Determination of binding sites of replication protein A (RPA) on the promoter of glycogen phosphorylase 2 gene in Dictyostelium discoideum

Abstract

Replication protein A (RPA) has multifunctional roles in the cell including DNA replication, DNA repair, recombination and regulation of transcription. RPA has been identified in numerous organisms having a highly conserved protein structure, composed of three subunits of around 70, 32, and 14 kDa. The cellular slime mold, Dictyostelium discoideum, is a social amoeba used as a model organism in studies including the ones aimed at understanding cell differentiation and cellular pathways involved in human diseases and this organism has a homolog of RPA (DdRPA). Glycogen phosphorylase 2 (encoded by gp2) is involved in cell differentiation and DdRPA has been suggested to bind a part of the gp2 promoter as shown by gel shift assays. In this study, in vitro footprint analysis has been used to identify the binding sites of the DdRPA protein, purified from amoeba and slug stages of the organism, on the gp2 promoter. The results indicate that DdRPA binds to the nucleotides in the C box region, the TAG box region, TA box-1 (-608bp) and upstream regions (-636bp). Thus, DdRPA behaves like a general DNA binding protein under these conditions, binding to several regions on the gp2 promoter. However, the TAG box region has been identified as the binding region for the DdRPA from amoeba cells, but not for the DdRPA from slug cells. This suggests that DdRPA could possibly be involved in the regulation of gp2 gene expression during cell differentiation in Dictyostelium discoideum.