A DNA Methyltransferase Can Protect the Genome from Postdisturbance Attack by a Restriction-Modification Gene Complex

Abstract

In prokaryotic genomes, some DNA methyltransferases form a restriction-modification gene complex, but some others are present by themselves. Dcm gene product, one of these orphan methyltransferases found in Escherichia coli and related bacteria, methylates DNA to generate 5-C m CWGG just as some of its eukaryotic homologues do. Vsr mismatch repair function of an adjacent gene prevents C-to-T mutagenesis enhanced by this methylation but promotes other types of mutation and likely has affected genome evolution. The reason for the existence of the dcm-vsr gene pair has been unclear. Earlier we found that several restriction-modification gene complexes behave selfishly in that their loss from a cell leads to cell killing through restriction attack on the genome. There is also increasing evidence for their potential mobility. EcoRII restriction-modification gene complex recognizes the same sequence as Dcm, and its methyltransferase is phylogenetically related to Dcm. In the present work, we found that stabilization of maintenance of a plasmid by linkage of EcoRII gene complex, likely through postsegregational cell killing, is diminished by dcm function. Disturbance of EcoRII restriction-modification gene complex led to extensive chromosome degradation and severe loss of cell viability. This cell killing was partially suppressed by chromosomal dcm and completely abolished by dcm expressed from a plasmid. Dcm, therefore, can play the role of a "molecular vaccine" by defending the genome against parasitism by a restriction-modification gene complex.Epigenetic methylation of DNA is a prominent phenomenon in many organisms. It occurs after passage of the replication fork and involves enzymatic transfer of a methyl group to specific bases on the DNA. The specific methylation of certain cytosine residues to 5-methylcytosine (5-m C) has been shown to affect gene expression in several organisms. In several eukaryotes, C-5 methylation is known to inactivate transposons and may have been maintained as a mechanism to defend the genome from genomic parasites (51). In the prokaryotes, many DNA methyltransferases are found as a component of a restriction-modification (RM) gene complex. Some methyltransferases are found paired with a restriction enzyme gene inactivated by a mutation (20,25,30), while others are not paired with a restriction enzyme homologue. Dcm in Escherichia coli and related bacteria is one of these "orphan" methyltransferases (39).In the E. coli chromosome, approximately 1% of the cytosine residues are methylated by Dcm to generate the sequence 5Ј-C m CWGG (W ϭ A or T) (39). 5-m C can deaminate to form thymine, which can lead to the production of C-to-T mutations. This mutation is prevented by the product of a contiguous gene, vsr, through the very-short-patch mismatch repair reaction (28,46). However, Vsr can increase other types of mutation under some other conditions, and the actions of Dcm and Vsr most likely have affected genome evolution (6,11,13,29,31). The reason for the existence of the dcm-vsr gene pair...

Genomics of DNA cytosine methylation in Escherichia coli reveals its role in stationary phase transcription

“…Dcm confers resistance against parasitism by the EcoRII R-M system. This is because, in the presence of a functional Dcm, EcoRII-target sites will be kept methylated even when the R-M system is disturbed, thus preventing post-segregational killing 24 . To our knowledge, this, an association between Dcm and Tn3 transposition 25, and lambda DNA recombination 26 , and a very recently discovered role in regulating ribosomal gene expression in stationary phase 27 , remain the only phenotypes associated with Dcm.…”

Maintenance Forced by a Restriction-Modification System Can Be Modulated by a Region in Its Modification Enzyme Not Essential for Methyltransferase Activity

“…Table 2 shows colony formation of E. coli strain BNH670 (⌬dcm) or GM31 (dcm-6) harboring a plasmid with various versions of the EcoRII RM gene complex. The cells carrying pNY30, which has an EcoRII RM gene complex with the L80P mutation in the methylase gene (65), showed a drastic decrease in viability at 42°C, while the cells possessing its r Ϫ version (pNY31) did not. We observed the same tendency in the absence of selective antibiotics with pUC derivatives (Table 2) and pACYC184 derivatives (data not shown).…”

“…The generation number was calculated from the viable cell count. The procedure has been detailed elsewhere previously (65).…”

“…Plasmids were constructed as follows. The ecoRIIRM genes were amplified from plasmid N3 using the primers RII1 and RII2 (65), which contain an XmaI site. The amplified fragment was digested with XmaI and inserted into the corresponding site of pUC19 or pHSG415 and designated pOS28 or pOS30, respectively.…”

“…In a similar way, postsegregational killing by an RM system would be prevented by a solitary methyltransferase that recognizes the same sequence. This was demonstrated for the EcoRII RM system, a type II system with a frequently occurring recognition sequence of 5Ј CCWGG (where W is A or T), and Dcm, a solitary methyltransferase found in the chromosome of Escherichia coli and related bacteria (65). Dcm was hypothesized to have evolved to serve as a molecular vaccine against parasitism by EcoRII isoschizomers.…”