, we now show that the enzyme sediments with the nucleoid of azacytidine-treated cells. Azacytidine treatment led to an increase in the amount of enzyme, and this increase required sequences in the ecoRIIM promoter region. Enzyme inducibility correlated with drug sensitivity: plasmids carrying the methyltransferase gene but lacking the wild-type promoter did not confer sensitivity. These results suggested that the ecoRIIM gene was under autogenous control. Transcriptional ecoRJIM'-lacZ fusions in E. coli were, therefore, constructed. They showed that expression from the ecoRIIM promoter was inhibited when EcoRU DNA (cytosine-5)methyltransferase was introduced into the cell in trans and inhibition was reversed by treating the cells with azacytidine. These results provide evidence that the expression of the ecoRIIM gene is under autogenous regulation and that cell death induced by azacytidine is due, in part, to the disruption of autoregulation.5-Azacytidine (azaCyd) is a nucleoside analog that selectively kills Escherichia coli when the bacteria carry plasmids that specify the EcoRII restriction-modification system (7). The killing is dependent on the presence of the methyltransferase (MTase) and not the restriction enzyme. This phenomenon was also demonstrated when other MTases were cloned and expressed in E. coli on multicopy plasmids (2, 16). The effect appeared to be dependent on the amount of MTase present in the cell (7). With the endogenous Dcm MTase there was little effect of the drug. When this MTase was cloned onto a multicopy plasmid and introduced into E. coli, the strain became sensitive to azaCyd (2). A similar but less dramatic effect was also noted when the MspI MTase, cloned in pBR322, was introduced into E. coli (16).Cells deficient in any of a number of different repair enzymes have enhanced sensitivity to azaCyd. These results imply that the drug causes DNA damage. Support for this conclusion includes the observations that the drug is a weak inducer of the SOS system and is a mutagen in E. coli (1, 16). Although the drug is known to undergo ring opening under mild conditions, it has not been shown that DNA strand breaks occur in vivo.We have shown that the drug inhibits DNA (cytosine-5)methyltransferases (5, 6) once the drug is incorporated into DNA in the sequence CC(A/T)GG (9). We proposed that the basis for cell death was the binding of the enzyme to azacytosine-containing DNA (azaC-DNA), which would inhibit the ability of the cell to repair DNA damage caused by azaCyd (13). In support of this suggestion, we demonstrated that DNA containing azaCyd will not undergo recA-mediated strand exchange if the EcoRII DNA (cytosine-5)methyltransferase (M.EcoRII) is boun...

Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion

“…The proposed sequence of expression of RMC genes-expression of M gene and C gene before that of R gene-is reminiscent of the orchestrated series of events that occur during bacteriophage infection, and especially during the establishment of lysogeny. RM systems without a C regulatory gene probably possess some alternative means of ensuring that M gene expression occurs prior to R gene expression (34)(35)(36)(37).…”

“…(iii) All of the RM systems are expected to have some mechanism to delay restriction during establishment in a host as discussed above (18,(34)(35)(36)(37). Such a regulatory machinery would allow evolution of mutual exclusion mediated by apoptosis if the machinery were associated with RMs of different sequence specificities.…”

Characterization of the intergenic region which regulates the MspI restriction-modification system

“…Inhibition was greater (73%) when M.MspI in trans was expressed from a multicopy plasmid. The repression is much less than the 96 to 97% repression caused by autoregulation of ecoRIIM-lacZ ϩ by M.EcoRII (2,12). M.BsuFI, an isoschizomer of M.MspI with a 56% amino acid homology (16) expressed by the plasmid pBW201, did not have any effect in trans on expression from the mspIM-lacZ ϩ fusion.…”

“…Appropriate regulation of the RM genes is important for the survival of the host. Such regulation has recently been demonstrated for several RM systems, including BamHI (4), PvuII (14), and EcoRII (2,12,13).…”

5-Azacytidine–Induced Methyltransferase-DNA Adducts Block DNA Replication In vivo

“…However, aza-C substitution at the target cytosine interferes with the reaction cycle and results in long-lived or irreversible MTase-DNA adducts (6)(7)(8). Evidence for adduct formation in vivo has been presented (9,10).…”