EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE 2 HUMAN PRIMASESUBUNITS AND TRUNCATED COMPLEXES FROM ESCHERICHIA-COLI

Eukaryotic DNA replication is primed by small RNA primers synthesized by the two-subunit primase complex, p58 and p49, where the p49 subunit contains the catalytic activity. The cDNA's for these two human DNA p rimase subunits were amplified, sequenced, and overexpressed in Escher ichia coli. Specific assays for initiation revealed that although the smaller subunit contains catalytic function, initiation requires the p resence of the larger subunit. A two-plasmid system was developed for the coexpression of both subunits in E. coli. This system was exploite d to express and study truncations of the larger, human p58 subunit in order to investigate its role in primer formation. Analysis of the co mplexes formed between the truncated human p58 subunits and the native human p49 subunit revealed that protein-protein contacts between thes e two subunits occur over several regions of the human p58 subunit. Of four primase complexes containing different truncated p58 subunits on ly one complex supported initiation as measured by the formation of di nucleotides. All complexes supported the extension of oligoA-primed po lydT, suggesting that the intrinsic RNA polymerase activity residing i n the smaller subunit was not affected. These results suggest that sev eral regions of the human p58 subunit are in contact with the human p4 9 subunit during the initiation of primer synthesis. (C) 1997 Academic Press.