Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase alpha-primase complex and their gene expression during cell proliferation and the cell cycle

Abstract

The DNA polymerase alpha-primase complex purified from mouse FM3A cells is composed of four polypeptides with molecular masses of 180, 68, 54, and 46 kDa. The largest subunit has DNA polymerase activity, the two smallest subunits have DNA primase activity, and the function of the 68-kDa subunit is unknown. We have isolated the cDNAs of the four subunits by low stringency hybridization and reverse transcription polymerase chain reaction and determined their nucleotide sequences. The predicted amino acid sequence of the 180-kDa subunit shows 88, 38, 34, and 32% identity to those of the catalytic subunits of human, Drosophila melanogaster, Schizosaccharomyces pombe, and Saccharomyces cerevisiae DNA polymerase alpha, respectively, and contains seven regions whose orders and sequences are highly conserved among viral and other eukaryotic DNA polymerases. The deduced amino acid sequence of the 68-kDa subunit shows 25% identity to that of the 73-kDa subunit of D. melanogaster DNA polymerase alpha-primase, shows no significant sequence similarity to any other protein in the data bases, but contains a potential phosphorylation site(s) for cdc2 kinase. The amino acid sequence of the 54-kDa subunit shows 32% identity to that of the large subunit of S. cerevisiae DNA primase. During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In growing mouse FM3A cells, the transcripts of the four subunits are present throughout the cell cycle and increase slightly prior to the S phase.