Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

Abstract

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K, k, k, and K for single-stranded and hemimethylated substrates, revealing discrimination of 10-fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

“…Lon exhibited an ∼14-fold higher affinity to DNA than to CcrM. Recent studies of DNA recognition by CcrM reported an equilibrium dissociation constant of 108 ± 20 nM for double-stranded DNA (22). A quantitative Western blot was performed to determine the in vivo concentration of CcrM and Lon at 120 min into the swarmer cell cycle, using purified CcrM and Lon to calibrate a standard curve.…”

Cell cycle-controlled clearance of the CcrM DNA methyltransferase by Lon is dependent on DNA-facilitated proteolysis and substrate polar sequestration

“…DNA Binding by CcrM is a prerequisite for its chromosome methylation activity. Given that DNA-bound Lon protease is active throughout the cell cycle, we hypothesized that CcrM, which has been shown to processively move on DNA (Berdis et al, 1998; Woodcock et al, 2017), could be recognized by Lon bound to DNA. To test this hypothesis, we attempted to reconstitute this interaction in vitro using three different DNA probes.…”

“…Thus, there is a ~14-fold-weaker affinity between LonS674A * and CcrM than between LonS674A * and DNA Probe 1. Recent studies on DNA recognition by CcrM reported an equilibrium dissociation constant of 108 ± 20 nM for double-stranded DNA (Woodcock et al, 2017). We performed a quantitative Western blot to determine the concentration of CcrM in vivo at the 120 minutes-post-synchrony timepoint, using purified CcrM to calibrate a standard curve (Figure S3D).…”

“…Although Lon is present and active throughout the cell cycle, CcrM transcription and translation is confined to the pre-divisional cell (Schrader et al, 2016; Zhou et al, 2015). At this time in the cell cycle, CcrM wins the race between synthesis and degradation and CcrM proceeds to processively methylate GANTC sites on the chromosome (Kozdon et al, 2013; Woodcock et al, 2017). When CcrM synthesis stops, Lon continues to clear CcrM from the cell.…”

The impact of DNA methylation in Alphaproteobacteria

“…This technique revealed that most GANTC motifs are efficiently methylated by CcrM on the C. crescentus genome, highlighting the efficiency of this enzyme (Kozdon et al ., ; Gonzalez et al ., ). It is unclear whether CcrM is a processive enzyme that remains attached to DNA and methylates multiple GANTC motifs before dissociating from the DNA (Berdis et al ., ; Shier et al ., ; Woodcock et al ., ) or a distributive enzyme that dissociates after each methylation reaction (Albu et al ., ; Maier et al ., ).…”