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Effect of Cycloheximide on Regulatory Protein for Initiating Mammalian DNA Replication at the Nuclear Membrane

Laboratory of Medical Biophysics, Kobe University School of Medicine, Ikuta-ku, Kobe 650, Japan

Treatment of partially synchronized L5178Y murine leukemic cells with cycloheximide (CHX), 100 µg/ml, during the S period allows DNA synthesis to continue for only 3 hr; this is a consequence of drastic inhibition of protein synthesis, irrespective of times at which CHX was added. Alkaline sucrose sedimentation of the DNA from synchronized cells that were labeled with thymidine-³H for 3 hr revealed that CHX prevents the production of discontinuous 4 S segments but does not prevent normal chain growth up to 76 S of replicating DNA the synthesis of which has been already initiated. In addition, CsCl equilibrium density centrifugation of both density- and radioactively labeled DNA elucidates the mechanism of restriction of in vivo DNA synthesis by CHX. It selectively inhibits the initiation of DNA synthesis but not the continuation of synthesis of individual DNA units in a replicon-equivalent length, the completion of which requires nearly 3 hr. Furthermore, M-band technique shows that this specific inhibition of the initiation event may occur at specific sites on the nuclear membrane. Thus, these results suggest that in mammalian cells a regulatory protein exists that controls only initiation of DNA replication at the nuclear membrane, in addition to proteins as structural elements for nuclear replication that have been synthesized during the G₁ period.

Received 3/20/72. Accepted 6/15/72.