The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

Abstract

The specificity and processivity of DNA methyltransferases have important implications regarding their biological functions. We have investigated the sequence specificity of CcrM and show here that the enzyme has a high specificity for GANTC sites, with only minor preferences at the central position. It slightly prefers hemimethylated DNA, which represents the physiological substrate. In a previous work, CcrM was reported to be highly processive [Berdis et al. (1998) Proc. Natl Acad. Sci. USA 95: 2874–2879]. However upon review of this work, we identified a technical error in the setup of a crucial experiment in this publication, which prohibits making any statement about the processivity of CcrM. In this study, we performed a series of in vitro experiments to study CcrM processivity. We show that it distributively methylates six target sites on the pUC19 plasmid as well as two target sites located on a 129-mer DNA fragment both in unmethylated and hemimethylated state. Reaction quenching experiments confirmed the lack of processivity. We conclude that the original statement that CcrM is processive is no longer valid.

Bacteriophage Orphan DNA Methyltransferases: Insights from Their Bacterial Origin, Function, and Occurrence

“…Initially identified in Caulobacter crescentus, the cell cycle regulator methyltransferase (CcrM) targets the recognition sequence GANTC (38,39) in a nonprocessive manner (40), in contrast to earlier findings (39). The CcrM methylase is essential for the proper control of the life cycle of Caulobacter, as methylation of the ori directs initiation by DnaA (41).…”

DNA Methylation

“…However, it is instructive to briefly review DNA methylation in Caulobacter crescentus , a member of the alpha-Proteobacteria which has defined morphological stages. The DNA methyltransferase in this organism is CcrM which methylates adenine in the sequence 5′-GANTC-3′ [56] in a distributive manner [245]. Unlike Dam, CcrM is essential for the life of the organism, is cell cycle regulated [246], and is not present at all stages of the life cycle.…”

DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria

“…After the introduction of m6A marks in the context of GAnTC sequences [5] CcrM is proteolyzed prior to cell division by the Lon protease [7]. How the m6A marks, introduced by CcrM, affect transcription is unclear, but the marks are transient as DNA replication converts the (full) methylation on both DNA strands to the hemi-methylated state, until strands are re-methylated in a distributive manner [18] once CcrM has accumulated at the end of S-phase. The time a given genomic locus spends in the hemi-methylated state is thus pre-determined by its physical proximity to the origin of replication [19], a feature that might be exploited to couple activation of certain promoters, such as ctrA P1, with replication progression [19].…”