Abstract
The enzymatic mechanism of primase was investigated using Escherichia coli and baculoviral overexpressed mouse primase subunits, p49 and p58. Neither of the singly purified primase subunits displayed primase activity alone, but the p49 subunit was able to extend a riboprimer, indicating that this subunit contains an RNA polymerase activity. The p58 subunit cooperated with the p49 subunit in binding the initiating purine to form the initial dinucleotide. After initiation, the p49 subunit alone was sufficient to extend the growing primer, but both the rate of p49 primer extension and its stability were influenced by the p58 subunit. The Km(ATP) in primer synthesis on poly(dT) of the p49-p58 heterodimeric primase complex was 10-fold higher than the Km(ATP) of the single p49 subunit in a ribo(A) primer extension assay. In addition, labeled ATP cross-linked to both of the individually purified subunits but with a striking difference in affinities; cross-linking was 11-fold more efficient to the p49 subunit. The interaction of the two primase subunits with polymerase alpha was also investigated. Immunoprecipitation experiments indicate that only the p58 subunit directly contacts the p180 subunit of DNA polymerase alpha. Competition experiments in the coupled primase-polymerase assay with a catalytically inactive mutant of DNA polymerase alpha and the Klenow fragment suggest that the DNA polymerase alpha-primase complex does not dissociate from the primer during the transition from RNA to DNA synthesis.
Highlights
The enzymatic mechanism of primase was investi- and 49 kDa containing the associated primase activity
Amino acid alignment between the yeast p49 and the mouse p49reveals a 34% identity, where the greatest amount of similarity occurs in the amino-terminal halfof the two proteins (6). the original mouse p58 cDNA sequence has never been published’ (9),the cDNAs for all four subunits of the mouseFM3A polymerase a-primase complexhave recently been described (11).This mouse p58 amino acid sequence is 32% identical to the yeast homolog
In an attempt tiodentify the active sites of primase activity, It has been just over 1 decade since primase activity was identified in eukaryotic cells as an activity associated with the DNA polymerase a complex (1).Much progress has been accomplished in the localization and characterization of this labeled ribonucleotide triphosphate was specificallyUV crosslinked only to the smaller subunit of both the calf thymus (16) andSaccharomyces cerevisiue primases (17).in the case of the yeast primase complex, affinity labeling was further applied to include adenine nucleotide derivatives conactivity
Summary
CsCl gradient-purified transfer vectors extensive washing in 1 N HCl. In all reactions with high ATP carrying these p49 and p58 inserts were cotransfected into insect cells concentrations the MgCl, levels were maintained at a ratio of 2-fold with Bsu36I-linearized Acj3Gal baculoviral DNA as described (19). For 1h at 37 “C.After 1h isopropyl-l-thio-j3-D-galactopyranosiwdeas Photocross-linking-Isolated primase subunits were cross-linked added to a final concentration of 1mM, and cultures were grown an by either ultraviolet light at 254 nm similar to that described in (8, additional 2.5 h a t 30 “C.Cells were harvested and resuspended in 20 16) or with a UV Stratalinker 1800 (Stratagene) at 254 nm and 120 ml of 50 mM Tris.HC1, pH 8.0, 150 mM NaC1, 1%Triton X-100, 3 mg/ml lysozyme, and 2 pg/ml aprotinin. The polymerase-primase coupled assay mix contained polymerase a as well as the corresponding primase subunit for synthesis
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