The isolated 48,000-dalton subunit of yeast DNA primase is sufficient for RNA primer synthesis.
The monoclonal antibody (mAb) 21A6, which specifically inhibits yeast DNA primase activity, has been used to verify whether only one of the two polypeptides of heterodimeric DNA primase (48 and 58 kDa) was responsible for DNA primase function in vitro. Immunoaffinity chromatography of a crude extract from cells of Saccharomyces cerevisiae on a mAb 21A6 protein A-Sepharose 6B column allowed the purification of the p48 primase polypeptide in an isolated form. This polypeptide was not derived through the dissociation of the four-subunit DNA polymerase alpha-primase complex, which can be purified from the same extract by affinity chromatography with a mAb recognizing the DNA polymerase alpha polypeptide. Therefore, free p48 was already present in the yeast extract and, possibly, within the cell. Isolated p48, devoid of any detectable p58 subunit, was sufficient for RNA primer synthesis, although free primase appeared to extend RNA primer-monomers to primer-multimers less efficiently. Primase activity associated with free p48 was highly unstable, indicating that although p48 bears the catalytic site, its association with the other polypeptides of the polymerase-primase complex plays an important role in stabilizing enzyme activity.
Santocanale
C
C
Dipartimento di Genetica e di Biologia dei Microrganismi, Milano, Italy.
Foiani
M
M
Lucchini
G
G
Plevani
P
P
D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D004257:DNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D019915:DNA Primase; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D009500:Neutralization Tests; D012313:RNA / Q000096:biosynthesis*; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; C034477:RNA primers; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II