DNA-Protein Interactions

Our research is focused on two fields:

1) Development of research tools for targeted DNA methylation

Targeted DNA methylation, a technique designed to methylate predetermined genomic sites, is a potentially useful approach to study the role of DNA methylation in the regulation of eukaryotic gene expression. Unfortunately, current versions of the method are not sufficiently specific, i.e. non-addressed sites get also methylated. We study the sources of off-target methylation in E. coli and in vitro using two CG specific bacterial DNA methyltransferases (M.MpeI and M.SssI), and try to develop methods, which allow higher methylation specificity. In a related project we try to alter the specificity of M.MpeI and/or M.SssI to obtain mutant enzymes whose specificity has changed from CG to CA and/or CC. The desired enzymes would be valuable research tools for studying non-CG specific DNA methylation in eukaryotes.

2) A simple E. coli assay system to detect sequence specific DNA-protein binding

We have developed a simple method, called I-Block assay, which can detect sequence-specific binding of proteins to DNA in E. coli. The method works by measuring the result of competition between the tested protein and RNA polymerase for binding to overlapping target sites in a modified lacI promoter. Binding of the tested protein to its target site interferes with transcription of the lacI gene leading to increased expression of β-galactosidase. The I-Block assay can, in its current state, test whether a protein can bind to a particular DNA sequence in E. coli. We are trying to develop the method into a high throughput technique, which can be used to determine target sites of sequence specific DNA binding proteins.

For further information please visit http://sysbiol.brc.hu/kiss-antal-lab-index.html