TY - JOUR
T1 - The binding of ORC2 to chromatin from terminally differentiated cells
AU - Xu, H.
AU - Lu, Z. H.
AU - Leno, G. H.
N1 - Funding Information:
We thank Ron Laskey for providing antibodies to XORC2, Peter Jackson for the GST-xCyclin A and GST-hCdk2 expression plasmids, Susan Wellman for purified H10, Paul Fisher and Miguel Berrios for the T7 RNA polymerase and antiserum, Asmita Kumar for helpful discussions, and Jennifer Johns for technical assistance throughout this study. This work was supported by the National Science Foundation (MCB-9506280 to G.H.L.).
PY - 2002
Y1 - 2002
N2 - Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the pre-replication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.
AB - Nuclei from terminally differentiated Xenopus erythrocytes lack essential components of the pre-replication complex, including the origin recognition complex (ORC) proteins XORC1 and XORC2. In Xenopus egg extract, these proteins are able to bind erythrocyte chromatin from permeable nuclei, but not from intact nuclei, even though they are able to cross an intact nuclear envelope. In this report we use both permeable and intact erythrocyte nuclei to investigate the role of cyclin-dependent kinase activity in modulating the binding of XORC2 to chromatin. We find that elevating the level of cyclin A-dependent kinase in egg extract prevents the binding of XORC2 to chromatin from permeable nuclei and that kinase inhibition reverses this effect. We also observe a nuclear transport-dependent accumulation of H1 kinase activity within intact nuclei incubated in the extract. However, inhibiting this kinase activity does not facilitate the binding of XORC2 to chromatin, suggesting that other molecules and/or mechanisms exist to prevent association of XORC proteins with replication origins within intact nuclei from terminally differentiated cells.
KW - Chromatin
KW - Cyclin-dependent kinase
KW - Erythrocyte nuclei
KW - Nuclear envelope
KW - Origin recognition complex
KW - Terminal differentiation
KW - Xenopus egg extract
UR - http://www.scopus.com/inward/record.url?scp=0036349529&partnerID=8YFLogxK
U2 - 10.1006/excr.2002.5484
DO - 10.1006/excr.2002.5484
M3 - Article
C2 - 11900493
AN - SCOPUS:0036349529
SN - 0014-4827
VL - 274
SP - 334
EP - 341
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 2
ER -