Cloning and Characterization of Replication Protein A from Dictyostelium discoideum

Abstract

The gene encoding the Dictyostelium replication protein A large subunit (DdRPA1) has been cloned by screening of an EcoR I partial genomic library and a Hind III genomic sub-library. The complete nucleotide sequence, including the promoter region of the gene has been obtained by sequencing. Though the DdRPA1 protein has a size shift during development, 62 kDa in undifferentiated cells and 81 kDa in differentiated cells; they are the products of the same gene. Northern blot analysis revealed that the expression level of the DdRPA1 was constant throughout differentiation and the size of mRNA is the same at all stages, corresponding to a 81 kDa protein. Thus, it seems that the size change between the 62 kDa and 81 kDa is probably due to posttranslational modification, most likely, proteolytic cleavage. The transcription start site for both sizes of DdRPA1 has been identified at 306 bp upstream of the coding sequence by primer extension reaction.

A PCR fragment representing 27% of the gene encoding the DdRPA middle size subunit (DdRPA2) has been generated by using the degenerate primers. This PCR fragment has been cloned and sequenced. The mRNA for this subunit corresponds to a protein of about 35 kDa. A decrease of the DdRPA2 mRNA expression level during differentiation was found by comparison between undifferentiated and differentiated cells.

In Dictyostelium, replication protein A is a heterotrimeric protein that can bind with specific DNA sequences in a stage-dependent pattern. These DNA sequences were identified as the cis-acting regulatory sites in differentiation-related genes, including the glycogen phosphorylase 2 gene (gp2). Therefore, it is possible that DdRPA is not only a single-stranded DNA binding protein that is used in multiple essential DNA metabolic processes, such as DNA replication, repair and recombination in undifferentiated cells, but also involved in the transcriptional regulation process during differentiation.