Shier V K, Hancey C J, Benkovic S J
Pennsylvania State University, Department of Chemistry, University Park, Pennsylvania 16802, USA.
J Biol Chem. 2001 May 4;276(18):14744-51. doi: 10.1074/jbc.M010688200. Epub 2001 Feb 8.
Caulobacter crescentus contains one of the two known prokaryotic DNA methyltransferases that lacks a cognate endonuclease. This endogenous cell cycle regulated adenine DNA methyltransferase (CcrM) is essential for C. crescentus cellular viability. DNA methylation catalyzed by CcrM provides an obligatory signal for the proper progression through the cell cycle. To further our understanding of the regulatory role played by CcrM, we sought to investigate its biophysical properties. In this paper we employed equilibrium ultracentrifugation, velocity ultracentrifugation, and chemical cross-linking to show that CcrM is dimeric at physiological concentrations. However, surface plasmon resonance experiments in the presence of S-adenosyl-homocysteine evince that CcrM binds as a monomer to a defined hemi-methylated DNA substrate containing the canonical methylation sequence, GANTC. Initial velocity experiments demonstrate that dimerization of CcrM does not affect DNA methylation. Collectively, these findings suggest that CcrM is active as a monomer and provides a possible in vivo role for dimerization as a means to stabilize CcrM from premature catabolism.
新月柄杆菌含有两种已知的缺乏同源内切核酸酶的原核DNA甲基转移酶之一。这种内源性细胞周期调控的腺嘌呤DNA甲基转移酶（CcrM）对新月柄杆菌的细胞活力至关重要。由CcrM催化的DNA甲基化提供了细胞周期正常进展的必要信号。为了进一步了解CcrM所起的调节作用，我们试图研究其生物物理特性。在本文中，我们采用平衡超速离心、速度超速离心和化学交联来表明CcrM在生理浓度下是二聚体。然而，在S-腺苷同型半胱氨酸存在下的表面等离子体共振实验表明，CcrM作为单体与含有典型甲基化序列GANTC的特定半甲基化DNA底物结合。初速度实验表明，CcrM的二聚化不会影响DNA甲基化。总的来说，这些发现表明CcrM作为单体具有活性，并为二聚化作为一种稳定CcrM以免过早分解代谢的手段提供了可能的体内作用。
