The CpG-specific methylase Sssl has topoisomerase activity in the presence of Mg²⁺

Abstract

A prokaryotic CpG-specific methylase from Spiroplasma, SssI methylase, is now widely used to study the effect of CpG methylation in mammalian cells, and can processively modify cytosines in CpG dinucleotides in the absence of Mg2+. In the presence of Mg2+, we found (i) that the methylation reaction is distributive rather than processive as a result of the decreased affinity of SssI methylase for DNA, and (ii) that a type I-like topoisomerase activity is present in SssI methylase preparations. This topoisomerase activity was still present in SssI methylase further purified by either SDS-polyacrylamide or isoelectric focusing gel electrophoresis. We show that methylase and topoisomerase activities are not functionally interdependent, since conditions exist where only one or the other enzymatic activity is detectable. The catalytic domains of SssI methylase and prokaryotic topoisomerases show similarity at the amino acid level, further supporting the idea that the topoisomerase activity is a genuine activity of SssI methylase. Mycoplasmas, including Spiroplasma, have the smallest genomes of all living organisms; thus, this condensation of two enzymatic activities into the same protein may be a result of genome economy, and may also have functional implications for the mechanism of methylation.

Evaluation of affinity-based genome-wide DNA methylation data: Effects of CpG density, amplification bias, and copy number variation

“…An attenuation of promoter microarray signal was observed at the highest CpG density regions, and notably, the attenuation seems to occur at a higher local CpG density for the MethylMiner-based procedure than MeDIP. As MeDIP recovers single-stranded DNA and MBDCap recovers double-stranded DNA, it is possible that topoisomerase activity from M.SssI may compromise amplification of DNA recovered from MeDIP relative to MBDCap (Matsuo et al 1994). However, we found that enrichment levels of methylated LNCaP DNA were also favored by MBDCap in comparison to MeDIP for CpG dense regions, albeit at reduced levels (Supplemental Fig.…”

Oxygen‐regulated erythropoietin gene expression is dependent on a CpG methylation‐free hypoxia‐inducible factor‐1 DNA‐binding site

“…simian virus 40 promoter and three concatamerized copies of the erythropoietin 3′ HBS (Fig. 3A), was methylated in vitro with the CpG-specific bacterial enzyme SssI methylase [30]. Unmeth-Erythropoietin expression in HepG2 cells is related to the degree of CpG methylation of the erythropoietin 3′ HBS.…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…An ATP addition does not influence the topoisomerase activity of М.SssI. Since type II topoisomerases need ATP for the enzymatic activity, М.SssI can be regarded as a type I topoisomerase [62]. The MTase and the topoisomerase activities of М.SssI are functionally independent.…”

“…Spiroplasma belongs to mycoplasmas -cellular organisms which have the smallest genome (from 600 to 1800 kbp). For a comparison, the T4 bacteriophage genome consists of 165 kbp and the E. coli genome -of 4600 kbp [62].…”