Expression, purification, and characterization of the two human primase subunits 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 primase subunits were amplified, sequenced, and overexpressed in Escherichia coli. Specific assays for initiation revealed that although the smaller subunit contains catalytic function, initiation requires the presence of the larger subunit. A two-plasmid system was developed for the coexpression of both subunits in E. coli. This system was exploited to express and study truncations of the larger, human p58 subunit in order to investigate its role in primer formation. Analysis of the complexes formed between the truncated human p58 subunits and the native human p49 subunit revealed that protein-protein contacts between these two subunits occur over several regions of the human p58 subunit. Of four primase complexes containing different truncated p58 subunits only one complex supported initiation as measured by the formation of dinucleotides. All complexes supported the extension of oligoA-primed polydT, suggesting that the intrinsic RNA polymerase activity residing in the smaller subunit was not affected. These results suggest that several regions of the human p58 subunit are in contact with the human p49 subunit during the initiation of primer synthesis.
Copeland
W C
WC
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D019915:DNA Primase; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D018076:DNA, Complementary; C034477:RNA primers; D011994:Recombinant Proteins; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases