Generation of poly(ADP-ribosyl)ation deficient mutants of the transcription factor, CTCF.

Generation of the mutated versions of proteins deficient for poly(ADP-ribosyl)ation (PARylation) is a major challenge because there is no clearly defined consensus site for PARylation. In this chapter, we describe possible strategies to produce such mutants, demonstrated by the example of CTCF, a transcription factor. To achieve this in our study, the protein domain modified by PARylation was mapped and the amino acids, which can be potentially PARylated, selected. Mutations of such individual amino acids as either single or combinatorial mutations were introduced by site-directed mutagenesis, using mutagenic primers and the wild-type sequences as a template. Mutants were validated by DNA sequencing and assessed for the presence of the PARylation mark. The latter was achieved by ectopic expression of mutated proteins in cells, followed by immunoprecipitation with the polyclonal anti-PAR antibody and Western analysis with a protein-specific antibody. The PARylation-deficient CTCF mutant was identified and compared with the wild-type protein. Based on several general characteristics (nuclear distribution/localisation, stability and levels of expression in the cell), the PARylation-deficient mutant was comparable with the wild-type CTCF.