TAILIEUCHUNG - Báo cáo khoa học: Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells

It was shown previously [Riedinger, H. J., van Betteraey-Nikoleit, M & Probst, H. (2002)Eur. J. , 2383–2393] that initiation ofin vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of repli-cation proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G1by starvation | Eur. J. Biochem. 270 3880-3890 2003 FEBS 2003 doi Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells Maria van Betteraey-Nikoleit Karl-Heinz Eisele Dirk Stabenow and Hans Probst Physiologisch-Chemisches Institut der Universitat Tubingen Germany It was shown previously Riedinger H. J. van Betteraey-Nikoleit M Probst H. 2002 Eur. J. Biochem. 269 2383-2393 that initiation of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of replication proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself we developed a protocol to extend these studies using the human bladder carcinoma cell line T24 which was presynchronized in G1 by starvation. Concomitantly with stimulation of the cells by medium renewal hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the hypoxic preinitiation state ready to initiate replication as soon as normal pO2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin including DNA-bound replication proteins while unbound proteins were removed. The usefulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol analyses of chromatin-bound MCM2 MCM3 Cdc6 and cdk2 suggests that the .

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