Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

Abstract

HP0593 DNA-(N6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5′-GCAG-3′and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

Determination of cell uptake pathways for tumor inhibitor lysyl oxidase propeptide

“…Fluorescence emission was collected by using Tristar LB 941 plate reader at 592 nm. From a modified Stern‐Volmer plot (Banerjee and Rao, 2011) it was determined that an 80 times higher concentration of CML‐BSA‐QSY9 quenched 95% of Atto565 fluorescence of rLOX‐PP‐Atto565 and chicken egg white lysozyme‐Atto565 independent of cell size in all cell lines tested (data is not shown). To test efficacy of quenching method, DU145 and PC3 cells were next treated with 0.2 μM rLOX‐PP‐Atto565 or 0.2 μM chicken egg white lysozyme‐Atto565 for 3 h washed with PBS for 3 times and treated with 16 μM CML‐BSA‐QSY9 for 15 min at 4 °C and replenished media with PBS and imaged using Zeiss Axiovert 100M inverted fluorescence microscope (Figure S5B–C).…”

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…For example, MTases of H. pylori are known to selectively alter transcript levels of some genes, e.g., the genes of the dnaK operon and catalase (210,212). Similarly, the target sites of an acid-adaptive MTase (HP0593) have been found to be in the promoter regions of physiologically important genes (213). Recent studies have also shown a regulatory role for the phase-variable modH gene of a type III R-M system in H. pylori (214).…”

N6-Methyladenine DNA Modification in Human Genome

“…In vitro methylation reactions were performed as previously described with minor modification (Banerjee and Rao, 2011 The underlined A in the four oligos were mutated to G and produced the A>G mutant oligos.…”