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J Biol Chem, Vol. 275, Issue 18, 13699-13707, May 5, 2000
From the Wellcome/CRC Institute, University of Cambridge, Tennis
Court Road, Cambridge CB2 1QR and the Department of Zoology,
University of Cambridge, Cambridge,CB2 3EJ United Kingdom
Initiation of human DNA replication is
investigated in vitro, using initiation-competent nuclei
isolated from cells arrested in late G1 phase by a 24-h
treatment with 0.5 mM mimosine (Krude, T. (1999) Exp.
Cell Res. 247, 148-159). Nuclei isolated from mimosine-arrested HeLa cells initiate semiconservative DNA replication upon incubation in
cytosolic extracts from proliferating human cells. Initiation occurs in
the absence and presence of a nuclear membrane. The cyclin-dependent kinase (Cdk) inhibitors roscovitine and
olomoucine inhibit initiation of DNA replication, indicating a
dependence of initiation on Cdk activity. Cell fractionation shows that
cyclins A, E, and Cdk2 are bound to nuclei from mimosine-arrested
cells. Exogenously added human cyclin A·Cdk2 and cyclin E·Cdk2
complexes, but not cyclin B1/Cdk1 or cyclin D2/Cdk6, can overcome
inhibition of initiation by roscovitine in vitro. Depleting
Cdk2 from cytosolic extract does not prevent initiation, demonstrating
that cyclin·Cdk2 complexes are not required in the soluble extract,
but are provided by the nuclei. Initiation depends further on an
essential and soluble activity present in cytosolic extracts from
proliferating cells, but not from mimosine-arrested cells, acting
together with nuclear cyclin/Cdk2 activity.
Initiation of Human DNA Replication in Vitro Using
Nuclei from Cells Arrested at an Initiation-competent State*
*
This work was supported by the Royal Society and the Cancer
Research Campaign.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Royal Society University Research Fellow. Tel.: 44-1223-334109;
Fax: 44-1223-334089; E-mail: tk1@mole.bio.cam.ac.uk.
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