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J. Biol. Chem., Vol. 268, Issue 35, 26179-26189, Dec, 1993
WC Copeland and TS Wang
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.
Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication
Department of Pathology, Stanford University School of Medicine, California 94305.
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