Cloning of the Arabidopsis thaliana poly (Adp-ribose) polymerase 2 cdna gene in Saccharomyces cerevisiae

Abstract

 Poly(ADP-ribose)polymerase (PARP) catalyzes the synthesis of covalently attached ADP-ribose polymers to acceptor proteins. The donor of the residues of ADP-ribose is NAD+. The prominent roles of PARP is a DNA damage sensor. PARP1, in poly(ADP-ribosyl)ated form binds to DNA breaks and engages proteins of  DNA repair to this sites. Recently, it has been shown that PARP poly(ADP-ribosyl)ates both proteins and DNA breaks. However, there is no direct evidence of  poly(ADP-ribosyl)ated  DNA adducts in vivo in animals and plants.

PARP activity is absent in Saccharomyces cerevisiae cells, despite the fact that it has been identified in organisms ranging from archaebacteria to mammals. Due to the simplicity of manipulation and genetic interpretation S. cerevisiae yeasts have been used to analyze the functions of proteins from animal and plant cells. Therein, the goal of our work is the cloning and heterologous expression of the AtPARP2 gene from the Arabidopsis thaliana in yeasts.

By using genetic engineering  methods, we have constructed a recombinant plasmid with cDNA of the AtPARP2 gene under the control of the inducible promoter GAL10 from episomal vector pESC-LEU2. A recombinant S.cerevisiae strain capable for expressing the cDNA of the AtPARP2 gene was obtained by genetic transformation method. By using Western blotting with the applying of polyclonal anti-AtPARP2 antibodies, it was revealed that the product of gene expression in yeast is a globular protein weighing 72kDa, consisting  637 amino acids (pI 5.92). A slight inhibition of growth was shown in FF 18733 yeast cells which express AtPARP2. Induction of  AtPARP2 expression in yeast revealed PARylating  activity of protein in vivo. Auto-poly(ADP-ribosyl)ation activity of AtPARP2 was shown in yeasts in response to double-stranded  DNA breaks induced by bleomycin treatment of cells. 3-AB, inhibitor of PARP suppressed the amount of poly(ADP-ribosyl)ated protein products.

Key words: Poly(ADP-ribose)polymerase, AtPARP2, poly(ADP-ribosyl)ation, Arabidopsis thaliana, Saccharomyces cerevisae.