title: Analysis in Escherichia coli of the effects of in vivo CpG methylation catalyzed by the cloned murine maintenance methyltransferase.
abstract: Due in part to the complexity of mammalian systems, some of the proposed biological influences of mammalian DNA methylation have not been fully established. Escherichia coli cells, which normally contain negligible CpG methylation, exhibited progressive slowing of replication and lengthened generation times when expressing the murine DNA maintenance methyltransferase. Genomic analysis indicated significant amounts of CpG methylation in expressing cells which was absent from control cells. Expressing cells exposed to the cytosine demethylating agent, 5-azacytidine, rapidly reverted to propagation levels of controls. Substitution of cysteine with alanine in the carboxyl-terminal region proline-cysteine dipeptide of the methyltransferase completely inactivated methylating activity and cells expressing the inactive enzyme replicated as well as controls. These findings strongly implicate a role of epigenetic de novo CpG methylation in modulating cellular propagation, demonstrate that the maintenance methyltransferase can de novo methylate in vivo, and show that the methyltransferase requires an active site cysteine for activity.
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599, USA. trygve@med.unc.edu
authors.forename: T O
authors.initials: TO
authors.lastname: Tollefsbol
authors.forename: C A
authors.initials: CA
authors.lastname: Hutchison
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D051379:Mice
chemical_list: D004269:DNA, Bacterial; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D003545:Cysteine; D001374:Azacitidine