Páginas: 34 (8485 palabras) Publicado: 13 de noviembre de 2011
Research Article


Mammalian nuclei become licensed for DNA replication during late telophase
Daniela S. Dimitrova1,*,§, Tatyana A. Prokhorova2,‡, J. Julian Blow2, Ivan T. Todorov3 and David M. Gilbert1
1Department 2CRC

of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA Chromosome Replication Research Group, TheWellcome Trust Building, University of Dundee, Dundee, DD1 5EH, UK 3Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center, 1500 Duarte Road, Duarte, CA 91010, USA
*Present address: Department of Biological Sciences, Cooke Hall, North Campus, SUNY at Buffalo, Buffalo, NY 14260 ‡Present address: Department of Biological Chemistry and Molecular Pharmacology, HarvardMedical School, Boston, MA 02115 §Author for correspondence (e-mail:

Accepted 3 October 2001 Journal of Cell Science 115, 51-59 (2002) © The Company of Biologists Ltd

Summary Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatinfor replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized atvarious times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites ofinitiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in thepreservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place duringtelophase, several hours prior to the specification of replication origins at the DHFR locus.

Key words: Mammalian nuclei, Mcm proteins, Replication licensing, ODP, Cell cycle

Introduction The minichromosome maintenance proteins (Mcm 2-7) have been shown to be key participants in the mechanism that limits eukaryotic replication to once-per-cell-cycle (Tye, 1999). In particular, the association ofMcm proteins with chromatin appears to be the final step in the formation of pre-replication complexes (pre-RCs) in a process referred to as replication licensing (Rowles and Blow, 1997). The molecular details of pre-RC assembly are beginning to emerge (Kelly and Brown, 2000; Leatherwood, 1998). The origin recognition complex (ORC) recruits Cdc6 which, along with Cdt1 (Maiorano et al., 2000b;Nishitani et al., 2000; Tada et al., 2001), is required for the subsequent loading of the Mcm 2-7 complex (Coleman et al., 1996; Romanowski et al., 1996; Rowles et al., 1996; Tanaka et al., 1997). The formation of pre-RCs takes place during late mitosis and early G1-phase and is prevented by the presence of cyclin-dependent kinase (CDK) activity (Coverley et al., 1996; Noton and Diffley, 2000) and...
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